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  • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
• • 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
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  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
  • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
  • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
  • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
  • C12Y305/04005—Cytidine deaminase (3.5.4.5)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention is concerned with the control of cell proliferation, especially the control of haemopoiesis and/or granulopoiesis.
• the present invention is concerned with the use of cytidine deaminase and/or its regulators to control cell proliferation.
• stem cell systems are the haemopoietic system in bone marrow and the epithelial and epidermal systems .
• GRE mature granulocytes and granulocyte extract
• Regulation of cell proliferation by stimulation or inhibition continues to be of interest as a treatment for diseases or conditions where natural control of cell division has malfunctioned, for example in cancer (in particular leukemia) or AIDS.
• cytidine deaminase is responsible for the conversion of cytosine to uracil in mammalian cells. This reaction is also catalysed by other enzymes, including deoxycytidylate deaminase and a sequence specific cytidine deaminase involved in the editing of apolipoprotein B mRNA.
• CDD has been isolated from human tissues, and sources include the liver, spleen and placenta (see Ho, Cancer Res 22.-- 2816-2820 (1973) and Cacciamani et al, Arch Biochem Biophys 290: 285 (1991)) . Additionally, Chabner et al . (J Clin Invest 52.: 922-931 (1974)) reported the partial purification and characterisation of CDD from normal and leukemic granulocytes. A highly purified form of CDD (E.C. 3.5.4.5) has been isolated from human placenta (see Laliberte et al, Cancer Chemother Pharmacol 20.: 7-11 (1992)) .
• CDD is known to be a 52 kD protein composed of four subunits which are currently thought to be identical. CDD continues to be of interest in the field of cancer therapy since this enzyme is responsible for the deamination of the widely used anti-cancer agent cytidine arabinoside (Ara-C) .
• the product of this deamination reaction is uridine arabinoside (Ara-U) which, besides being much less effective therapeutically than Ara-C, is thought to cause neurotoxicity.
• Ara-C chemotherapy the active agent is continually degraded by cytidine deaminase, high dosages of Ara-C are often administered in order to maintain that compound at a therapeutically effective level in the body.
• THU tetrahydrouridine
• CDD cytidine deaminase
• the cytidine deaminase is in an at least partially purified form.
• Substantially purified cytidine deaminase is also preferred.
• cytidine deaminase is inhibitory although the degree of inhibition depends on the concentration present.
• a reduced inhibitory effect has been observed at high concentrations of CDD ie. a bell-shaped dose-response curve is obtained.
• the cytidine deaminase used may be derived from any convenient source.
• the enzyme may be isolated from bodily organs such as the liver or spleen (see Ho (1973) , Chabner (1974) and Cacciamani (1991) supra) . More conveniently, however, the enzyme may be isolated from cell cultures - the cells either producing CDD naturally or as a result of transformation with recombinant DNA.
• the enzyme CDD is produced from cells grown in culture which have been transformed or transfected with a DNA vector coding for cytidine deaminase, the cytidine deaminase gene being controlled by appropriate promoter and/or regulator sequences.
• Harvesting and purification of cytidine deaminase may be by any suitable method. Suitable purification and separation techniques are well-known to those skilled in the art and include centrifugation, precipitation, dialysis, chromatography, including affinity chromatography and column chromatography.
• Human granulocytes contain large amounts of CDD, 9- 10 times more than mononuclear cells. The amount drops to 36% of the normal value in the granulocytes with chronic myelogen leukemia. The reduction per cell is even greater with acute myelogen leukemia. Recent findings indicate that the amount of CDD in normal and leukemic cells can be approximately equivalent, however, CDD activity is reduced in the leukemic cells. The high number of leukemic cells can thus be explained by a reduction of CDD or active peptide groups from CDD. CDD may therefore be used in the treatment of the symptoms.
• Thymidine is required as a co-factor for CDD as has been previously observed for crude granulocyte extract (see Helgestad et al, supra and B ⁇ yum et al, Eur J. Haematol 40_: 119 (1988) ) . In agar cultures with human granulocyte cells the inhibitory effect of CDD was detectable with a thymidine concentration of 10 ⁇ 6 M.
