WO2004110457A1 - Matrix metalloproteinases inhibitors for the stimulation and protection of bone marrow stem cells - Google Patents

Matrix metalloproteinases inhibitors for the stimulation and protection of bone marrow stem cells Download PDF

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WO2004110457A1
WO2004110457A1 PCT/EP2004/006028 EP2004006028W WO2004110457A1 WO 2004110457 A1 WO2004110457 A1 WO 2004110457A1 EP 2004006028 W EP2004006028 W EP 2004006028W WO 2004110457 A1 WO2004110457 A1 WO 2004110457A1
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mmp
stem cells
bone marrow
csf
cells
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PCT/EP2004/006028
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French (fr)
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Frauke Alves
Hans-Willi Krell
Katalin Vehmeyer
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F. Hoffmann-La Roche Ag
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A61K31/515—Barbituric acids; Derivatives thereof, e.g. sodium pentobarbital
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to the use of matrix metalloproteinases for the stimulation and protection of stem cells, preferably during the high dose cytotoxic 5 therapy and radiotherapy.
  • a better curative effect of tumor patients e.g. longer disease-free interval could be achieved through intensification of chemotherapy (high dose chemotherapy).
  • high dose chemotherapy Most 0 important limitation of this therapy is a continuous, therapy-induced exhaustion of the stem cell pool in the bone marrow. Especially the repeated application of strong cytotoxic substances causes a severe depletion of bone marrow stem cells. The bone marrow stem cell pool deficiency and lack of mature peripheral cells is associated with frequent infections, higher incidence of bleeding, anemia etc.
  • the high dose chemotherapy is performed with simultaneously transplanted hematopoietic stem cells.
  • Stem cells are collected either from the patients (autologous stem cell transplantation) or from the bone marrow or peripheral blood of an allogenous donors (allogeneic stem cell transplantation) (To, L.B., et al., Blood 89 (1997) 2233-2258).
  • cytokines growth hormones
  • G-CSF granulocyte colony-stimulating factor
  • peripheral blood stem cells In steady state conditions the number of peripheral blood stem cells (PBSC) is very rare.
  • PBSC peripheral blood stem cells
  • G-CSF stimulating both the growth of bone marrow stem cells and their transit into the peripheral blood
  • PBSC bone marrow stem cells
  • stem cells could be mobilized either with cytokines such as G-CSF alone or with G-CSF in combination of a cytotoxic therapy, for example with a cytotoxic agent such as cyclophosphamide.
  • cytotoxic agents such as cyclophosphamide.
  • the repeated application of cytotoxic substances especially in combination with G-CSF could lead to a very strong depletion of bone marrow stem cells (van Os, R., et al., Blood 92 (1998) 1950-1956 and Stem Cells 18 (2000) 120-127). For this reason a simultaneous protection of bone marrow stem cell pool is needed in order to be able to intensify cytotoxic therapies. Since previous efforts for the protection of stem cells by MIPIa or TGF- ⁇ failed to fullfill the initial expectation (Lord, B.I., et al., Blood 79 (1992) 2605-
  • MMPs Matrix metalloproteases
  • ECM extracellular matrix
  • basement membrane Egeblad, M., and Werb, Z., Nat. Rev. Cancer 2 (2002) 161- 174; Overall, CM., and Lopez-Otin, C, Nat. Rev. Cancer 2 (2002) 657-672). They are believed to have pivotal roles in embryonic development and growth
  • MMPs can increase the bioavailability of VEGF (Bergers, G., et al., Nat. Cell Biol. 2 (2000) 737-744) but also generate angiogenesis inhibitors such as angiostatin by cleavage of plasminogen (Dong, Z., et al., Cell 88 (1997) 801-810).
  • MMPs are thought to be involved in the mobilization of bone marrow stem cells (Janowska-Wieczorek, A., et al., Blood 93 (1999) 3379-3390). High concentration of MMP9 was observed during the G-CSF induced HPC mobilization (Carstanjen, D., et al., Transfusion 42 (2002) 588-596).
  • the object of the invention is to establish a suitable method for the stimulation and protection of bone marrow stem cells.
  • trioxopyrimidine-based MMP inhibitors which are highly selective for MMP-2 and MMP-9 and MMP- 14 are useful in the stimulation and/or protection of hemopoietic stem cells.
  • the object of the invention is a method for stimulation and/or protection of hemopoietic stem cells characterized by treatment of said stem cells with a trioxopyrimidine compound having an inhibitory activity against MMP-I, MMP-2,
  • MMP-3, MMP-9, and MMP- 14 defined as
  • IC 50 value of less than 5 ⁇ M for MMP-2, MMP-9 and MMP- 14 each; b) a ratio of more than 100 for the IC 50 values of MMP-l:MMP-2, MMP-I: MMP-9, MMP-1.-MMP-14; and c) a ratio of more than 10 for the IC 50 values of MMP-3:MMP-2, MMP-3:
  • MMP-9, MMP-3:MMP-14, IC 5 Q values being measured by an in vitro assay for MMP enzymatic activity.
  • the stimulation and/or protection of the stem cells according to the invention is performed preferably in combination with a cytokine, preferably with a colony- stimulating factor, and more preferably with G-CSF, during a cytokine primed growth of the stem cells.
  • a cytokine preferably with a colony- stimulating factor, and more preferably with G-CSF
  • the protection of stem cells is performed in combination with a cytokine, preferably G-CSF.
