NZ612450B2 - Method for enhancing engraftment of haematopoetic stem cells - Google Patents

Abstract

Disclosed is a composition comprising at least one prostacyclin analogue together with an unspecific cAMP activating agent and stimulated haematopoietic stem cells. Also disclosed is a method for enhancing engraftment of haematopoietic stem cells (HSCs) by an ex vivo pretreatment of the HSCs which comprises the following steps: a. using a sample containing haematopoietic stem cells; b. admixing to said sample at least one prostacyclin analogue to obtain a mixture; c. incubating said mixture for a period of time sufficient to stimulate G alpha-s-signalling in said cells. Further disclosed is the use of a composition comprising at least one prostacyclin analogue in the production of a composition for the treatment of individuals suffering from bone marrow disease transplanted with haematopoietic stem cells prepared according to the above method. h comprises the following steps: a. using a sample containing haematopoietic stem cells; b. admixing to said sample at least one prostacyclin analogue to obtain a mixture; c. incubating said mixture for a period of time sufficient to stimulate G alpha-s-signalling in said cells. Further disclosed is the use of a composition comprising at least one prostacyclin analogue in the production of a composition for the treatment of individuals suffering from bone marrow disease transplanted with haematopoietic stem cells prepared according to the above method.

Description

Method for enhancing engraftment of haematopoetic stem cells The present invention provides a novel method for enhancing engraftment of haematopoetic stem cells by an ex-vivo atment comprising the steps of ing a sample ning haematopoetic stem cells and admixing a prostacyclin analogue to obtain a mixture, incubating said mixture for a period of time sufficient to stimulate G alphas-signalling in said cells and optionally ing said stimulated cells. r, a composition comprising Treprostinil for use in the treatment of individuals undergoing haematopoetic stem cell transplantation is provided.

Hematopoietic stem cells (HSCs) are primitive cells capable of regenerating all blood products throughout the life of an individual, balancing their self-renewal with y differentiation. HSCs transition in on during development and circulate in s throughout life, moving into and out of the tream to engage bone marrow niches in sequential steps of homing, engraftment and retention. Homing is the process by which the donor stem cells find their way to the bone marrow, engrafting of stem cells means their growth in bone marrow.

Hematopoietic stem cells have therapeutic potential as a result of their capacity to restore blood and immune cells in transplant recipients. Furthermore, HSCs have the potential to generate cells for other tissues such as brain, muscle and liver. Human autologous and allogeneic bone marrow transplantation methods are currently used as therapies for diseases such as leukemia, lymphoma, and other life-threatening diseases. For these procedures, a large amount of donor bone marrow must be isolated to ensure that there are enough HSCs for engraftment.

Haematopoetic stem cells need a Gas-transduced signal in vivo to populate the bone marrow niche (1). These recent findings confirm earlier in vitro experiments, which showed that the activation of Goas promote the al and differentiation of haematopoetic stem cells (2,3). Goas is the guanine nucleotide binding oc-subunit of the heterotrimeric G protein that stimulates all 9 isoforms of membrane-bound mammalian adenylyl cyclase. Goas can be constitutively ted ex vivo/in vitro by treating the cells with cholera toxin, because a toxin APD-ribosylates the catalytic arginine residue (R186’187/201/202, the precise number of the arginine depends on the splice t of Gocs); an intact arginine residue is required for GTP-hydrolysis and the resulting deactivation of Goas (4). Enhanced engraftment can indeed be observed after pretreatment of haematopoetic stem cells with cholera toxin: there were about twice as many (Lin') precursor cells in the bone marrow, if the stem cell ation had been pretreated with cholera toxin (1). r, for stem cell preparation for patients undergoing bone marrow transplantation, cholera toxin would be highly disadvantageous. Cholera toxin (CT), esp. its non-toxic B subunit pentamer moiety (CTB) is a mucosal adjuvant having strong immunomodulatory properties both in vivo and in vitro and is one of the most potent mucosal immunogens. Cholera toxin and CTB ate a strong intestinal lgA antibody response and long-lasting immunological memory. Based on this, CTB has become an important component in recently developed oral vaccines against cholera and diarrhea caused by enterotoxigenic E. coli. The strong genicity of CT and CTB can to a large extent be explained by their ability to bind to receptors on the intestinal mucosal surface.

Additionally, during the l course of the infection the pentameric part B of the toxin molecule binds to the surface of inal epithelium cells and is rapidly tosed together with the A subunit. Once endocytosed, the catalytically active A—subunit detaches from the pentameric part B and enters the cell via a pore that is formed by the B-subunit. Once inside the cell, it permanently ribosylates the Gs alpha subunit of the heterotrimeric G protein ing in constitutive cAMP production. This in turn leads to secretion of H20, Na“, K“, Cl', and HC03' into the lumen of the small intestine resulting in rapid dehydration and other factors associated with cholera. lf ed intravenously, cholera toxin is taken up by most cells (only cells such as the blood brain barrier are shielded by specific barriers). Accordingly, it will raise cAMP in most body cells and cause a large range of side effects (ranging from ardia to vasodilation, muscle tremor, hyperglycaemia etc.).

Thus, these effects make cholera toxin unfeasible for human use with regard to stimulation of haematopoetic stem cells.

The therapeutic nce of stimulating stem cells is however important when undergoing heterologous bone marrow lantation (i.e., haematopoetic stem cells harvested from immunocompatible donors) and is a standard procedure that is used for the treatment of people suffering from leukaemia, for the treatment of people with genetic defects in the blood cell compartment (e.g., haemoglobinopathies such as thalassaemia; defects in phil granulocyte function etc.).

It is also ant as gous bone marrow transplantation is a standard procedure that is used to increase the therapeutic window of cytotoxic drugs and thus to allow for high dose intensity chemotherapy (5,6).

Haematopoetic stem cells express all four glandin E receptors (EP1-4). The pretreatment of haematopoetic stem cells with (dimethylated) prostaglandin E2 enhances their engraftment (7,8). This effect is mediated by cal Gocs- dependent signalling, because the nduced activation of protein kinase A (PKA) synergizes with Wnt-dependent signals to stabilize B-catenin (9).

Implantation of autologous bone marrow (BM) mononuclear cells (MNCs) could be enhanced by Beraprost sodium in a rabbit model according to Otsuka et al. The aim of the study was to support artery development in peripheral and myocardial ischemia. Haemopoietic stem cells, however, are a specific type of cells within the bone marrow cells (16).

A combination of stem cell therapy and pharmacological treatments is described by Madonna et al., wherein prostacyclin is tested in ADSC myocardial engrafting after intracoronary administration (17). lshii M. et al. disclose that sustained release of prostacyclin ed the proangiogenic function of mesenchymal stem cells and muscle cell growth in ischemic tissue (18).