• the present invention also provides a combination of cytidine deaminase or a functional fragment thereof and one or more cofactors which enhance the activity of cytidine deaminase, for use in the regulation of cell growth, in particular haemopoiesis, especially granulopoiesis, for example in chronic myelogen leukaemia.
• cofactors include nucleosides or analogues thereof, in particular pyrimidine nucleosides and analogues thereof, especially those selected from the list consisting of thymidine, deoxycytidine, deoxyuridine and their phosphate derivatives. Thymidine is especially preferred. Conveniently the thymidine is present at a concentration range of 5xl0" 3 to lxl0 "6 M.
• the thymidine or other cofactor may be admini-stered simultaneously or sequentially with the CDD.
• Thymidine is of course naturally present in the body and it may therefore not be essential for thymidine to be administered at all for inhibition of cell division to be achieved.
• Ensminger et al (Cancer Res ___7: 1857 (1977)) reported a median thymidine concentration of 0.2 x 10" 6 M in human plasma. Whilst this concentration appears lower than that required in vitro, it is believed that thymidine concentration at particular sites in the body, for example in the bone marrow, may be locally elevated. In the bone marrow for example large numbers of erythroid nuclei are extruded from normoblasts and then engulfed by macrophages in which DNA degradation takes place, thus generating inter alia thymidine.
• Thymidine has itself been reported to cause inhibition of cell proliferation (see Blumenreich et al, in Cancer Research 44.: 2203-2207 (1984) , Chiuten et al, in Cancer Research 4JQ.: 818-822 (1980) and Leyva et al, in J. Cancer Res. Clin. Oncol. 107: 211-216 (1984)) .
• relatively high dosages of thymidine giving millimolar concentrations in the plasma
• did cause some remission in cancer patients it was not shown to be an effective anti-cancer therapy and induced side-effects such as nausea, vomiting and hepatotoxicity.
• the inhibitory effect of thymidine on cell division was believed to be due to allosteric inhibition of the enzyme ribonucleotide reductase.
• ribonucleotide reductase would prevent or reduce the conversion of cytidine to deoxycytidine.
• CDD deaminates deoxycytidine, again lowering the concentration of this metabolite. Whilst thymidine and CDD separately only have a marginal effect in isolation, their combination could cause a significant deficiency in deoxycytidine triphosphate (dCTP) (which is one of the precursors of DNA) leading to failure of cells to divide.
• dCTP deoxycytidine triphosphate
• CDD binds to a specific receptor on the cell surface, the binding or subsequent steps in the mechanism being enhanced by the co-factor thymidine.
• the binding of CDD to its receptor may then cause transmission of a signal which prevents or inhibits cell division.
• Thymidine thus acts to enhance the effect of cytidine deaminase and at higher concentrations, for example 3-6 x 10 "5 M, thymidine causes CDD to exhibit a much stronger inhibitory effect ie. 50-90% suppression of normal cellular proliferation.
• Other regulators which enhance the CDD-mediated inhibition of cellular proliferation include the nucleosides deoxycytidine and deoxyuridine. Both deoxycytidine and deoxyuridine may replace thymidine in enhancing the inhibitory action of CDD. Additionally, a mixture of the nucleosides deoxycytidine and thymidine act synergistically in promoting the inhibition of cell division exhibited by CDD.
• Thymidine phosphates such as thymidine monophosphate and thymidine triphosphate act in the same way as thymidine.
• dCMPD deoxycytidine monophosphate deaminase
• other inhibitors of dCMPD such as 5-fluorodeoxyuridine (5-FdU) and 5-fluoro- deoxyuridine monophosphate (F-dUMP) act similarly.
• CDD and its potentiators could be used in combination therapy with anti-cancer agents.
• CDD and CDD potentiators By use of CDD and CDD potentiators, cells of interest, for example the bone marrow, could be taken out of cycle and thus be protected (ie. made less sensitive) to the effects of anti-cancer agents.