  • the stem cells are preferably bone marrow stem cells. Stimulation and/or protection of stem cells can be performed ex vivo or in vivo. The protection of a stem cell pool, preferably a bone marrow stem cell pool, is performed (preferably beginning shortly before) during cytotoxic treatment. Such a cytotoxic treatment can be radiotherapy or chemotherapy.
  • the invention comprises a method for the protection of stem cells against cytotoxic agents or gamma-radiation, characterized in that said cells are treated before treatment with said cytotoxic agent or said gamma-radiation with a trioxopyrimidine compound according to the invention and preferably in combination with a cytokine.
  • Trioxopyrimidines useful for the invention are compounds from a well-known structural class. Such compounds are described in, for example, US Patent Nos. 6,242,455 and 6,110,924; WO 97/23465, WO 98/58915, WO 01/25217, which are incorporated herein by reference, and Grams, F., et al., Biol. Chem. 382 (2001) 1277-1285, and are effective and highly selective for MMP-2, MMP-9 and MMP- 14.
  • MMPs induce increased colony growth of both differentiated progenitor cells and undifferentiated stem cells such as high proliferating potential cells (HPPC) in the presence of cytokines in vitro and in vivo.
  • HPPC high proliferating potential cells
  • the invention establishes a suitable method for the mobilization and simultaneously for the protection of bone marrow stem cells from exhaustion.
  • the advantages of the application of MMP inhibitors are easy handling, low side effects, low costs and preferably the capability of protecting the bone marrow stem cell pool.
  • Such an invention prevents severe side effects and shortens the time for clinical care for tumor patients during and after cytotoxic treatment.
  • MMP inhibitors are well-known in the state of the art and are, for example, trioxopyrimidine compounds as described in US Patent No. 6,110,924.
  • Thiol group-containing amide or peptidylamide-based metalloproteinase inhibitors are known from, for example, WO 95/12389, WO 96/11209, WO 00/71514 and US Patent No. 4,595,700.
  • Hydroxamate group-containing MMP inhibitors are disclosed in a number of published patent applications, such as WO 95/29892, WO 97/24117, WO 97/49679 and EP 0 780 386. Such inhibitors are carbon-backboned compounds.
  • Hydroxamate group-containing MMP inhibitors having a peptidyl backbone or peptidomimetic backbone are described in WO 90/05719, WO 93/20047, WO 95/09841, WO 96/06074; Schwartz, M.A., and Van Wart, H.E.,
  • a “hemopoietic stem cell” is defined as a cell with extensive self-renewal and proliferative potential coupled with the capacity to differentiate into progenitors of all blood cell lineages. Such cells comprise a pool of very early primitive stem cells allowing the differentiation of all types of mature hemopoietic cells such as leukocytes, erythrocytes, thrombocytes, lymphocytes. Progenitors are cells which are committed for a given hemopoietic differentiation.
  • Such primitive stem cells comprise also the high proliferative potential cells (HPPC) that show high proliferation (self-renewal) and differentiation capability.
  • HPPC high proliferative potential cells
  • Primitive stem cells are essential for the reconstitution of hemopoiesis.
  • cytotoxic therapy such as radiotherapy or chemotherapy and application of cytokines such as G-CSF the hemopoiesis is highly stimulated giving rise to a short time enhancement of primitive stem cells in both bone marrow and peripheral blood. This process is depending on the presence of an intact stem cell pool.
  • cytotoxic therapy especially in combination with cytokines often leads to the exhaustion of bone marrow stem cell pool. Due to the bone marrow failure the management of an adequate chemotherapy is highly limited.
  • the MMP inhibitors positively influences the progeny of stem cells in the bone marrow and results partly in their retain in the bone marrow microenvironment, thereby saving the bone marrow stem cell pool from the exhaustion.
  • the invention provides a method of protecting bone marrow stem cells, preferably during the cytotoxic treatment and cytokine application. The method contributes to quick recovery of patients receiving repeatedly cytotoxic therapy.
  • the MMP inhibitor is used preferably in combination with a cytokine.
  • Cytokines such as G-CSF are extensively used for the supportive care of patients with hematological and malignant diseases.
  • G-CSF is widely used for the mobilization of hemapoietic stem cells (see, e.g., Hubel, K., and Engert, A., Ann. Hematol. 82 (2003) 207-213).
  • the cytokine preferably G-CSF
  • is used according to this well-known application for stem cell mobilization see also Lemoli, R.M., et al., Blood 102 (2003) 1595-1600; De Propris, M.S., et al., Acta Hematol.
  • G-CSF-mobilized peripheral stem cells will be collected and transplanted in order to support a quick recovery of hemopoiesis.
  • the collection of a sufficient amount of autologuos stem cells assumes also the availability of an intact stem cell pool. Renewed applications of cytotoxic therapy without saving of the bone marrow stem cell pool make the mobilization and collection of stem cells often difficult or impossible.
  • the invention described here offers a possibility to avoid the bone marrow failure during the course of high dose cytotoxic treatment and repeated chemotherapy.
  • the MMP inhibitor is administered daily, beginning a few days (2 to 5 days preferably) before chemotherapy is started. Administration will be continued during the whole regime of chemotherapy including all single courses of chemotherapy at a dose of 0.5 to 200 mg/kg body weight per day .
  • the MMP inhibitor is administered 2 to 3 days before each course of chemotherapy, during the single course of chemotherapy and 3 to 5 days thereafter (excluding intervals between single courses of chemotherapy) with similar doses.
  • Figure IA Inhibitory effect of BB-94 (comparison compound) on stem cell growth using bone marrow of four patients without hematological disorders.
  • Figure 2B Effect of MMP inhibitor on stem cell growth in vitro using bone marrow of four patients without hematologic disorders.