W02006/O17169 describes an implatable sensor with a patible coating for controlling tissue growth which may contain, amongst , prostacyclin-analogs.

The inhibitory effect of Cicaprost or lloprost on the synthesis of tissue factor, tumor is factor and interleukin-1% in human THP1 cells is described in Crutchley D. et al. (19).

The localization of stem cells ing transplantation is a critical determinant of success of the transplantation. At present, a high number of stem cells is needed for transplantation because stem cells are not successfully engrafted in the bone marrow and there is a long period of bone marrow aplasia leading to a decrease of mature blood cells.

It is thus still an unmet need to provide methods and compositions to stimulate HSCs to increase homing, engraftment and retention of isolated HSCs to bone marrow niches of subjects undergoing bone marrow transplantations.

The problem is solved by the embodiments of the present invention.

Brief description of the invention: It has been surprisingly shown that the synthetic prostacyclin l2 (PGIZ) analogues like for example Treprostinil, lloprost, Beraprost and Cicaprost are capable of increasing cAMP levels in haematopoetic stem cells which leads to increased engraftment of stem cells in bone marrow.

A prostacyclin analogue like Treprostinil offers several advantages over prostaglandin E2: (i) It is a stable analogue of cyclin/PGIZ, which also stimulates EP2— und EP4- receptors (10). Thus it has the potential to stimulate le pled ors while not engaging inhibitory (i.e., Gi-coupled) EP3-receptors, which inhibit cAMP accumulation. For the latter, dimethyl-PGEZ is a full agonist (1). In contrast, Treprostinil is only a low ty agonist at EP3-receptors. (ii) Because they are lically more stable than natural prostacyclins, Treprostinil, st, Beraprost and Cicaprost can elicit more long lasting effects if applied in vivo and thus support more efficient bone marrow engraftment. (iii) Prolonged/repeated stration of a prostacyclin analogue, specifically of Treprostinil, lloprost, Beraprost and ost are well tolerated.

The t invention provides a novel method for enhancing engraftment of haematopoetic stem cells by an ex-vivo pretreatment which comprises the steps of a. obtaining a sample containing haematopoetic stem cells and b. admixing at least one prostacyclin analogue to obtain a mixture c. incubating said mixture for a period of time sufficient to stimulate G alphassignalling in said cells and optionally d. isolating and optionally purifying said stimulated cells. ing to an embodiment of the invention, the prostacyclin analogue is selected from the group of Treprostinil, lloprost, Cicaprost and Beraprost or pharmaceutically acceptable salts thereof. ing to the inventive method, a mixture of stimulated and unstimulated stem cells can also be provided.

Specifically, the sample is bone marrow. The stem cells can generally be of any source known, ically they can be HSCs isolated from eral blood, cord blood or bone .

According to the invention, stinil can be a derivative selected from the group of acid derivatives, prodrugs, polymorphs or isomers.

Similarly, lloprost, Cicaprost or Beraprost can be derivatives from the group of acid derivatives, prodrugs, polymorphs or isomers therefrom.

According to an alternative embodiment of the invention, the sample may be admixed with at least one prostacyclin analogue together with an unspecific cAMP activating agent, preferably ed from cholera toxin and lin.

According to a specific embodiment of the ion, a composition comprising a prostacyclin analogue is provided for use in the treatment of individuals suffering from bone marrow diseases which may undergo haematopoetic stem cell transplantation.

According to a specific embodiment of the invention, the prostacyclin analogue of the composition is selected from the group of Treprostinil, lloprost, Cicaprost or Beraprost or pharmaceutically acceptable salts thereof.

According to a preferred embodiment, the ition comprises Treprostinil.

Specifically, the individuals are suffering from leukaemia, a defect of the blood cell compartment, bone marrow transplantation after chemotherapy or irradiation.

According to a further embodiment of the invention, the defect of the blood cell compartment is haemoglobinopathy or a defect in neutrophil ocyte function.

The composition of the invention can also be used for the treatment of individuals ing from a bone marrow disease which are undergoing haematopoetic stem cell transplantation by administering a prostacyclin analogue for at least seven days after bone marrow transplantation. Specifically, a cyclin analogue selected from the group of Treprostinil, lloprost, Cicaprost and Beraprost is used, more specifically, Treprostinil.

According to a further embodiment, a ition comprising a prostacyclin analogue and stimulated haematopoetic stem cells is ed.

According to a further embodiment, a composition comprising Treprostinil or a pharmaceutically acceptable salt thereof and stimulated haematopoetic stem cells is provided.

Specifically for use in s or animal studies, the compositions of the invention can further comprise forskolin or cholera toxin.

The inventive compositions can be pharmaceutical compositions.

The inventive composition can be administered by all routes known in the art, ically it is ed for intravenous or subcutaneous administration.

The cyclin analogue can be provided in an orally available form selected from the group of sustained release forms, tablets or es.

The amount of the cyclin analogues depends on the therapeutic application or method for preparing stimulated HSCs. Very specifically, for therapeutic applications, the effective amount of Treprostinil is at least 1.0 ng/kg of body weight.

Figures: Figure 1: Characterization of the Lin+ cell population retained by the magnetic beads.

The x-axis (labelled APC-C7-A) denotes the fluorescence recorded by the mixture of antibodies ed against e markers: CD33 for T—cells, CD45R(=8220) for B- cells, CD11b and Ly-6G/Ly-6C (Gr-1) for the myeloid (monocytic/granulocytic) lineage, Ter-1 19 for the erythroid lineage. The y-axis is the fluorescence recorded for the dy against the murine stem cell factor receptor c-Kit. It is t that the upper left quadrant (denoting c-Kit+ and lineage marker-negative cells) is depleted of cells.

Figure 2: Characterization of the Lin' cell population that did not bind to the magnetic beads. As in Fig. 1, the x-axis (labelled APC-C7-A) denotes the fluorescence recorded by the mixture of antibodies directed against lineage markers: CD33 for T— cells, CD45R(=8220) for B-cells, CD11b and Ly-6G/Ly-6C (Gr-1) for the myeloid ytic/granulocytic) lineage, Ter-1 19 for the erythroid lineage. The y-axis is the fluorescence recorded for the dy against the murine stem cell factor receptor c- Kit. It is evident that the lower quadrant (denoting c-Kit-negative and lineage marker- positive cells) is depleted of cells and that cells are predominantly found in the left upper nt where the c-Kit-positive and lineage marker-negative cells are to be expected.

