JPH03505575A - stem cell inhibitors - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] stem cell inhibitors The present invention relates to stem cell inhibitors, particularly for the management of cancer chemotherapy. Regarding improvement.

Cytotoxic drugs active in the cell cycle during cancer chemotherapy Bone marrow aplasia represents a limiting factor for clinical use. is well known. Current methods of chemotherapy target hematopoietic stem cells during treatment with cytotoxic drugs. It has been recognized for many years that if it is possible to protect cells, improvements can be made. However, despite extensive research in this field, only moderate amounts of No specific inhibitor has been found so far. One reason for this lack of progress is that There are difficulties with suitable in vitro assays to control cell regulation. It is believed that.

Designed to protect stem cells against cell cycle-specific drugs used in cancer chemotherapy Certain macrophage cell lineages have the ability to interact with blood stem cells. The present inventors have been able to isolate and identify the protein-like substance obtained from 1ine). This is now possible.

Therefore, the present invention provides a hematopoietic stem cell inhibitor characterized by having the following properties. do: i.e.; 1. Inhibitor activity is sensitive to degradation by trypsin, and therefore the inhibitor is proteinaceous. It's like that.

2. When the inhibitor is partially purified by treatment with an anion exchanger, the molecular weight It exhibits a molecular weight in the range of 45-60 Kd on analytical resin.

3. Inhibitors were analyzed in a single batch on polyacrylamide gel electrophoresis using reducing conditions. When purified to a molecular weight range of 8-10 Kd, the shows a slightly higher molecular weight.

4. The inhibitors are as follows: (i) inhibitor activity is preserved after heating at 37°C, 55°C and 75°C for 1 hour; (if) the inhibitor activity is preserved after heating at 100°C for 10 min, as in It is qualitative.

5. The inhibitor is combined with an anion exchanger and contains between 0.26 and 0.28 mol NaC It is eluted with a salt gradient of 1.

6. Inhibitor is heparin/Sepharose/affinity chromatography resin and can be eluted from the resin with 1 molar sodium chloride buffer.

7. Inhibitor is added to Blue Sepharose affinity chromatography resin It binds strongly and can be eluted from the resin with 5M magnesium chloride.

8. When cycling stem cells are treated with a partially purified inhibitor, cytotoxic Becomes resistant to the action of synarabinoside. Treated stem cells are then treated with cytotoxic drugs Once washed with saline buffer to remove substances and inhibitors, the surviving stem cells remain in culture. It shows normal growth in the nutrient substrate.

Isolation and identification of inhibitors is facilitated by the development of in vitro assays that control stem cell regulators. It was easy for the first time. Progenitor cells in the cell cycle (not stem cells, but more mature cells) cells) are not affected by the inhibitor. Directly apply purified inhibitor to this assay. When added, it results in inhibition of macroscopic colony expression, but other Does not affect colonies.

In vitro stem cell (CFU-A) assay For detection of in vitro stem cells, 25% Supplemented alpha variant containing bovine or horse fetal serum and 0.3% agar 104 bone marrow cells in 4 mA of minimal essential medium (MEM) were placed in a 5 cm Petri dish. Conditioned L929 cells in 0.6% agar, 10% ) Medium (L929CM.

source of growth factor C3F-1) and 10% API-19 T cell conditioned medium (API, -1000MX growth factor GM-C3F and other unspecified sources of stem cell growth factors) inoculated on top of a lower layer of the same medium containing The culture medium was 10% 002.5% 02. It was incubated at 37°C in a well-humidified atmosphere containing 85% N2. Ko INT2-(p-iodophenyl)-3-(p-nitrophenyl)-5 - Stained overnight with hydrated salt of phenyltetrazolium chloride. CFU-A assay Although there were colonies with diameters smaller than two irises in After performing preliminary experiments with arabinosides, this value was selected as a useful cut-off point. I chose. We found that in individual dishes, colonies with diameters smaller than two-fold shoulders were predominantly However, colonies larger than two wings are the smallest proliferating cells. It was found that it was induced from cells. Only colonies larger than 2 Reifumi have these The assay was scored.

Inhibitors of the invention can be obtained from several macrophage cell lines. At the beginning Various known macrophage cells derived from bone marrow, typically such macrophages Murine bone marrow is of interest because it is a major source of cell lineages. Like that At least two populations of cell lines can be screened to produce the inhibitors of the invention. Two known cell lineages were identified.