• thymidine was used at rather low concentrations, but nevertheless yielded 20-30% inhibition (p ⁇ 0.01) (4-8xl0 "5 M thymidine) in combination with CDD in a granulocyte extract (GRE) from 1.6xl0 5 granulocytes (/ml) .
• GRE granulocyte extract
• thymidine 8xlO" 5 M yielded no inhibition.
• FdUMP had itself an antiproliferative effect at the highest concentrations, but this effect was partly abolished when combined with thymidine.
• the most striking effect of FdUMP was the ability to induce inhibition also together with a high GRE concentration.
• human serum contains CDD that induces inhibition of colony formation when thymidine is added. Accordingly it was shown by thin layer chromatography that CDD activity could easily be detected in fresh human serum. No appreciable activity was found in fetal calf serum. However these sera have been subjected to freezing/thawing which tends to reduce CDD activity. In any case, these finding may suggest the CDD plays a physiological role in regulation of white cell production.
• THU may be used in the treatment of some diseases where there is a need for stimulating cell proliferation by inactivating CDD.
• thymidine and other CDD enhancers may be used in certain diseases to enhance the inhibitory effect of CDD in tissue fluid.
• thymidine and CDD may be used in combination. Large doses of thymidine have been used in the treatment of malignant diseases, with little success.
• thymidine dosage may enhance the inhibitory effect of CDD whereby production of granulocytes may be maintained at a lower level.
• CDD inhibitors may be of use in the regulation of leukopenic situations in general and more particularly after anti-cancer treatment or bone marrow transplantations or in connection with the treatment of infections. CDD inhibitors are believed to increase the number of white blood cells and/or their activity.
• dCMPD is antagonist of CDD and thus acts as a CDD inhibitor.
• the present invention thus provides the use of CDD inhibitors in the regulation of cell proliferation, in particular cell proliferation mediated by CDD.
• CDD inhibitors such as THU
• the present invention provides the use of one or more CDD inhibitors in the preparation of a medicament for the mobilization of haematopoietic stem cells to the blood.
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising (a) cytidine deaminase or a functional fragment thereof optionally in combination with a CDD potentiating nucleoside or analogue thereof or (b) an inhibitor of cytidine deaminase.
• compositions according to the present invention the usual pharmaceutically acceptable inert carriers, diluents, additives, flavourings and/or colourings may of course be present as required.
• Suitable adjuvants and excipients will be known to those skilled in the art.
• the present invention provides a method of treatment of the human or non-human animal body to regulate cell proliferation, said method comprising administering to said body cytidine deaminase or a functional fragment thereof.
• the cytidine deaminase or functional fragment thereof may also be used in combination with one or more cofactors which enhance the activity of cytidine deaminase.
• a further method of treatment to regulate cell proliferation comprises administration of one or more cytidine deaminase inhibitors.
• Fig. 1 illustrates the effect of different concentrations of deoxycytidine, alone or in combination with thymidine in inhibiting colony formation in the presence of granulocyte extract (GRE) ;
• Fig. 2 shows how colony formation is affected by addition of GRE, with and without thymidine or deoxyuridine;
• Fig. 3 depicts the relationship between colony number and dose of GRE at different concentrations of thymidine
• Fig. 4 shows the inhibition of colony formation at varying concentrations for different Mono Q fractions of GRE
• Fig. 6 is a bar chart showing how colony number is affected by the concentration of azacytidine with and without GRE;
• Fig. 7 illustrates how colonies of mouse BMC and human blood cells treated with GRE and affected at different concentrations of THU:
• Fig. 9 illustrates the results of MTT-assay for different Mono Q fractions of GRE.
• Fig. 10 shows the results of MTT-assay for fraction 5 (of the Mono Q purification of GRE) at different concentrations.