Figure 3 shows the characterization of the Lin+ (Fig. 3A) and Lin' cell tions (Fig. 3B) for the presence of Sca-1 and c-Kit. Panels 3A and SB show the cells retained on magnetic beads (characterized in Fig. 1) and that appeared in the flow through (characterized in Fig. 2). These were stained as outlined in the s section and analyzed simultaneously for the presence of c-Kit (PerCP-Cy5A, y- axis) and of Sca-1 (PE-Cy7-A, x-axis). It is evident that the lineage positive cells analyzed in panel A are found in the left lower quadrant, i.e. they are devoid of both, c-Kit and sSca-1. In st, cells in Panel B are predominantly in the upper quadrants, i.e. they have high levels of either c-Kit (left upper quadrant) or of both, c- Kit and Sca-1 (upper right quadrant) as expected for the haematopoetic stem cell population and of non-committed progenitors.

Figure 4: Cyclic AMP accumulation in (Lin', c-Kit”, Sca-1+) haematopoetic stem cells after stimulation by forskolin, Treprostinil, lloprost Berapost or the combination of forskolin and prostanoids. Lin' cells /assay) were incubated in the presence of [3H]adenine to metabolically prelabel the adenine nucleotide pool as outlined under Methods. The cells were incubated in the absence (left hand column labelled O) and presence of the ted compounds. The accumulated [3H]cAMP was purified by sequential chromatography on Dowex AG50-X8 and alumina columns and quantified by liquid scintillation counting. Data are means i S.D. (n=4).

Figure 5: Concentration-response curve for Treprostinil-induced cAMP accumulation in (Lin', c-Kit”, Sca-1+) haematopoetic stem cells. Assay conditions were as outlined in the legend to in Fig. 4. Data are means i S.D. (n=2).

Figure 6. EX vivo treatment of haematopoetic stem cells with Treprostinil results in ed engraftment of bone marrow in ly ated mice. Haematopoetic stem cells prepared as in Fig. 3 were pretreated with the indicated compounds (FSK, lin) as outlined under Methods. White blood cell count was detemined by FACS. The number of animals investigated is indicated.

Figure 7: Ratio of Ly5.2 and Ly5.1 positive blood cells 16 weeks after bone marrow transplantation. Ly5.1 cells were ated with CTX or Treprostinil and Forskolin.

Figure 8: Ratio of Ly5.2 and Ly5.1 positive blood cells 16 weeks after bone marrow transplantation. Ly5.2 cells were pretreated with CTX or Treprostinil and Forskolin.

Detailed description of the invention: Providing methods and means to increase homing and ting of HSCs to the bone marrow environment has strong biologic and medical implications. The localization of stem cells following transplantation is highly important for clinical procedures as currently massive numbers of stem cells are required in al transplantation thus leading to the need of high amounts of donor cells. Such methods are also highly useful because a significant number of autologous donor transplants contain insufficient numbers of stem cells, or HSCs. Likewise, patients are often unable to find histocompatible donors, emphasizing the need for methods and itions for reducing the number of HSCs needed for successful transplantation. The ability to improve homing and engrafting of HSCs in vitro or ex vivo allows the collection of fewer cells from , thereby reducing the time and discomfort associated with bone marrow/peripheral stem cell harvesting, and increasing the pool of willing HSC donors.

The present ion thus provides a novel method for enhancing engraftment of HSCs by an ex-vivo pretreatment of the HSCs comprising the steps of a) obtaining a sample containing opoetic stem cells and b) admixing at least one prostacyclin analogue to obtain a mixture, c) incubating said mixture for a period of time sufficient to stimulate G alphas-signalling in said cells and d) optionally isolating said stimulated cells or using said mixture containing said ated cells for r use, for e for treatment or transplantation. ically, the prostacyclin analogue is selected from the group of stinil lloprost, Beraprost and Cicaprost or pharmaceutically acceptable salts thereof.

Treprostinil is a synthetic analogue of prostacyclin. Treprostinil is marketed as RemodulinTl". Treprostinil is a (1 R,2R,3aS,9aS)-[[2,3,3a,4,9,9a-hexahydro-2—hydroxy- 1-[(38)—3-hydroxyoctyl]—1H-benz[f]indenyl] oxy]acetic acid monosodium salt. lloprost is marketed as “llomedine” and is a 5-{(E)-(1S,5S,6R,7R)hydroxy-6[(E)- (38, 4R8)hydroxymethylocteniny|]-bi-cyclo[3.3.0]octan e}pentanoic acid. ost is a 2,3,3a,8b-tetrahydrohydroxy(3-hydroxy methyloctenynyl)-1H—cyclopenta(b)benzofuranbutanoic acid.

Cicaprost is a 2—[(2E)—2—[(3aS,4S,5R,6aS)hydroxy[(38,4S)—3-hydroxy- 4-methylnona-1,6-diynyl]—3,3a,4,5,6,6a-hexahydro-1H-pentalen-2—ylidene] ethoxy]acetic acid.

According to an inventive embodiment, at least two, specifically at least three different prostacyclin analogues can be used for said . Alternatively, two, three, four, five or six or even more different prostacyclin analogues can be used for said method.

This method advantageously provides stimulated stem cells which can directly be stered to individuals and which do not show any unwanted side effects due to high amounts of unselective cAMP activating . .

According to a further embodiment of the invention a method for enhancing engraftment of haematopoetic stem cells by an ex-vivo pretreatment is provided which comprises ing steps: a. obtaining a sample containing haematopoetic stem cells and b. admixing a cyclin analogue and forskolin to obtain a mixture c. ting said e for a period of time sufficient to stimulate G alphas- signalling in said cells and optionally d. isolating said stimulated cells and optionally e. purifying and/or concentrating said stimulated cells.

According to a specific embodiment of the invention, the ratio of prostacyclin analogue and forskolin may be about 1:3. The HSCs treated with forskolin and prostacyclin analogues may be purified before being reimplanted, however, these HSCs may also be re-implanted without further purification steps as low amounts of forskolin may be present but may not cause any negative side effects.

WO 95511 2012/050484 Alternatively, a combination of Treprostinil with one of lloprost, Beraprost or Cicaprost can be added to the stem cells. Alternatively, Treprostinil can be admixed in combination with more than one, for example with two, three, four or five of other prostacyclin analogues, for example, but not limited to lloprost, Beraprost or Cicaprost or physiologically acceptable salts thereof.

According to a specific embodiment for use in animal studies or treatment of animals, a cAMP enhancer like forskolin and/or cholera toxin can be additionally admixed to the HSCs or HSC/Treprostinil, lloprost, Cicaprost or Beraprost mixture before incubation.

The period of time which is needed to stimulate the G alphas-signalling in said cells can be measured ing to known methods, for example by using cAMP ements of which there are many ions : RIA, Fluorescence Resonance Energy Transfer (FRET) with EPAC (epac1) (Ponsiouen B. et al., EMBO s, 5, 12, 1176-1180 (2004)), radiochemical methods etc. Stimulated cells wherein G alphas-signalling is occurring can be selected or discriminated or isolated from unstimulated cells by methods known in the art like a FRET-based cAMP reporter. ing to an embodiment of the invention the incubation time is about 1 to 60 min, preferably about 2 to 30 min.