In the examples illustrating the invention, such known macrophage cell lines Isolation from is described.

As an alternative to using known macrophage cell lines, bone marrow cells can be retrorowed. Transformation of bone marrow cells using virus transforming agents to generate suitable macrophage cells It is also possible to produce strains.

The inhibitors of the invention can be prepared by culturing inhibitor-producing macrophage cell lines under conventional conditions, e.g. It can be produced extremely easily by culturing in an appropriate growth medium at ℃. Ru. 5% v/v diacid in air until the cell concentration is approximately 106 per mA'. The use of aerobic conditions with carbonized carbon provides an ideal growth environment. At this stage Note that the growing cells are separated from the growth medium, for example by centrifugation or preferably membrane filtration, The inhibitor can then be recovered from the supernatant. First supernatant to demonstrate inhibitor activity It is desirable to concentrate it using membrane dialysis, for example, to a concentration of about 20 times. then The inhibitor can be isolated from the concentrated supernatant by chromatographic techniques. can.

The first step in the purification is to pass the concentrated supernatant through an anion exchange resin and pass the concentrated supernatant through an anion exchange resin. 25 - eluting the fractions with a 0.30M NaCl solution.

The second step of purification is to transfer the product from the first step to a Sepharose-heparin column. Elute the fractions using a low (both IM and IM) molar NaCI solution. including.

The third step of purification is to pass the product from the second step through Sepharose Blue to reduce the Elute the fragments using a MgCl2 solution with a molar concentration of at least 3M. including. This eluent was isolated by polyacrylamide gel electrophoresis under reducing conditions. It gives a product showing one band.

The primary use of the inhibitors of the invention is in clinical practice, as described in more detail below. in which the inhibitor or immunogenically active fraction thereof immunizes the host animal. , some or all of the inhibition can be achieved by recovering antibodies from host animals or antibody-producing cells. It can also be used as an immunogen to collect antibodies that recognize harmful agents. These antibodies For example, it can be produced using rabbits that can collect polyclonal antibodies from rabbit serum. can be used as an immunogen for the production of monoclonal antibodies by conventional techniques. Mi can be used.

The invention also extends to DNA sequences encoding the inhibitors of the invention. Ru. Such DNA is of interest for the production of inhibitors by recombinant DNA technology. like this These techniques can include two different approaches. both approaches Messengers from macrophage cell lines that naturally produce inhibitors as a first step Requires the production of a gene library from ginger RNA. This is a bacteria or It involves the production of complementary DNA that can be expressed in other host cells.

Once the cDNA library has been produced, the first approach is to generate DNA probes. Includes production and use. Probes and hybrid forms by limited sequence analysis of inhibitors DNA library to identify these cDNAs in the resulting library. Enables the synthesis of oligonucleotides that can be used to investigate global inhibitors It will be possible to identify Messenger RNAs encoding . macropher An alternative method for examining gene libraries derived from humans is to examine gene libraries of human origin. of human origin, in which the library can be expressed in host cell lines using currently well-known techniques. can be examined to identify and isolate DNA encoding an inhibitor of .

Preliminary experiments on the biological properties of the inhibitor indicate that its effect on stem cells during the cell cycle is It is indicated that it is reversible. Chemotherapy agents used in cancer chemotherapy The majority of these drugs have relatively low in vivo half-lives, usually less than 24 hours; The inhibitory effect of light inhibitors is at least effective while the chemotherapeutic agent is active in vivo. maintained for most of the time.

The inventor's prediction is that the normal physiological mechanisms in the body will interfere with stem cells during the cell cycle. This would limit the duration of effective activity of the harmful agent.

In clinical use, it is desirable to target the inhibitors of the invention to hematopoietic tissues. . This goal is usually achieved by injecting the inhibitor via infusion or intravenous porous administration. This invention can be achieved by combining the inhibitor and a suitable diluent or such parenteral The invention also includes pharmaceutical compositions containing carriers suitable for clinical administration.

The interest in the inhibitors of the present invention is not limited to hematopoietic tissue-specific stem cells, but also to other stem cells. related to reducing the side effects of cytotoxic drug treatments on cells, e.g. bone marrow cells. In addition to producing inhibitors that are relevant for the treatment of solid tumors. It has also spread to epithelial stem cells. Normal stem cell proliferation is prevented and therefore increased. Proliferating leukemia cells can now be treated with cytotoxic drugs, and leukemia bone marrow cells can be treated with cytotoxic drugs. Inhibitors can also be used to treat leukemia as they are treated in vitro or in vivo. It is also of interest.