• Thymidine, cytidine, deoxycytidine, deoxyuridine, 5-azacytidine, 5-aza-2 ' -deoxycytidine, and cytosine arabinoside were obtained from Sigma. Tetrahydrouridine was obtained from Calbiochemic (La Jolla, Ca) . Deoxy[5- 3 H] -cytidine, specific activity 1.07 TBq/mmol, was obtained from Amersham. McCoy's 5A medium, RPMI 1640 and CMRL 1066 medium were obtained from Flow. The CMRL 1066 medium was used with additives (Helgestad et al, supra) . Lymphoprep was provided by Nycomed AS, Oslo. Murine interleukin 3 was obtained from Genzyme (Cambridge, Ma) and murine granulocyte- macrophage colony-stimulating factor (GM-CSF) from Pepro Tech Inc. (Rocky Hill, NJ) .
• Genzyme Cambridge, Ma
• Bone marrow cells were obtained from the femurs of female B 6 D 2 mice (Bomholdt gaard, Denmark) . Mononuclear cells and granulocytes from human blood and buffy coat samples were separated with Lymphoprep (B ⁇ yum et al, Scand J. Immuno 697 (1991)) . A slow centrifugation (60g, 10 min) was included to remove platelets from the mononuclear cells. Contaminating erythrocytes in the granulocyte fractions were lysed by incubating the cells in 0.83% NH 4 C1 for 7 minutes at room temperature. After centrifugation (600 g, 7 min) , the supernatant was removed, the cells resuspended in 0.9%
• NFS 60 cells an early murine myeloid cell line, the C6 cell line, a fibroblast type murine cell, and a human bladder carcinoma cell line (5637) were kindly provided by Dr. Andrew King, SmithKline Beechham (Pa, USA) . These cells, and mouse L-cells (NCTC Clone 929) , were cultured in RPMI 1640 medium with 10% FCS, and were subcultured once or twice weekly. The supernatant from L-cells was used as source of macrophage CSF. The medium conditioned by the carcinoma cell line (CM 5637) is a rich source of human G-CSF (Welte et al, PNAS £2.: 1526 (1985)) . The NFS 60 cells were cultured in the presence of 2% (v/v) CM 5637.
• Granulocyte extract The granulocyte pellet obtained after NH 4 C1 treatment and washing was suspended in water for 4-5 minutes, at a concentration of 200 x 10 6 cells/ml . The supernatant was collected after centrifugation and stored at -20°C until used.
• GM-CFC assay Mouse BMC (5 x 10 4 per plate) were cultured in 0.3% agar (Bacto-agar, Difco) in CMRL 1066 medium and 16% fetal calf serum. CM 5637 was used (0.1 ml per 1 ml culture plate) as stimulator. After 7 days of incubation at 37°C and with 7.5% C0 2 in humidified air, the colonies (>50 cells) were counted. Mononuclear human blood cells (5 x 10 5 /ml) were cultured for 14 days in 0.3% agar, in McCoys' s 5A or CMRL 1066 medum, with 16% FCS. No stimulator was added. Aggregates of more than 40 cells were counted as colonies. The cultures were run in triplicate.
• MTT-assay Proliferation of established cell lines was measured by a colorimetric method (Mosmann, J Immunol Meth 6 _: 55 (1983)) .
• the cells were seeded in microtitre plates (Costar 3596, Cambridge, Mass.) at a concentration of 2 x 10 4 cells/well in 100 ⁇ l of CMRL 1066 medium with 15 x 10" 5 M thymidine and 5% FCS (Hyclone, Logan, Utah) . Fifty ⁇ l of test samples in various dilutions was added to each well .
• Cytidine deaminase assays Reaction mixtures contained 50 mM Hepes, pH 7.5; 0.1 mM deoxy[5- 3 H] -cytidine (specific activity 18.5 MBq/mmol) ; and protein fractions to a final volume of 5 ⁇ l, and was incubated for 30 minutes at 37°C. The reaction was stopped by addition of unlabelled deoxycytidine and deoxyuridine in equimolar amounts (1 ⁇ l of 5 mM each) followed by application on a Polyethyleneimine thin layer sheet (plastic backing, Schleicher & Sch ⁇ ll) . The chromatograms were developed in isopropanol/0.1M HCl
• GRE Protein chromatography: GRE (4 ml, approximately 40 mg protein) was diluted with 6 ml buffer A (50 mM NaCl; 50 mM Hepes, pH 7.5; 1 mM EDTA) and applied to a DEAE- Sephacel (Pharmacia) column (1.3 x 2.6 cm) preequilibrated with buffer A. The column was washed with 10 ml of buffer A and eluted with 6 ml of 400 mM NaCl (50 M Hepes, 1 mM EDTA) .