The cAMP-dependent pathway is an essential pathway for promoting engraftment of haematopoetic stem cells. It has been shown by the inventors that a prostacyclin analogue can trigger cAMP elevation in haematopoetic stem cells. It does so by activating multiple ors, i.e. IP- and EP-receptors thus g to increased G alphas-signalling. Accordingly, prostacyclin analogues like Treprostinil, lloprost, Cicaprost or Beraprost are more effectively raising cAMP levels.

According to a very specific embodiment, Trepostinil is a preferred prostacyclin analogue used according to the method of the present invention.

Alternatively, in certain methods and compositions of the invention, at least one agent selected from a cyclic AMP (cAMP) enhancer or a ligand to a glandin EP receptor may also be added. Examples of cAMP enhancers include, but are not limited to, dibutyryl cAMP (DBcAMP), phorbol ester, forskolin, sclareline, 8—bromo- cAMP, cholera toxin (CT), aminophylline, 2,4 dinitrophenol (DNP), norepinephrine, epinephrine, isoproterenol, ylmethyl-xanthine (IBMX), ne, theophylline (dimethylxanthine), dopamine, rolipram, prostaglandin E1, prostaglandin E2, pituitary adenylate cyclase ting polypeptide (PACAP), and vasoactive intestinal polypeptide (VIP), among others known in the art can be added to the stem cells or the stem cells/Treprostinil or stem cells/Treprostinil, lloprost, Cicaprost and/or Beraprost mixture before incubation. Examples of cAMP enhancers also include cAMP and analogs of cAMP, such sp-5,6-DCl-B|MPS (BIMPS), among others. ing to a specific embodiment of the invention, forskolin and/or cholera toxin or the A subunit of cholera toxin are additionally d to the stem cells or the stem cells/Treprostinil mixture before incubation.

In a specific embodiment, said cAMP enhancers are used for performing stem cell treatment of animal cells or for performing animal studies in view of stem cell engraftment.

In reference to prostacyclin ues, according to the t invention the term ”prostacyclin ues” es also derivatives and analogues of said substances.

The terms "analogue" or "derivative" relate to a chemical molecule that is similar to another chemical substance in structure and function, often differing structurally by a single element or group, which may differ by cation of more than one group (e.g., 2, 3, or 4 groups) if it retains the same function as the parental chemical. Such modifications are routine to skilled persons, and include, for example, onal or substituted chemical moieties, such as esters or amides of an acid, protecting groups such as a benzyl group for an alcohol or thiol, and tert-butoxylcarbonyl groups for an amine. Also included are modifications to alkyl side chains, such as alkyl substitutions (e.g., methyl, dimethyl, ethyl, etc.), cations to the level of saturation or unsaturation of side chains, and the addition of modified groups such as substituted phenyl and y. Derivatives can also include ates, such as biotin or avidin moieties, enzymes such as horseradish peroxidase and the like, and radio-labeled, bioluminescent, chemoluminescent, or fluorescent moieties. Further, moieties can be added to the agents described herein to alter their pharmacokinetic properties, such as to se half-life in vivo or ex vivo, or to increase their cell penetration properties, among other desirable properties. Also included are prodrugs, which are known to enhance us desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.).

The term ative" also includes within its scope alterations that have been made to a parent sequence including additions, deletions, and/or substitutions that provide for functionally equivalent or functionally improved molecules. ing to a specific embodiment of the invention, the Treprostinil derivative is ed from the group of acid derivatives of Treprostinil, prodrugs of Treprostinil, rphs of stinil or isomers of Treprostinil.

Similarly, lloprost, Cicaprost or Beraprost can be derivatives from the group of acid derivatives, gs, polymorphs or isolmers rom.

The term “haematopoetic stem cells” (HSCs) or the more general term “stem cells” are understood as equivalent terms in the description of the present invention, and generally relate to either pluripotent or multipotent "stem cells" that give rise to the blood cell types, including myeloid (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid es (e.g., T-cells, B- cells, NK—cells), and others known in the art.

"Stem cells" are usually characterized by their ability to form multiple cell types (i.e. being multipotent) and their ability for self-renewal. However, oligopotent and unipotent progenitors may be included also. "Haematopoiesis" refers generally to the process of cellular differentiation or formation of specialized blood cells from an HSC.

During development, hematopoiesis translocates from the fetal liver to the bone , which then remains the site of haematopoiesis throughout adulthood. Once established in the bone , HSCs are not distributed randomly throughout the bone cavity. Rather, HSCs are typically found in close proximity to the endosteal surfaces. The more mature stem cells increase in number as the distance from the bone surface increases.

Haematopoetic tissues contain cells with long-term and term regeneration capacities, as well as committed multipotent, oligopotent, and unipotent progenitors.

The sample containing HSCs specifically can be bone .

HSCs can be obtained by known techniques from any source known to contain HSCs, specifically from eral blood, umbilical cord or cord blood, placenta and bone marrow. atively, also sources like fetal liver, fetal spleen, and aorta- gonad-mesonephros) of animals are possible. HSCs from human origin being are preferred for the methods and compositions of the invention.

For example, HSCs may be found in the bone marrow of adults, including femurs, hip, ribs, sternum, and other bones. HSCs may be obtained directly by removal from the hip using a needle and syringe, or from the blood, often following pre-treatment with cytokines, such as G-CSF (granulocyte colony-stimulating factors), that induce cells to be released from the bone marrow tment.

HSCs may be fied according to n phenotypic or genotypic markers. For example, HSCs may be identified by their small size, lack of lineage (lin) markers, low staining (side tion) with vital dyes such as rhodamine 123 (rhom) or Hoechst 33342, and presence of various antigenic markers on their e, many of which belong to the cluster of differentiation series (e.g., CD5, CD11b, CD34, CD38, CD90, CD133, CD105, CD45, GR-1 (=Ly-6G/C), 7-4, Ter—1 19 and c-kit). HSCs are mainly negative for the markers that are typically used to detect lineage commitment, and, thus, are often ed to as lin(-) cells. Most human HSCs may be characterized as CD5+, CD45R (8220)+, CD11+, GR-1”, CD34“, CD59“, Thyl/CD90+,CD38'°/“, C-kit/CD117+, and lin('). However, not all stem cells are covered by these ations, as certain HSCs are CD347+ and CD33“. Also some studies suggest that earliest stem cells may lack c-kit on the cell surface.