  • Figure 3A Effect of MMP inhibitor on stem cell growth in vivo in the bone marrow of six mice.
  • Figure 3B Effect of MMP inhibitor on stem cell growth in vivo in the spleen of six mice.
  • TACE inhbitors MMP inhibitor selective for TACE
  • MNC Mononuclear cells including hematopoietic stem/progenitor cells have been purified from healthy individuals (without any hematopoietic disease) by LymphorepTM -separation.
  • Cells have been cultivated with a combination of growth factors Interleukin 3 (11-3), stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) with and without Batimastat (BB-94), a broad-specific MMP-inhibitor.
  • SCF stem cell factor
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • MNC mononuclear cells
  • Batimastat 1 10 und 50 ⁇ M. Growth factors and Batimastat are given in a previously prepared 0.5% agar layer (under layer). Colonies were evaluated under a microscope after incubation for 3 weeks at 37°C in 7.5% CO 2 (Pragnell, I.B., et al., Blood 72 (1988) 196-201). The colonies are cell conglomerates consisting of >50 cells. Colony forming unit HPPC (CFU-HPPP) are colonies showing diameter of 4-
  • Batimastat showed a strong inhibition of colony growth in all probes analyzed (Fig. IA). Similar effects have been found using an MMP-1/TACE selective MMP inhibitor which shows at 5 and 10 ⁇ M also an inhibitory effect (Fig. IB). This compound is described in WO 99/01428.
  • MNC including hematopoietic stem/ progenitor cells have been purified from healthy individuals (without any hematopoietic disease) using Lymphorep ® - centrifugation. Cells have been cultivated with a combination of growth factors IL- 3, SCF, G-CSF, GM-CSF and with or without a highly selective MMP -inhibitor.
  • MNC mononuclear cells
  • FCS+HS 25% serum
  • IL-3 growth factors
  • GM-CSF GM-CSF
  • G-SCF growth factors
  • 20 ng/ ml 20 ng/ ml with and without Compound I 1, 10 und 50 ⁇ M.
  • Growth factors and highly selective MMP- inhibitors are given in a previously prepared 0.5% agar layer (under layer). Colonies were evaluated under a microscope after incubation for 3 weeks at 370C in
  • the colonies are cell conglomerates consisting of >50 cells.
  • Colony forming unit HPP are colonies showing diameter of 4-5 mm. 13 probes were investigated.
  • Compound I showed at the concentration of 10 ⁇ M in 9 of 13 probes investigated a stimulatory effect of colony growth. Not only the number but also the size of colonies were affected. The number of large colonies (CFU-HPPC) were increased until 60%. In four of investigated cases Compound I showed either no effect or only a less increased colony growth.
  • MMP inhibitor in combination with G-CSF was also in vivo investigated.
  • the combined administration of G-CSF and the MMP inhibitor Compound I resulted in a higher CFU-GM and HPPC level in the bone marrow compared with the control group.
  • CFU-GM progenitor colony growth
  • the number of CFU-HPPC was hardly affected.
  • Compound I day 1-4).
  • the second group received G-CSF subcutaneously, 250 ⁇ g/kg/day, day 2-4.
  • the third group was orally treated with Compound I (22.5 resp. 45 mg/kg/day, day 1-4).
  • the fourth group received both Compound I, day 1- 4, and G-CSF on day 2-4.
  • HPPCs was doubled by Compound I and G-CSF compared to the control group.
  • inhibitors of MMPs selective for MMP-2, MMP-9 and MMP- 14 induce an increased colony growth of bone marrow stem cells in the presence of growth hormones in vitro.
  • HPPC high proliferating potential cells
  • Figure 2 these cells are known to be important for a long lasting reconstitution of the hematopoiesis (Perez-Oteyza, J., et aL, J. Exp. Hematology 25 (1997) 516-520).
  • Compound I With Compound I the egress of stem cells from the bone marrow into the peripheral blood was significant reduced. This could mean that the gelatinase inhibitor Compound I prevents the complete exhaustion of the bone marrow stem cell pool during the G-CSF mobilization and also during the cytotoxic therapy.
  • Inhibitors were tested in a modified fluorescence-assay as described by Stack, M.S., and Gray, R.D., J. Biol. Chem. 264 (1989) 4277-4281.
  • Human MMP-I, MMP-2, MMP-3, MMP-9 and MMP-14 are commercially available (e.g. Calbiochem).
  • the pro-enzymes were activated with 1 mM APMA (incubation for 30 min at 37°C) immediately before testing.
  • Activated enzyme is diluted to 100 ng/ml in incubation buffer (50 mM Tris, 100 mM NaCl, 1OmM CaCl2, pH 7.6).
  • the compounds were dissolved in 100% DMSO. For IC 50 determination a minimum of 8 dilution steps between 0.5 - 1000 nM have been prepared.
  • DNP-substrate (Bachem M1855,
  • the test tube contains 970 ⁇ l incubation buffer, lO ⁇ l inhibitor solution and lO ⁇ l enzyme solution. The reaction was started by adding the lO ⁇ l substrate solution.
  • IC 5 o's are defined as the concentration of inhibitor that gives a signal that is 50% of the positive enzyme control.

Abstract

A method for the stimulation and/or protection of hemopoietic stem cells, characterized by treatment of said stem cells with a substance having an inhibitory activity against MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14 defined as a) an IC50 value of less than 5 µM for MMP-2, MMP-9 and MMP-14 each; b) a ratio of more than 100 for the IC50 values of MMP-I:MMP-2, MMP-1: MMP-9, MMP­ I:MMP-14; and c) a ratio of more than 10 for the IC50 values of MMP-3:MMP-2, MMP-3: MMP-9, MMP-3:MMP-14.