• buffer A 50 mM NaCl
• Hepes pH 7.5
• 1 mM EDTA DEAE- Sephacel
• the eluate was subjected to buffer exchange to buffer A with a PD-10 column (Pharmacia) , and applied to a Mono Q anion exchange column (0.7 x 5.5 cm, FPLC system, Pharmacia) .
• the column was washed with 20 ml of buffer A and eluted with a linear gradient to 1000 mM NaCl at a flow rate of 0.25 ml/min.
• Fractions (1 ml) were sterile filtered and tested for inhibitory activity in the GM-CFC assay or for cytidine deaminase activity as indicated above.
• Active fractions were further purified by gel filtration (1.5 x 25 cm Ultrogel AcA 34) and eluted with buffer B (400 mM KC1; 50 mM Hepes, pH 7.5; 1 mM EDTA, 25% glycerol, 1 mM mercaptoethanol) . Fractions (1 ml) were sterile filtered and assayed for GM-CFC inhibitor and cytidine deaminase activity as described above. The gel column was precalibrated with myoglobin (17.5 kD) , ovalbumin (47 kD) , and blue dextran (reflecting void volume) under identical flow conditions.
• buffer B 400 mM KC1; 50 mM Hepes, pH 7.5; 1 mM EDTA, 25% glycerol, 1 mM mercaptoethanol
• GM-CFC granulocyte-macropha ⁇ e colony-forming cells
• thymidine and deoxycytidine had an additive effect (p ⁇ 0.05) . It is also shown that deoxycytidine could replace thymidine as a co-factor for the granulocyte extract at a concentration of 2 x 10" 4 M ie. at a lOx higher concentration than the concentration of thymidine required to produce strong inhibition.
• GM-CFC Human blood GM-CFC were grown in agar cultures. Thymidine or deoxyuridine, alone or with 10 6 GRE (granulocyte extract) were added in McCoy's medium. The inhibitory effect on GM-CFC was observed. McCoy's medium is nucleoside free. A comparison with CMRL 1066 medium (contains 4.0 x 10" 5 M thymidine) was also performed.
• thymidine at high concentration (5 x 10" 4 M) exerted an inhibitory effect in the absence of granulocyte extract.
• BMC Mouse bone marrow cells
• Granulocyte extract was added in increasing doses .
• Mean values ( ⁇ SD) were obtained from 3 replicate cultures.
• Mouse bone marrow cells have previously been shown to be sensitive to GRE and thymidine (See B ⁇ yum et al, Eur J. Haematol 4_0: 119 (1988)) .
• thymidine See B ⁇ yum et al, Eur J. Haematol 4_0: 119 (1988)
• 4 x 10' 5 M thymidine was sufficient to cause strong (approximately 80%) inhibition of cells cultured in methylcellulose.
• the low density fraction of mouse BMC separated with Lymphoprep were cultured in McCoy's medium with 13xlO ⁇ 5 M thymidine.
• Recombinant IL-3 (100 units/ml) rGM-CSF (10 ng/ml) , L-CSF (0.1 ml/plate) and CM 5637 (0.1 ml/plate) were used as stimulators.
• Colony number Colony number Stimu- in control as a percentage of control value lator per 5xl0 4 BMC
• GM-CFC from human blood were cultivated and the inhibitory effects of GRE, Ara-C and GRE with Ara-C were investigated. The results are shown in Figure 5.
• TNU tetrahydrouridine
• the cells were cultured in agar in CMRL 1066 medium with either 3.3 x 10" 5 M (human cells) or 13 x 10" 5 M (mouse cells) thymidine. GRE from 2 x 10 s cells were added per culture plate.
• Figure 7 gives the results with mean values (+SD) from 4 mouse cell experiments and 3 human cell experiments.