For purification of lin(-) HSCs by flow cytometry, or FACS, an array of mature blood- lineage marker antibodies may be used to deplete the lin(+) cells or late multipotent progenitors (MPP), including, for example, antibodies to CD3epsilon, CD5, CD45R, CD11b, CD16, GR-1, 7-4 and Ter—119, CD 13, CD32 and CD33, CD71, CD 19, CD61, Mac-1 (CDl lb/CD18), Gr—l, 117Ra, CD3, CD4, CD5, and CD8 among others known in the art. Additional purification methods are known in the art, for e, methods that use the particular signature of the 'signaling cyte activation les' (SLAM) family of cell surface molecules.

HSCs, whether from cord blood, bone marrow, peripheral blood, or other source, may be grown or expanded in any suitable, commercially available or custom defined medium, with or without serum. HSCs from human source are preferred embodiments of the invention. For ce, in certain embodiments, serum free medium may utilize albumin and/or transferrin. Further, cytokines may be included, such as Flt-3 ligand, stem cell factor (SCF), and thrombopoietin (TPO), among others. HSCs may also be grown in vessels such as bioreactors. A suitable medium for ex vivo expansion of HSCs may also comprise HSC supporting cells, such as l cells (e.g., lymphoreticular stromal cells), which can be derived, for instance, from the disaggregation of lymphoid tissue, and which have been show to support the in vitro, ex vivo, and in vivo maintenance, growth, and differentiation of HSCs, as well as their progeny.

"Cord blood" or ical cord blood" relates generally to a vely small amount of blood (up to about 180 ml) from a newborn baby that returns to the neonatal circulation. Cord blood is rich in HSCs and may be harvested and stored for later use according to techniques known in the art The terms "ex vivo" or “in vitro”refer to activities that take place outside an organism, such as experimentation or measurements done in or on living tissue in an artificial environment outside the organism, ably with minimum alteration of the natural conditions. In certain embodiments, such tissues or cells can be collected and frozen, and later thawed for ex vivo treatment. Tissue culture experiments or procedures lasting longer than a few days using living cells or tissue are typically considered to be "in vitro" though this term can be used interchangeably with ex vivo. The recitations "ex vivo stration, ex vivo treatment," or "ex vivo therapeutic use," relate generally to medical procedures in which one or more organs, cells, or tissues are obtained from a living or recently deceased subject, optionally purified/enriched, exposed to a treatment or procedure to treat the stem cells (e.g., an ex vivo administration step that es incubating the cells with a composition of the present invention to enhance ting capabilities of HSCs), and then stered to the same or different living subject after that optional treatment or procedure.

Such ex vivo therapeutic applications may also include an optional in vivo ent or procedural step, such as by administering a composition of the ion one or more times to the living subject after administration of the organ, cells, or tissue. Both local and systemic stration is contemplated for these embodiments, according to well-known techniques in the art. The amount of Treprostinil or the amount of mixture containing Treprostinil and stimulated stem cells optionally together with further agents administered to a subject depend on the characteristics of that subject, such as general health, age, sex, body weight, and tolerance to drugs, as well as the degree, severity, and type of reaction to stinil and/or cell transplant.

WO 95511 2012/050484 According to a specific embodiment of the invention, a composition comprising a prostacyclin analogue is provided for use in the treatment of individuals undergoing haematopoetic stem cell transplantation.

According to a red embodiment, the composition comprises prostacyclin analogue selected from the group of Treprostinil, lloprost, Cicaprost and Beraprost or ceutically acceptable salts thereof, more specifically it comprises Treprostinil.

The inventive composition can also comprise Treprostinil together with one or more of lloprost, ost or Beraprost. Alternatively the composition can comprise lloprost in ation with one or more of Treprostinil, Cicaprost or Beraprost or pharmaceutically acceptable salts thereof. Alternatively the composition can comprise Beraprost in combination with one or more of Treprostinil, Cicaprost or lloprost or pharmaceutically acceptable salts thereof. Alternatively the composition can comprise Cicaprost in combination with one or more of Treprostinil, ost or st or pharmaceutically acceptable salts f.

Said subjects can suffer from any bone marrow disease, Le. a disease wherein the normal bone marrow architecture is displaced by malignancies, aplastic anaemia, or infections leading to a decrease in the production of blood cells and blood ets.

Said bone marrow disease can be for example leukaemia, a defect of the blood cell tment or a need for bone marrow transplantation after chemotherapy or irradiation treatment.

More specifically, the defect of the blood cell compartment can be lobino- pathy like thalassaemia or s in neutrophil ocyte function or a defect in neutrophil granulocyte function.

The use for the treatment of individuals suffering from bone marrow diseases, for example due to chemotherapy or irradiation and thus undergoing haematopoetic stem cell transplantation by administering at least one prostacyclin analogue for a limited period of time after bone marrow transplantation is also covered by the present invention.

The treatment of subjects undergoing bone marrow transplantation using at least one prostacyclin analogue, at least one prostacyclin analogue together with one or more, specifically two, more specifically three cAMP enhancers or a mixture comprising at least one, specifically two, more specifically threee prostacyclin analogues and stimulated stem cells optionally er with r agents like one or more CAMP ers is covered by the t invention as well.

More specifically, the cAMP enhancer can be forskolin.

At least one prostacyclin analogue can be used for enhancing the engraftment of human HSCs during bone marrow transplantations or upon reconstitution of the bone marrow by using HSCs. Accelerated engraftment shortens the period at which subjects are susceptible to potentially lethal infections, bleeding and other serious complications. Hence, a prostacyclin analogue ought to be a useful therapeutic option to pretreat donor bone marrow to enhance bone marrow engraftment (i.e., by reducing the number of cells required and ning the duration of bone marrow aplasia).

Specifically, the prostacyclin analogue is selected from the group of Treprostinil, lloprost, Cicaprost or Beraprost or pharmaceutically acceptable salts thereof. More specifically, Treprostinil is the preferred prostacyclin analogue to be used.

Continuous treatment of subjects for several days after bone marrow transplantation with a prostacyclin analogue ought to result in ed clinical outcome by improving engraftment (i.e., by reducing the number of cells required and shortening the duration of bone marrow aplasia).

Thus, according to a specific ment, the treatment is performed at least five days after transplantation, more specifically for at least 10 days, more specifically for at least 14 days after transplantation.

According to an alternative embodiment of the ion, a composition comprising one or more than one prostacyclin analogue and stimulated opoetic stem cells is covered.

Alternatively, in certain embodiments, an agent selected from a cyclic AMP (cAMP) enhancer or a ligand to a prostaglandin EP receptor may be added to the composition. ing to a specific embodiment, the inventive composition can also comprise lin.

Specifically, the compostions of the invention are pharmaceutical compositions.

Further pharmaceutically acceptable excipients as known in the art can be contained in said compositions.

The amount of the prostacyclin analogue depends on the therapeutic or method for preparing ated HSCs. Very specifically, for eutic applications, the ive amount of Treprostinil is at least 1.0 ng/kg of body weight The inventive composition can be administered to the subject by any mode applicable and known in the art. More specifically, intravenous or subcutaneous administration is provided.