Description

MATRIX METALLOPROTEINASE INHIBITORS FOR THE STIMULATION AND PROTECTION
OF BONE MARROW STEM CELLS
Field of the Invention
The present invention relates to the use of matrix metalloproteinases for the stimulation and protection of stem cells, preferably during the high dose cytotoxic 5 therapy and radiotherapy.
Background of the Invention
A better curative effect of tumor patients e.g. longer disease-free interval could be achieved through intensification of chemotherapy (high dose chemotherapy). Most 0 important limitation of this therapy is a continuous, therapy-induced exhaustion of the stem cell pool in the bone marrow. Especially the repeated application of strong cytotoxic substances causes a severe depletion of bone marrow stem cells. The bone marrow stem cell pool deficiency and lack of mature peripheral cells is associated with frequent infections, higher incidence of bleeding, anemia etc. In order to 5 reduce undesirable side-effects, the high dose chemotherapy is performed with simultaneously transplanted hematopoietic stem cells. Stem cells are collected either from the patients (autologous stem cell transplantation) or from the bone marrow or peripheral blood of an allogenous donors (allogeneic stem cell transplantation) (To, L.B., et al., Blood 89 (1997) 2233-2258).
0 Proliferation and differentiation of stem cells are under the control of specific growth hormones (cytokines). To these hormones belongs among others the granulocyte colony-stimulating factor (G-CSF) (Metcalf, D., Science 229 (1985) 16- 22; Metcalf, D., Immunol. Cell Biol. 76 (1998) 441-447) facilitating primarily the growth of progenitor cells with granulocytic differentiation. The clinical use of 5 cytokines in mobilizing bone marrow stem cells into the blood is well documented
(Simmons, P.J., et al., Stem Cells 12, Suppl. 1 (1994) 187-201, discussion 201-202).
In steady state conditions the number of peripheral blood stem cells (PBSC) is very rare. For the mobilization and subsequent harvest of PBSC by leukopheresis, patients receive usually G-CSF stimulating both the growth of bone marrow stem cells and their transit into the peripheral blood (Simmons, P.J., et al., Stem Cells 12, Suppl. 1 (1994) 187-201, discussion 201-202).
Mobilized stem cells from the peripheral blood will be collected and stored under controlled conditions until their re-transfusion according to a given therapy protocol. In the last years the transplantation of PBSC receives more and more importance and acceptance. PBSC allow a faster reconstitution of the hematopoesis compared to the transplantation of bone marrow stem cells (BMSC) directly harvested from bone marrow. The harvest of PBSC of patients having previously repeated and intensive cytotoxic therapy is often difficult or ineffective because of their exhausted stem cell pools in the bone marrow. Patients with bone marrow depression are "poor mobilizers" (Micallef, I.N., et al., Hematol. J. 1 (2000) 367- 373; Drake, M., et al., Br. J. Haematol. 98 (1997) 745-749).
For the purpose of peripheral stem cell transplantation stem cells could be mobilized either with cytokines such as G-CSF alone or with G-CSF in combination of a cytotoxic therapy, for example with a cytotoxic agent such as cyclophosphamide. On the other hand, the repeated application of cytotoxic substances especially in combination with G-CSF could lead to a very strong depletion of bone marrow stem cells (van Os, R., et al., Blood 92 (1998) 1950-1956 and Stem Cells 18 (2000) 120-127). For this reason a simultaneous protection of bone marrow stem cell pool is needed in order to be able to intensify cytotoxic therapies. Since previous efforts for the protection of stem cells by MIPIa or TGF-β failed to fullfill the initial expectation (Lord, B.I., et al., Blood 79 (1992) 2605-
2609), compounds which allow effectively the preservation of bone marrow stem cells during the course of intensive chemo- or radiotherapy are urgently required.
Matrix metalloproteases (MMPs) are a family of zinc- and calcium-dependent proteases that are capable of degrading the extracellular matrix (ECM) and basement membrane (Egeblad, M., and Werb, Z., Nat. Rev. Cancer 2 (2002) 161- 174; Overall, CM., and Lopez-Otin, C, Nat. Rev. Cancer 2 (2002) 657-672). They are believed to have pivotal roles in embryonic development and growth
(Holmbeck, K., et al., Cell 99 (1999) 81-92; Vu, T.H., et al., Cell 93 (1998) 411-422) as well as in tissue remodeling and repair (Shapiro, S.D., Curr. Opin. Cell Biol. 10 (1998) 602-608; Lund, L.R., et al., EMBO J. 18 (1999) 4645-4656). Excessive or inappropriate expression of MMPs may therefore contribute to the pathogenesis of many tissue-remodelling processes, including tumor progression (Egeblad, M., and Werb, Z., Nat. Rev. Cancer 2 (2002) 161-174; Overall, CM., and Lopez-Otin, C, Nat. Rev. Cancer 2 (2002) 657-672) and aneurysm formation (Carmeliet, P., et al., Nat. Genet. 17 (1997) 439-444). MMP effects are far from being restricted to ECM degradation (Chang, C, and Werb, D., Trends Cell Biol. 11 (2001) S37-43). Peptide growth factors that are sequestered by ECM proteins become available once degraded by MMP-9 (Manes, S., et al., J. Biol. Chem. 274 (1999) 6935-6945). MMPs can increase the bioavailability of VEGF (Bergers, G., et al., Nat. Cell Biol. 2 (2000) 737-744) but also generate angiogenesis inhibitors such as angiostatin by cleavage of plasminogen (Dong, Z., et al., Cell 88 (1997) 801-810).