• THU GM-CFC was completely abolished by 4 x 10 "6 M THU.
• THU is known to be a specific inhibitor of CDD and these results therefore indicate that CDD is responsible for the inhibitory effect observed.
• the growth inhibitor and CDD activity were found to copurify, both eluting in a single peak of M r 50 kD which corresponds to the molecular weight of human CDD reported earlier by Cacciamani et al in Arch Biochem Biophys 2 0.: 285 (1991) .
• THU at a concentration of 4 x 10" 5 M neutralized the inhibitory and deaminating ability in all active fractions.
• NFS 60 and C6 cells were cultured in microtitre plates (20,000 cells/well) for four days and the cell growth was measured with a colorimetric method (MTT) .
• GM-CFC was monitored with the agar colony assay.
• Fractions obtained after anionic exchange chromatography (Mono Q) were added to NFS 60 and GM-CFC cultures after 800 fold final dilution in growth medium and to the C6 cells after 50 fold dilution.
• Figures 9 gives the results from the mean ( ⁇ SD) of triplicate cultures from one representative experiment. The results were confirmed twice, in one experiment with a different set of GRE fractions.
• Example 9 The most active fraction from Example 9 (Fraction 5 - see Figure 9) was tested at several concentrations for its ability to inhibit cell replication for murine GM- CFC, NFS 60 and C6 cells.
• NFS 60 cells and C6 cells were cultured in microtiter plates (20,000 cells/well) for four days and the cell growth was measured with the MTT assay.
• GM-CFC was monitored with the agar colony assay.
• Figure 10 gives the results of mean values (+ SD) of triplicate cultures from one representative experiment. The results were confirmed in two additional experiments.
• Figure 10 shows the dose-response curve for fraction 5 at different dilutions.
• the inhibitory effect on GM-CFC and NFS 60 cells was partially alleviated at high concentrations of Fraction 5 GRE, thus giving a bell- shaped response curve.
• Bone marrow cells were cultured for 7 days in McCoy's medium and then the colonies were counted.
• Two granulocyte extracts were prepared, the first from 1.6x10 s granulocytes per ml and the second from 8x10 s granulocytes per ml.
• T thymidine
• FdUMP 5-fluoro-2-deoxyuridine- monophosphate
• GRE granulocyte extract
• GRE 1 extract from 1.6xl0 5 granulocytes/ml .
• GRE 2 extract from 8xl0 6 granulocytes/ml.
• FdUMP itself had an antiproliferative effect at high concentrations, but this effect was abolished when thymidine was also present.
• Mononuclear human blood cells were cultured in fetal calf serum (FCS) or autologous serum from the donor of the blood cells. The cells were cultured at two different thymidine concentrations (3.3xl0 *5 M thymidine or 16xl0 "5 thymidine) . Aggregates with more than 40 cells were counted as colonies. THU and/or GRE were added and the effect on colony number was evaluated as a percentage of the control values.
• FCS fetal calf serum
• Colony number as a percentage of control values with FCS and low thymidine concentration.
• TNU tetrahydrouridine
• a dose of 0.6 mg per mouse was injected intraperitoneally at time -4 hours (THUxl) or time -20 and -4 hours (THUx2) , and blood was collected by heart puncture at time 0 hours.
• White blood cell number was determined and 1x10 s cells were seeded in 1 ml 0.33% agar in CMRL 1066 medium and incubated at 37°C at 7.5% C0 2 in air for the indicated time period, The results are shown in Table 4.
• THU haematopoietic progenitor cells

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  • 1994-06-01 WO PCT/GB1994/001190 patent/WO1994027632A1/en not_active Application Discontinuation
  • 1994-06-01 EP EP94916343A patent/EP0703787A1/en not_active Ceased
  • 1994-06-01 JP JP7500426A patent/JPH08510385A/ja active Pending
  • 1994-06-01 AU AU68036/94A patent/AU6803694A/en not_active Abandoned
• 1995
  • 1995-11-30 NO NO954880A patent/NO954880L/no unknown

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