Said composition can be in an orally available form ed from the group of sustained release forms, tablets or capsules.

The ing description will be more fully understood with reference to the following examples. Such examples are, however, merely representative of methods of practicing one or more embodiments of the present invention and should not be read as limiting the scope of invention.

Examples: Example 1 Materials and Methods Isolation of bone marrow stem cells Ten mice (C57BL/6) were sacrificed by cervical dislocation. The long bones of the hind limbs (i.e., femora and tibiae) were freed of muscle and connective tissue and flushed with RPMI medium using a syringe and 271/2 G needle. The cell suspension was freed from visible connective tissue, collected and transferred to centrifuge tubes. Cells were harvested by centrifugation (1,200 rpm/~100 g for 5 min) and resuspended in 3 mL ocyte lysis buffer (0.15 M NH4CI, 10 mM KHCO3, 0.1 mM EDTA, pH adjusted to 7.2 to 7.4). The cell suspension was ted for 2 min at °C followed by 4 min on ice. Thereafter, RPMI (10 mL) was added and the cells were harvested by centrifugation and counted. The typical yield of cells was 3*107/mouse.

Cells were resuspended in ice-cold PBS (phosphate buffered ) containing 2% FCS (fetal calf serum) at a cell density of 2.5 * 108 cells/mL to which a cocktail of biotinylated antibodies ("Lineage Cell Depletion Kit" of Miltenyi Biotec) containing e-specific antibodies directed against CD5, CD45R (B220), CD11b, GR-1 (=Ly- 6G/C), 7-4, and Ter-119 at a ratio of 0.1 mL antibody solution per 108 cells. Cells were incubated for 20 min on ice with the antibodies and pelleted by centrifugation.

After resuspension (3.3 *1 O8 cells/mL) the second iotin-coated MicroBeads (0.2 mL/1O8 cells, provided with the ("Lineage Cell Depletion Kit" of Miltenyi Biotec) was added to the cell sion and the mixture was incubated for 15 min on ice.

Thereafter, the sample was diluted in MACS-buffer (30 mL), the cells were ted by centrifugation and resuspended in 6 mL of uffer. This suspension was loaded onto prepacked LS columns, which contain ferromagnetic beads coated with a ompatible plastic material. Typically three columns were employed for (2 mL cells suspension/column). The hrough contained the lineage marker-negative cells (Lin' cells), while the lineage ted cells were retained on the column. Cells were pelleted by centrifugation and resuspended in mL PBS.

The typical yield was 7*105 Lin' cells/mouse.

Fluorescence-activated cell sorting (FACS) analysis The antibodies employed for staining of cell surface markers were from the following sources: The mouse lineage panel antibodies were from Becton Dickinson Biosciences (BD 559971, containing in biotinylated from anti-CD33, anti-CD11b, anti- CD45R, anti-Ly-6G/Ly-6c, anti-Ter-119), the affinity purified rat ouse CD16/CD32 (mouse FCYll/ln block, BD 553142) and the fluorescent dye streptavidin- allophycocyanin-Cy7 (streptavidin APC-Cy7, BD554063) were also from Becton Dickinson Biosciences. Phycoerythrin(PE)-Cy7-labelled anti-mouse Ly6A/E (=stem cell antigen -1 = Sca1) PE-Cy7 ogue no. 2582) and PE-Cy5 ouse CD117 (c-Kit) (catalogue no. 1581) were from eBiosciences.

Directly after MACS, 1*106 lineage positive (Lin+) and negative (Lin-) cells were transferred into FACS tubes and stored on ice in 50 uL PBS. In the me, the following FACS antibodies were d (1:50) and mixed in PBS: anti CD16/CD32 purified (to block Fc—receptors), biotinylated anti-CD33, ylated anti-CD1 1 b, biotinylated anti-CD45R, biotinylated anti-Ly-6G/Ly-6C, and biotinylated anti-Ter-119, streptavidin-labelled APC-Cy7, PE-Cy7-labeled anti-Sca-1, PE-Cy5-labeled anti-c-kit.

This master mix (50 uL) was added to each sample, which were then mixed by gentle vortexing and incubated at 4°C in the dark for 15 min. Thereafter, the cells were harvested by centrifugation, washed in 2 mL PBS and resuspended in PBS. Samples were analysed in a FACS Canto ll (Becton Dickinson). The gating procedure was as follows: the gates for living cells were set by recording fonnard and sideward scatter.

The living cells were further discriminated based on the expression of lineage markers (i.e., CD11b, CD45R, Ly-6G/Ly-6C, Ter-119). This allowed for defining the gates for Lin' cells, which were r analyzed for the expression of Sca-1 and c-Kit. [3H]cAMP accumulation assay Lin‘ /sca1+-cells were incubated in 1 mL of stem cell medium (Stem Span SFEM, Stem Cell Tech #09650) containing 0.5 mg/L each, benzylpenicillin and omycin, 50 ng/mL each murine stem cell factor , human Flt-3, hlL-11 50 ng/mL, m-lL3 (10 ng/mL) (all from PreProTech) 10 ug/mL adenosine deaminase (Roche) and [3H]adenine (Perkin Elmer, 1 uCi/mL). The preincubation lasted for 4 h at 37°C. The cells were then stimulated with the compounds (forskolin, treprostinil and other prostanoids; cholera toxin) for 1 h. The cells were then pelleted (5 min at 100 g), the medium was removed and the pellet was lysed in ice-cold 2.5% oric acid (0.9 mL) containing 0.1 mM cAMP, held on ice for 1 h and neutralized with 4.2 M KOH (0.1 mL)). Lineage marker-positive cells were preincubated in a r manner but the medium ned RMPI instead of serum-free stem cell medium. ATP and cAMP were separated by tial chromatography on s containing Dowex AG50-X8 and neutral alumina (12).

The fact that differences can only be seen in the ce of forskolin is a technical problem — the signal is too low in the absence of forskolin-sensitization to see any differences between the different compounds, i.e. ilo-, beraprost and treprostinil.

Sensitization means that cells are sensitized for receptor response as these cells contain extremely low cAMP.

According to the concentration-response curve (Fig. 5) for Trepostinil it can be clearly shown that increase of cAMP is icant without addition of forskolin.

Bone marrow transplantation: lsogenic recipient mice were subjected to lethal irradiation. If not rescued by intravenous administration of haematopoetic stem cells, these mice died within the 2012/050484 first two weeks. Lin' (Sca1+ and c-Kit”) haematopoetic stem cells were prepared as outlined above and were pretreated ex vivo in the absence and presence of 10 uM Treprostinil, the combination of Treprostinil + 30 uM forskolin (FSK), 10 ug/mL cholera toxin for 1 h at 37°C. Thereafter, the cells /mouse) were injected via the tail vein. White blood cell count was determined by FACS. White blood cell counts were determined every 5 days starting on day 9 (where blood cell count was ~1 G/L.