MMPs are thought to be involved in the mobilization of bone marrow stem cells (Janowska-Wieczorek, A., et al., Blood 93 (1999) 3379-3390). High concentration of MMP9 was observed during the G-CSF induced HPC mobilization (Carstanjen, D., et al., Transfusion 42 (2002) 588-596).
The object of the invention is to establish a suitable method for the stimulation and protection of bone marrow stem cells.
Summary of the Invention
It was surprisingly found that trioxopyrimidine-based MMP inhibitors which are highly selective for MMP-2 and MMP-9 and MMP- 14 are useful in the stimulation and/or protection of hemopoietic stem cells.
The object of the invention is a method for stimulation and/or protection of hemopoietic stem cells characterized by treatment of said stem cells with a trioxopyrimidine compound having an inhibitory activity against MMP-I, MMP-2,
MMP-3, MMP-9, and MMP- 14 defined as
a) an IC50 value of less than 5 μM for MMP-2, MMP-9 and MMP- 14 each; b) a ratio of more than 100 for the IC50 values of MMP-l:MMP-2, MMP-I: MMP-9, MMP-1.-MMP-14; and c) a ratio of more than 10 for the IC50 values of MMP-3:MMP-2, MMP-3:
MMP-9, MMP-3:MMP-14, IC5Q values being measured by an in vitro assay for MMP enzymatic activity.
The stimulation and/or protection of the stem cells according to the invention is performed preferably in combination with a cytokine, preferably with a colony- stimulating factor, and more preferably with G-CSF, during a cytokine primed growth of the stem cells. Preferably, the protection of stem cells is performed in combination with a cytokine, preferably G-CSF.
The stem cells are preferably bone marrow stem cells. Stimulation and/or protection of stem cells can be performed ex vivo or in vivo. The protection of a stem cell pool, preferably a bone marrow stem cell pool, is performed (preferably beginning shortly before) during cytotoxic treatment. Such a cytotoxic treatment can be radiotherapy or chemotherapy.
Therefore, the invention comprises a method for the protection of stem cells against cytotoxic agents or gamma-radiation, characterized in that said cells are treated before treatment with said cytotoxic agent or said gamma-radiation with a trioxopyrimidine compound according to the invention and preferably in combination with a cytokine.
Trioxopyrimidines useful for the invention are compounds from a well-known structural class. Such compounds are described in, for example, US Patent Nos. 6,242,455 and 6,110,924; WO 97/23465, WO 98/58915, WO 01/25217, which are incorporated herein by reference, and Grams, F., et al., Biol. Chem. 382 (2001) 1277-1285, and are effective and highly selective for MMP-2, MMP-9 and MMP- 14.
According to the invention, the following compounds are particularly preferred:
5-Biphenyl-4-yl-5- [4- (4-nitro-phenyl) -piperazin- 1 -yl] pyrimidine-2,4,6-trione (Compound I)
5-(4-Phenoxy-phenyl)-5-(4-pyrimidin-2-yl-piperazin-l-yl)-pyrimidine-2,4,6- trione (Compound II) 5-[4-(4-Chloro-phenoxy)-phenyl]-5-(4-pyrimidin-2-yl-piperazin-l-yl)- pyrimidine~2,4,6-trione (Compound III) 5- [4- (3,4-Dichloro-phenoxy) -phenyl] -5- (4-pyrimidin-2-yl-piperazin- 1 -yl) - pyrimidine-2,4,6-trione (Compound IV) 5-[4-(4-Bromo-phenoxy)-phenyl]-5-(4-pyrimidin-2-yl-piperazin-l-yl)- pyrimidine-2,4,6-trione (Compound V).
It was surprisingly found that this type of matrix metalloproteinase inhibitors effectively increases the cytokine-primed growth of stem cells and simultaneously protects the bone marrow stem cell pool in vivo.
Surprisingly, MMPs induce increased colony growth of both differentiated progenitor cells and undifferentiated stem cells such as high proliferating potential cells (HPPC) in the presence of cytokines in vitro and in vivo.
Detailed Description of the Invention
The invention establishes a suitable method for the mobilization and simultaneously for the protection of bone marrow stem cells from exhaustion. The advantages of the application of MMP inhibitors are easy handling, low side effects, low costs and preferably the capability of protecting the bone marrow stem cell pool. Such an invention prevents severe side effects and shortens the time for clinical care for tumor patients during and after cytotoxic treatment.
"Matrix metalloproteinase inhibitors (MMP inhibitors)" according to the invention are well-known in the state of the art and are, for example, trioxopyrimidine compounds as described in US Patent No. 6,110,924.
Thiol group-containing amide or peptidylamide-based metalloproteinase inhibitors are known from, for example, WO 95/12389, WO 96/11209, WO 00/71514 and US Patent No. 4,595,700. Hydroxamate group-containing MMP inhibitors are disclosed in a number of published patent applications, such as WO 95/29892, WO 97/24117, WO 97/49679 and EP 0 780 386. Such inhibitors are carbon-backboned compounds. Hydroxamate group-containing MMP inhibitors having a peptidyl backbone or peptidomimetic backbone are described in WO 90/05719, WO 93/20047, WO 95/09841, WO 96/06074; Schwartz, M.A., and Van Wart, H.E.,
Progr. Med. Chem. 29 (1992) 271-334; Rasmussen, H.S., and McCann, P.P., Pharmacol. Ther. 75 (1997) 69-75; and Denis, L. J., and Verweij, J., Invest New Drugs 15 (1997) 175-185.