The invention is further illustrated in the Examples below.

1774.2, which is widely available, was used. This is J, Immunol, (1975) 114.898, and Cancer Res, (1977) As described in 37.546.

Selection for serum-free growth in spinner culture J774.2 cells were initially grown in 10% beef tallow. were grown in modified Eagle's medium supplemented with infant serum. Cells were subcultured in the following medium. Darube Soko XIO50 has been cultivated! 5F12X10 50 proverbs ! Glutamine 200 wings M 10 irises! Sodium pyruvate M10 (bM) 5! Distilled water 840m1 Nutridoma-v (nptridoma) SP 10 ml Sodium Bicarbonate 7.5% 41ml Nudride vsP Behringer (Sussex, Lewes) 306hringer), catalog number 1011375; other reagents are Flow Laboratories UK and Give Manufactured by Gibco-Biokult LIK.

774.2 cells were subcultured in the above medium with tallow serum (5%) for one week. , and was further subcultured for three days in the same medium. The cells are further treated with 2.5% tallow blood for one week. subcultured in medium supplemented with serum and then incubated for an additional week until all serum was removed. The cells were subcultured in a medium supplemented with % tallow serum. Repeat every two days at this stage. subcultured and grown to a concentration of 8X10'/l, and 2X1 in subculture medium. Diluted to 0'/mA'.

The cells were then transferred to a stirred culture medium and subcultured in suspension every two days as described above. acclimatized to the growth of It is called J774.2(S) and is grown in serum-free suspension culture medium. This fertile cell line was then used for the production of inhibitors.

500ml (8X 10'/ml) of J774.2 (S) at 1500mA' The culture medium was inoculated into a large fermenter (Tecbne, 7/). Sky A gas containing 5% CO2 was supplied, and the mixture was stirred at 2 Orpm for 2 days at 37°C. 61 medium was then replenished and allowed to grow for 3 consecutive days.

The undiluted medium from 8 x 10' cells was transferred to a microporous microporous membrane with a 0.45 micron scale. Milipore Pellicon-Case tte) is separated from the cells using a device. The medium from which the cells were removed was then placed in an IOK cup. Concentrate in the same apparatus using a cut-off membrane to a final volume of approximately 400 1A. To biochemically purify this concentrate, G- 25HR16150 (Pharmacia) and anion exchange monoQHRI. O/10 column (Pharmacia, FPLC equipment) and finally salt gran Elute with Endo (0 to 1M NacI).

The active fraction of the inhibitor elutes between the range 26 and 0.28 NaCI.

Second stage. The impure inhibitor from the first step was directly removed from Sepharose-heparin Macia) column, HRIO/10. The inhibitor binds to the resin and binds to the protein. Part (more than 90%) of pH 7.6 area IMNaC]-0.02M) Lith HCI can be washed out of the resin using The inhibitor was then added to LMN at pH 7.6. aCl-eluted from the resin in 0,02M Tris HCI.

Third stage. The partially purified inhibitor from the second step is directly blue-coated without desalting. Apply to Sepharose (Pharmacia) column. The column is then inhibited in a second step. Wash with the buffer used to elute the agent to remove other proteins. strongly connected The purified inhibitor is 5MMgc12-0.02M) at pH 7.6) Eluted with CI.

Other biochemical properties of the inhibitor are a) thermal stability; b) Stable for 1 hour at 37°C, 55°C and 75°C.

molecule size The purified inhibitor was deposited on a polyacrylamide gel under reducing conditions to a molecular weight of 8-10. , d can be seen as a single band. Inhibitors are added to polyamines under non-reducing conditions. When subjected to acrylamide gel electrophoresis, it appears that it has a slightly higher molecular weight. I understand. These observations suggest that the inhibitor may have internal disulfide effects that affect electrophoretic mobility. This suggests that it is a single chain polypeptide with fido bonds.