Results Purification of haematopoetic stem cells: The MACS-based procedure retains lineage marker-positive (Lin+) cells on the magnetic beads and allows for recovery of lineage marker-negative cells (Lin'): The nature of the cell population was terized by FACS. The MACS-retained cell population was indeed enriched in cells that stained for lineage-specific markers and were devoid of the stem-cell receptor D117 (Fig. 1).

In contrast, cells that were recovered from the column flow-through were predominantly found in the upper left quadrant, i.e., they displayed high c-Kit levels and were depleted of 7-A fluorescence, which was indicative of depletion of lineage markers (Fig. 2).

The cell populations were further evaluated by staining for both c-Kit and Sca-1 (stem cell antigen-1): the lineage-positive cells were devoid of these two markers (Fig. 3A) whereas the lineage-negative fraction expressed high levels of c-Kit or the combination of c-Kit and Sca-1 (Fig. 3B).

Cyclic AMP accumulation in c-Kit+ and Sca1+ cells The adenine tide pool of the haematopoetic stem cells (c-Kit+ and Sca-1+ population, see Fig. 3B) was lically labelled with [3H]adenine and their se to Treprostinil and to other prostaglandin receptor agonists examined.

Because these cells have a very modest cAMP response, the enzyme was sensitized by using forskolin. This diterpene binds in the pseudosubstrate cleft between the catalytic Cl and CZ domains of adenylyl cyclase and renders the s isoforms of the enzyme more responsive to the stimulatory G protein Gds (13-15). As can be seen from Fig. 4, Treprostinil, Beraprost and lloprost per se caused a modest accumulation of cAMP that was comparable in magnitude to that ed by 30 uM forskolin. However, when combined with lin, Treprostinil caused an increase in cAMP levels that exceeded that caused by the lP-(l prostanoid) receptor-specific compounds st and Beraprost. This can be rationalized, if the action of treprostinil on EP (E noid)-receptors is taken into account (10). Treprostinil caused a concentration-dependent accumulation of cAMP in the range of 0.1 to uM (Fig. 5). The estimated E050 was in the range of 0.3 uM. Treprostinil failed to increase cAMP levels in the Lin+ opoetic cell fraction (data not shown).

Reconstitution of the bone marrow by (Lin', c-Kit”, Sca1+) haematopoetic stem cells Lethally irradiated mice were rescued by the intravenous injection of 3*105 Lin', c-Kit”, Sca1+ cells. The white blood cell count started to increase from a nadir on day 9 and slowly increased over the next several weeks. The level of white blood cells at day 60 after injection of haematopoetic stem cells was chosen as the relevant end point, because after 60 days circulating white blood cells could have only been produced from engrafted haematopoetic stem cells. As can be seen from Fig. 6, mice that had been ed with Treprostinil-pretreated haematopoetic stem cells had significantly higher levels of circulating white blood cells than those receiving vehicle- treated haematopoetic stem cells (p<0.05, t-test for unpaired data).

As additional controls, s were injected with (i) cells d with cholera toxin, because this is the most effective and persistent activator of Gas, (ii) lin, because — as mentioned above - it activates adenylyl cyclase isoforms directly (iii) the combination of forskolin and Treprostinil.

It is evident that Treprostinil was as effective as the positive control cholera toxin which was ished as effective ex vivo manipulation (1,2).

Example 2 Isolation of bone marrow stem cells C57BL/6 and B6SJL mice were sacrificed and the bone marrow stem cells were isolated as described in Example 1.

In vitro pre-treatment of isolated stem cells Bone marrow stem cells from C57BL/6 or C6SJL mice were pretreated in vitro with choleratoxin (CTX) or Treprostinil+Forskolin (FSK) and the stem cells are marked with Ly5.2 or Ly5.1.

In a first experiment, bone marrow stem cells from C57BL/6 mice were used without pretreatment and the stem cells were marked with Ly5.2. Bone marrow stem cells from C6JL were pretreated in vitro with choleratoxin or Treprostinil+Forskolin and stem cells were marked with Ly5.1.

For comparative studies, a 1:1 mix of Ly5.1+ and Ly5.2+ cells are uced into mice by bone marrow transplantation and the ratio of Ly5.2 and Ly5.1 positive blood cells was ed initially.Then 16 weeks after bone marrow transplantation blood cells outgrowth was measured. The results are shown in Figure 7 wherein it is clearly shown that treprostinil/FSK pretreated cells (Ly5.1+) show a icantly increased outgrowth compared to untreated cells and cells ated with CT.

In a second experiment, the bone marrow stem cells from C57BL/6 were pretreated in vitro with choleratoxin or stinil+Forskolin and stem cells were marked with Ly5.2. Bone marrow stem cells from C6JL mice, marked with Ly5.1 were used without any pretreatment.

Again, for comparative studies, a 1:1 mix of Ly5.1+ and Ly5.2+ cells were introduced into mice by bone marrow transplantation and the ratio of Ly5.2 and Ly5.1 positive blood cells was measured initially.Then 16 weeks after bone marrow transplantation blood cells outgrowth was measured. The results are shown in Figure 8 wherein it is again clearly proven that stinil/FSK pretreated cells (Ly5.2+) show a significantly increased outgrowth compared to untreated cells and cells pretreated with CT. Thus it can be shown that the effect of treprostinil and Forskolin is independent from the origin of the bone marrow cells.

A combination of Treprostinil and forskolin thus increases the tment of haematopoetic stem cells and is as or even more ive as pretreatment with cholera toxin.

References 1. Adams GB, Alley IR, Chung Ul, Chabner KT, Jeanson NT, Lo Celso C, Marsters ES, Chen M, Weinstein LS, Lin CP, Kronenberg HM, Scadden DT (2009) Haematopoietic stem cells depend on Gocs-mediated signalling to engraft bone marrow. Nature 459: 1 03-1 07. 2. Dexter TM, Whetton AD, Heyworth CM (1985) Inhibitors of cholera toxin-induced adenosine diphosphate ribosylation of membrane-associated proteins block stem cell differentiation. Blood 4-1548. 3. Long MW, Heffner CH, Gragowski LL (1988) Cholera toxin and phorbol diesters synergistically modulate murine poietic progenitor cell proliferation Exp Hematol. 16:195-200. 4. Freissmuth M, Gilman AG (1989) Mutations of Gso ed to alter the reactivity of the protein with bacterial toxins. Substitutions at ARG187 result in loss of GTPase activity. J Biol Chem 907-21914 . Aksentijevich l, Flinn l (2002) Chemotherapy and bone marrow reserve: lessons learned from autologous stem cell transplantation. Cancer Biother Radiopharm 17:399-403. 6. Awedan AA (2002) High intensity regimens with gous hematopoietic stem cell transplantation as treatment of le myeloma. Ann Transplant 7:38-43. 7. North TE, Goessling W, Walkley CR, Lengerke C, Kopani KR, Lord AM, Weber GJ, Bowman TV, Jang lH, Grosser T, Fitzgerald GA, Daley GQ, Orkin SH, Zon Ll (2007) Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis. Nature 447: 1 007-1 01 1. 8. t J, Singh P, Sampath J, Pelus LM (2009) Prostaglandin E2 es hematopoietic stem cell homing, survival, and proliferation. Blood 113:5444- 5455. 9. Goessling W, North TE, Loewer S, Lord AM, Lee S, Stoick—Cooper CL, Weidinger G, Puder M, Daley GQ, Moon RT, Zon Ll (2009) Genetic interaction of PGE2 and Wnt signaling regulates developmental ication of stem cells and regeneration. Cell 136:1136-1147.