A "hemopoietic stem cell" is defined as a cell with extensive self-renewal and proliferative potential coupled with the capacity to differentiate into progenitors of all blood cell lineages. Such cells comprise a pool of very early primitive stem cells allowing the differentiation of all types of mature hemopoietic cells such as leukocytes, erythrocytes, thrombocytes, lymphocytes. Progenitors are cells which are committed for a given hemopoietic differentiation.
Such primitive stem cells comprise also the high proliferative potential cells (HPPC) that show high proliferation (self-renewal) and differentiation capability.
Primitive stem cells are essential for the reconstitution of hemopoiesis. During cytotoxic therapy such as radiotherapy or chemotherapy and application of cytokines such as G-CSF the hemopoiesis is highly stimulated giving rise to a short time enhancement of primitive stem cells in both bone marrow and peripheral blood. This process is depending on the presence of an intact stem cell pool. The repeated application of cytotoxic therapy especially in combination with cytokines often leads to the exhaustion of bone marrow stem cell pool. Due to the bone marrow failure the management of an adequate chemotherapy is highly limited. According to the invention, the MMP inhibitors positively influences the progeny of stem cells in the bone marrow and results partly in their retain in the bone marrow microenvironment, thereby saving the bone marrow stem cell pool from the exhaustion. Thus, the invention provides a method of protecting bone marrow stem cells, preferably during the cytotoxic treatment and cytokine application. The method contributes to quick recovery of patients receiving repeatedly cytotoxic therapy.
The MMP inhibitor is used preferably in combination with a cytokine. Cytokines such as G-CSF are extensively used for the supportive care of patients with hematological and malignant diseases. G-CSF is widely used for the mobilization of hemapoietic stem cells (see, e.g., Hubel, K., and Engert, A., Ann. Hematol. 82 (2003) 207-213). According to the invention, the cytokine, preferably G-CSF, is used according to this well-known application for stem cell mobilization (see also Lemoli, R.M., et al., Blood 102 (2003) 1595-1600; De Propris, M.S., et al., Acta Hematol. 109 (2003) 57-63; Benboubker, L., et al., Exp. Hematol. 31 (2003) 89-97). In the course of high dose chemotherapy, G-CSF-mobilized peripheral stem cells will be collected and transplanted in order to support a quick recovery of hemopoiesis. The collection of a sufficient amount of autologuos stem cells assumes also the availability of an intact stem cell pool. Renewed applications of cytotoxic therapy without saving of the bone marrow stem cell pool make the mobilization and collection of stem cells often difficult or impossible. The invention described here offers a possibility to avoid the bone marrow failure during the course of high dose cytotoxic treatment and repeated chemotherapy.
According to the invention, the MMP inhibitor is administered daily, beginning a few days (2 to 5 days preferably) before chemotherapy is started. Administration will be continued during the whole regime of chemotherapy including all single courses of chemotherapy at a dose of 0.5 to 200 mg/kg body weight per day .
Alternatively, the MMP inhibitor is administered 2 to 3 days before each course of chemotherapy, during the single course of chemotherapy and 3 to 5 days thereafter (excluding intervals between single courses of chemotherapy) with similar doses.
The following examples, references and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.
Description of the Figures
Figure IA Inhibitory effect of BB-94 (comparison compound) on stem cell growth using bone marrow of four patients without hematological disorders.
Figure IB Effect of MMP-1/TACE selective MMP inhibitor 3-(N'-Isobutyl-
N'-methylsulfonyl-hydrazinocarbonyl)-5-methyl-2-(3-phenyl- allyl)~hexanoic acid hydroxyamide (comparison compound) on stem cell growth using bone marrow of four patients without hematological disorders. Figure 2A Effect of MMP inhibitor on HPPC using bone marrow of four patients without hematological disorders.
Figure 2B Effect of MMP inhibitor on stem cell growth in vitro using bone marrow of four patients without hematologic disorders.
Figure 3A Effect of MMP inhibitor on stem cell growth in vivo in the bone marrow of six mice.
Figure 3B Effect of MMP inhibitor on stem cell growth in vivo in the spleen of six mice.
Example 1 Inhibition of stem cell growth using broad-selective MMP inhibitors Batimastat
(in vitro) or MMP inhibitor selective for TACE (TACE inhbitors)
Mononuclear cells (MNC) including hematopoietic stem/progenitor cells have been purified from healthy individuals (without any hematopoietic disease) by Lymphorep™ -separation. Cells have been cultivated with a combination of growth factors Interleukin 3 (11-3), stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) with and without Batimastat (BB-94), a broad-specific MMP-inhibitor.
1.5 x 104 mononuclear cells (MNC) after Lymphoprep isolation were seeded in 4 ml 0.3% Agar medium (upper layer) containing 25% serum (FCS+HS) and growth factors (IL-3, GM-CSF, G-SCF 50 ng/ ml each and SCF, 20 ng/ ml with and without
Batimastat 1, 10 und 50 μM). Growth factors and Batimastat are given in a previously prepared 0.5% agar layer (under layer). Colonies were evaluated under a microscope after incubation for 3 weeks at 37°C in 7.5% CO2 (Pragnell, I.B., et al., Blood 72 (1988) 196-201). The colonies are cell conglomerates consisting of >50 cells. Colony forming unit HPPC (CFU-HPPP) are colonies showing diameter of 4-
5 mm. 13 probes were investigated.
Batimastat showed a strong inhibition of colony growth in all probes analyzed (Fig. IA). Similar effects have been found using an MMP-1/TACE selective MMP inhibitor which shows at 5 and 10 μM also an inhibitory effect (Fig. IB). This compound is described in WO 99/01428.
Example 2 Stimulation of stem cells with highly selective MMP inhibitors (in vitro)
MNC including hematopoietic stem/ progenitor cells have been purified from healthy individuals (without any hematopoietic disease) using Lymphorep®- centrifugation. Cells have been cultivated with a combination of growth factors IL- 3, SCF, G-CSF, GM-CSF and with or without a highly selective MMP -inhibitor.
1.5 x 104 mononuclear cells (MNC) after Lymphoprep® isolation were seeded in 4 ml 0.3% agar medium (upper layer) containing 25% serum (FCS+HS) and growth factors (IL-3, GM-CSF, G-SCF 50 ng/ ml each and SCF, 20 ng/ ml with and without Compound I 1, 10 und 50 μM). Growth factors and highly selective MMP- inhibitors are given in a previously prepared 0.5% agar layer (under layer). Colonies were evaluated under a microscope after incubation for 3 weeks at 370C in
7.5% CO2 (Pragnell, I.B., et al., Blood 72 (1988) 196-201). The colonies are cell conglomerates consisting of >50 cells. Colony forming unit HPP are colonies showing diameter of 4-5 mm. 13 probes were investigated.
Compound I showed at the concentration of 10 μM in 9 of 13 probes investigated a stimulatory effect of colony growth. Not only the number but also the size of colonies were affected. The number of large colonies (CFU-HPPC) were increased until 60%. In four of investigated cases Compound I showed either no effect or only a less increased colony growth.
Example 3 Stimulation of stem cells with highly selective MMP inhibitor (in vivo)
The effect of MMP inhibitor in combination with G-CSF was also in vivo investigated. Growth of hematopoietic stem/ progenitor cells in bone marrow and spleen of mice was analyzed. The combined administration of G-CSF and the MMP inhibitor Compound I resulted in a higher CFU-GM and HPPC level in the bone marrow compared with the control group. In the spleen a decreased progenitor colony growth (CFU-GM) was observed compared with the control group having only G-CSF. However the number of CFU-HPPC was hardly affected.
Methods:
In the experiments four groups of Balb/c mice (n=6 each) were included. One group was treated only with pysiological saline and Eudragit® (vehicle for
Compound I, day 1-4). The second group received G-CSF subcutaneously, 250 μg/kg/day, day 2-4. The third group was orally treated with Compound I (22.5 resp. 45 mg/kg/day, day 1-4). The fourth group received both Compound I, day 1- 4, and G-CSF on day 2-4.
24 h after the last application of the compound the blood of the animals have been taken retroorbital to receive a hemagram. Following this procedure the animals have been killed, both femurs and the spleen have been prepared to isolate stem cells.
Aliquots of cells have been stained with acridin orange and counted using a small Neubauer chamber. IxIO4 vital cells of bone marrow and 5 x 104 of spleen have been plated in 0.3% agar on a 0.5% agar layer. Growth factors (GM-CSF, G-CSF, IL-3 and M-CSF, 50 ng/ml, SCF 20 ng/ml) were given in the lower agar layer (0.5% agar). Colony growth was analyzed after 14 and 21 days of incubation.
In bone marrow G-CSF alone raised the number of colonies by 56% compared to untreated control (Fig. 3A). Number of HPPC colonies have been stimulated by
40%. However, the combination of G-CSF and gelatinase inhibitor Compound I resulted in a stimulation of the total colonie number by 126% and the number of HPPC colonies was raised by 80% compared to untreated controls. Treatment of Compound I alone did not show an effect on the number of colonies.
In summary, in bone marrow both the number of total colonies and the number of
HPPCs was doubled by Compound I and G-CSF compared to the control group.
In the spleen the number of differentiated colonies was about 20-30% less in mice treated with the combination of G-CSF with Compound I than in mice without Compound I treatment. The number of HPPC however was comparable in both groups (Fig. 3B).
The experiments show that Compound I prevent the exhaust of the bone marrow stem cell pool by retaining in part the stimulated stem cells in-situ.
Cell culture experiments confirmed the role of MMPs in regulation the cytokine- controlled growth of the hematopoietic stem cells. Using a broad-selective MMP inhibitor batimastat, growth and distribution of stem cells could be blocked even in the presence of growth factors (Figure 1).
Surprisingly, inhibitors of MMPs selective for MMP-2, MMP-9 and MMP- 14 induce an increased colony growth of bone marrow stem cells in the presence of growth hormones in vitro. Especially, the high proliferating potential cells (HPPC) are stimulated (Figure 2). Among others these cells are known to be important for a long lasting reconstitution of the hematopoiesis (Perez-Oteyza, J., et aL, J. Exp. Hematology 25 (1997) 516-520).
Similar to the in vitro observation, the in vivo application of Compound I in combination with G-CSF showed in mice an increased progenitor and HPPC colony growth in the bone marrow (Fig. 3A). In the spleen cultures there were less differentiated colonie observed in the group that have been treated with Compound I and G-CSF (Fig. 3B). As mobilized PBSC have been cleared in the spleen within a short time, analyses of spleen colony-forming cells in the spleen reflect the PBSC.