.Aronoff DM, Peres CM, Serezani CH, Ballinger MN, Carstens JK, Coleman N, Moore BB, Peebles RS, Faccioli LH, Peters-Golden M (2007) Synthetic prostacyclin analogs entially regulate macrophage function via distinct analog-receptor binding specificities. J Immunol 178:1628—1634. 11.Kiriyama M, Ushikubi F, Kobayashi T, Hirata M, Sugimoto Y, and Narumiya S (1997) Ligand binding specificities of the eight types and es of the mouse prostanoid ors expressed in Chinese hamster ovary cells. Br J col 122:217-224 12.Johnson RA, Alvarez, R, Salomon, Y. (1994) Determination of adenylyl cyclase catalytic activity using single and double tographic procedures. Methods in Enzymo/ogy 238:31-56 13.Tesmer JJ, Sunahara RK, Gilman AG, Sprang SR (1997) Crystal structure of the catalytic domains of adenylyl cyclase in a complex with Gso.GTPyS. Science 278:1907-1916. 14. Sunahara RK, Dessauer CW, Gilman AG (1996) Complexity and diversity of mammalian yl cyclases. Annu Rev Pharmacol l 36:461-480.

. Kudlacek O, Mitterauer T, Nanoff C, Hohenegger M, Tang WJ, Freissmuth M, Kleuss C (2001) Inhibition of adenylyl and guanylyl cyclase isoforms by the antiviral drug foscarnet. J Biol Chem 276:3010-3016 16. Otsuka H. et al., The cyclin analog beraprost sodium ts the efficacy of therapeutic angiogenesis induced by gous bone marrow cells, A...Vasc.Surg, 2006, 20:646-652 17. Madonna R., “Prostacyclin improves transcoronary myocardial delivery of adipose tissue-derived stromal , Europ. Heart Journ., 2006, Vol. 27, No. 17, 2054- 2061 18. lshii M. et al., “Mesenchymal stem cell-based gene therapy with prostacyclin synthase enhanced neovascularisation in hindlimb ischemia”, 2009, Vol. 206, No. 1, 109-118 19. Crutchley D. J. et al., ,,Effects of prostacyclin s on the synthesis of tissue factor, tumor necrosis factor-alpha and interleukin-16 in human monocytic THP-1 cells”, J.. . and Exp. Therap, 1994, Vol. 271, No. 1, 446-451

Claims (23)

Claims:

1. A method for enhancing engraftment of haematopoietic stem cells (HSCs) by an ex vivo pretreatment of the HSCs which comprises following steps: a. using a sample containing haematopoietic stem cells, b. admixing to said sample at least one prostacyclin analogue to obtain a mixture, c. incubating said e for a period of time sufficient to stimulate G alphassignalling in said cells.

2. A method according to claim 1, wherein said stimulated cells are isolated.

3. A method according to claim 1 or 2, wherein said prostacyclin analogue is ed from the group of Treprostinil, Iloprost, Cicaprost and Beraprost or pharmaceutically acceptable salts thereof.

4. A method according to claim 1 to 3, wherein said cyclin analogue is Treprostinil.

5. A method according to claims 1 to 4, wherein said stinil is selected from the group of acid derivatives of Treprostinil, polymorphs of Treprostinil and isomers of Treprostinil.

6. A method ing to any one of claims 1 to 5, wherein said sample is bone marrow.

7. A method according to any one of claims 1 to 6, wherein the sample is admixed with at least one prostacyclin analogue together with an unspecific cAMP activating agent.

8. A method according to claim 7, wherein the ific cAMP acting agent is cholera toxin and/or forskolin.

9. Use of a composition comprising at least one prostacyclin analogue in the production of a composition for the treatment of individuals suffering from bone marrow disease transplanted with haematopoietic stem cells prepared ing to the method according to claims 1 to 8.

10. Use according to claim 9, wherein said prostacyclin analogue is selected from the group of Treprostinil, Iloprost, Cicaprost and Beraprost or pharmaceutically acceptable salts thereof.

11. A use according to claims 9 or 10, wherein said prostacyclin analogue is a derivative of Treprostinil ed from the group of acid derivatives of Treprostinil, rphs of Treprostinil or isomers of Treprostinil.

12. Use according to any one of claims 9 to 11, wherein the bone marrow disease is leukaemia, a defect of the blood cell compartment, bone marrow diseases induced by chemotherapy or irradiation.

13. Use according to claim 12, wherein said defect of the blood cell compartment is haemoglobinopathy or a defect in phil granulocyte function.

14. Use according to any one of claims 7 to 11, for use in the treatment of subjects suffering from bone marrow disease by administering a prostacyclin analogue for at least 7 days after bone marrow transplantation.

15. Use according to claim 14, for use in the treatment of subjects suffering from bone marrow disease by stering a prostacyclin analogue for at least 10 days after bone marrow transplantation.

16. Use according to claim 14, for use in the ent of subjects suffering from bone marrow disease by administering a prostacyclin ue for at least 14 days after bone marrow transplantation.

17. A composition sing at least one cyclin analogue together with an unspecific cAMP activating agent and stimulated opoietic stem cells.

18. A composition according to claim 17, n the unspecific cAMP activity agent is choleratoxin and forskolin.

19. Use according to of claim 14 which is a pharmaceutical composition.

20. Use according to any one of claims 9 to 16 for intravenous or subcutaneous administration, or orally available form selected from the group of sustained release forms, tablets and capsules.

21. A method according to any one of claims 1 to 8 or use of a composition according to any one of claims 9 to 14, wherein said stem cells are derived from cord blood, donor bone marrow or placenta.

22. A method according to claim 1, substantially as herein described with reference to any one of the accompanying examples and/or figures thereof.

23. Use according to claim 8, substantially as herein described with reference to any one of the accompanying es and/or figures thereof. WO 95511 Q4-1