The results revealed a shift in the distribution of hematopoietic stem-progenitor- cells in the different compartments by Compound I. With Compound I the egress of stem cells from the bone marrow into the peripheral blood was significant reduced. This could mean that the gelatinase inhibitor Compound I prevents the complete exhaustion of the bone marrow stem cell pool during the G-CSF mobilization and also during the cytotoxic therapy. The combination of G-CSF and Compound I results in an insitu repopulation of the bone marrow by new generated stem cells while the number of harvestable stem/progenitor cells in the blood (spleen) is hardly affected. Thus, the reconstitutive capacity (quality) of stem cell preparations for transplantation is not altered. The suitability of selective inhibitors of matrix-metalloproteinase will be discussed for the co-stimulation and a protection of bone marrow stem cells in the course of G-CSF mobilization procedure and cytotoxic therapy. The usefulness of Compound I could be of special interest during the high dose chemotherapy and stem cell transplantation. Similar effects can be found with Compounds II - V.
Example 4
Determination of MMP enzymatic activity
Inhibitors were tested in a modified fluorescence-assay as described by Stack, M.S., and Gray, R.D., J. Biol. Chem. 264 (1989) 4277-4281. Human MMP-I, MMP-2, MMP-3, MMP-9 and MMP-14 are commercially available (e.g. Calbiochem). The pro-enzymes were activated with 1 mM APMA (incubation for 30 min at 37°C) immediately before testing. Activated enzyme is diluted to 100 ng/ml in incubation buffer (50 mM Tris, 100 mM NaCl, 1OmM CaCl2, pH 7.6). The compounds were dissolved in 100% DMSO. For IC50 determination a minimum of 8 dilution steps between 0.5 - 1000 nM have been prepared. DNP-substrate (Bachem M1855,
255μM) was dissolved in incubation buffer.
The test tube contains 970μl incubation buffer, lOμl inhibitor solution and lOμl enzyme solution. The reaction was started by adding the lOμl substrate solution.
Kinetics of activity were determined using excitation at 280 nm and emission at 346 nm measured on a FluoroMax™ (Spex Industries Inc., Edison, NJ, USA) over 120 sec. DMSO has been used as control instead of inhibitor solution.
IC5o's are defined as the concentration of inhibitor that gives a signal that is 50% of the positive enzyme control.
IC50 values (nM) are shown in Table 1.
Table 1
Figure imgf000014_0001
List of References
Benboubker, L., et al., Exp. Hematol. 31 (2003) 89-97
Bergers, G., et al., Nat. Cell Biol. 2 (2000) 737-744
Carmeliet, P., et al., Nat. Genet. 17 (1997) 439-444 Carstanjen, D., et al., Transfusion 42 (2002) 588-596
Chang, C, and Werb, D., Trends Cell Biol. 11 (2001) S37-43
Denis, L.J., and Verweij, L, Invest New Drugs 15 (1997) 175-185
De Propris, M.S., et al., Acta Hematol. 109 (2003) 57-63
Dong, Z., et al., Cell 88 (1997) 801-810 Drake, M., et al., Br. J. Haematol. 98 ( 1997) 745-749
Egeblad, M., and Werb, Z., Nat. Rev. Cancer 2 (2002) 161-174
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Janowska-Wieczorek, A., et al., Blood 93 (1999) 3379-3390
Lemoli, R.M., et al., Blood 102 (2003) 1595-1600
Lord, B.I., et al., Blood 79 (1992) 2605-2609
Lund, L.R., et al., EMBO J. 18 (1999) 4645-4656 Manes, S., et al., J. Biol. Chem. 274 (1999) 6935-6945
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Pragnell, I.B., et al., Blood 72 (1988) 196-201
Rasmussen, H.S., and McCann, P.P., Pharmacol. Ther. 75 (1997) 69-75
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WO 96/06074
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WO 99/01428

Claims

Patent Claims
1. A method for the stimulation and/or protection of hemopoietic stem cells, characterized by treatment of said stem cells with a trioxopyrimidine compound having an inhibitory activity against MMP-I, MMP-2, MMP-3, MMP-9 and MMP-14 defined as
a) an IC50 value of less than 5 μM for MMP-2, MMP-9 and MMP- 14 each; b) a ratio of more than 100 for the IC50 values of MMP-l:MMP-2, MMP- 1: MMP-9, MMP-1:MMP-14; and c) a ratio of more than 10 for the IC50 values of MMP-3:MMP-2, MMP-3: MMP-9, MMP-3:MMP-14.
2. A method according to claim 1, characterized in that said trioxopyrimidine compound is used in combination with a colony stimulating factor.
3. A method according to claim 1 or 2, characterized in that said colony stimulating factor is G-CSF.
PCT/EP2004/006028 2003-06-06 2004-06-04 Matrix metalloproteinases inhibitors for the stimulation and protection of bone marrow stem cells WO2004110457A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997023465A1 (en) * 1995-12-23 1997-07-03 Boehringer Mannheim Gmbh New barbituric acid derivatives, processes for their production and pharmaceutical agents containing these compounds
US5932577A (en) * 1996-05-15 1999-08-03 Bayer Corporation Substituted oxobutyric acids as matrix metalloprotease inhibitors
WO2002053138A2 (en) * 2001-01-02 2002-07-11 Elisabeth Shanahan-Prendergast Treatment for inhibiting neoplastic lesions using incensole and/or furanogermacrens

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997023465A1 (en) * 1995-12-23 1997-07-03 Boehringer Mannheim Gmbh New barbituric acid derivatives, processes for their production and pharmaceutical agents containing these compounds
US5932577A (en) * 1996-05-15 1999-08-03 Bayer Corporation Substituted oxobutyric acids as matrix metalloprotease inhibitors
WO2002053138A2 (en) * 2001-01-02 2002-07-11 Elisabeth Shanahan-Prendergast Treatment for inhibiting neoplastic lesions using incensole and/or furanogermacrens

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