Biological properties of inhibitors Reversible triggering of pluripotent stem cells outside the cell cycle (in vitro assay) CFU-A) Bone marrow cells as described in Sei were supplemented with 20% horse serum. Pairs containing 5X10' cells in IKl's Fischer's medium were incubated in test tubes. Inhibitor or alpha MEM is added to each tube. and Fuscher's medium was added to control tubes. Mixture at 37°C for 5 hours incubated (inhibition assay). during the last 60 minutes of incubation. Add 10-3M cytosine arabinoside to one tube and add an equal volume of medium to the other tube. was added to the test tube. Then analyzed by CFU-A assay as described above. Cells were washed twice before. Inhibitors reduce the number of cycling stem cells by an average of 30% to an average It was found that the reduction was up to 3%. Untreated cells are treated with inhibitors In contrast to cells, they were killed by cytotoxic drug treatment. Impure preparations of inhibitors have also been reported in the literature. CFU-A described in 1. When assayed in vivo using Reversibly induces sexual stem cells out of cycle.

Copy and translation of written amendment) Submission (Article 184-8 of the Patent Law) October 19, 1990 Day Commissioner of the Patent Office Toshi Ue Matsu 1. Display of patent application PCT/GB89100396 2. Name of the invention stem cell inhibitors 3. Patent applicant Address: 5RA Carlton, 1WY, London, United Kingdom terrace house 2 Name Cancer Research Campaign Technology Limited Representative: Foden, Susan 4. Agent〒107 Address: 1-9-15-5 Akasaka, Minato-ku, Tokyo, Date of submission of amendment June 18, 1990 6. List of attached documents This is a 10-line correction.

Preliminary experiments on the biological properties of the inhibitor indicate that its effect on stem cells during the cell cycle is It is indicated that it is reversible. Chemotherapy agents used in cancer chemotherapy The majority of these drugs have relatively low in vivo half-lives, usually less than 24 hours; The inhibitory effect of the chemotherapeutic agent is at least effective while the chemotherapeutic agent is active in vivo. maintained for most of the time.

The inventor's prediction is that normal physiological mechanisms within the body inhibit stem cells during the cell cycle. This would limit the duration of effective activity of the agent.

In clinical use, it is desirable to target the inhibitors of the invention to hematopoietic tissues. . This goal is usually achieved by injecting the inhibitor via infusion or intravenous porous administration. The present invention can be achieved by combining an inhibitor and a pharmaceutically acceptable diluent or Pharmaceutical compositions comprising a carrier, preferably a diluent or carrier suitable for parenteral administration It extends to

international search report Iww+a+, a+a+ Am&”+IU6M 1+e, PCT/Gself9 910029 et al.1+l#+OkiaAjl^ea1. tjl,aANa,s(mT/ G”　　a9/GO396

Claims (10)

[Claims] 1. Characteristics below: 1) Inhibitor activity is sensitive to degradation by trypsin, therefore the inhibitor is a protein It's like that. 2) When the inhibitor is partially purified by treatment with an anion exchanger, the molecular weight It exhibits molecular weight acids in the range of 45-60 Kd on analytical resins. 3) Inhibitors were isolated in single bands by polyacrylamide gel electrophoresis under reducing conditions. When purified to a shows a slightly higher molecular weight. 4) The inhibitors are as follows: (i) inhibitor activity is preserved after heating at 37°C, 55°C and 75°C for 1 hour; (ii) Inhibitor activity is preserved after heating at 100°C for 10 min, as shown in It is qualitative. 5) The inhibitor is combined with an anion exchanger and contains between 0.26 and 0.28 molar NaC It can be eluted with a salt gradient of 1. 6) Inhibitor is heparin/Sepharose/affinity chromatography resin and can be eluted from the resin with 1 molar sodium chloride buffer. 7) Inhibitor is added to Blue Sepharose affinity chromatography resin It binds strongly and can be eluted from the resin with 5M magnesium chloride. 8) When cycling stem cells are treated with a partially purified inhibitor, cytotoxic Becomes resistant to the action of synarabinoside. Treated stem cells are then treated with cytotoxic drugs Once washed with saline buffer to remove substances and inhibitors, the surviving stem cells remain in culture. It shows normal growth in the nutrient substrate. A hematopoietic stem cell inhibitor characterized by having the following. 2. The inhibitor according to 1 above, which is obtained from macrophage J774.2 cells. 3. The inhibitor according to 1 or 2 above together with a pharmaceutically acceptable carrier or diluent A pharmaceutical composition comprising: 4. 3. The composition according to 3 above for parenteral administration. 5. An antibody against the inhibitor described in 1 or 2 above. 6. an inhibitor or an immunogenically active fragment of an inhibitor according to 1 or 2 above; immunizing a host animal and recovering antibodies or antibody-producing cells from the host animal. A method for increasing antibodies, comprising: 7. A DNA encoding the inhibitor according to 1 or 2 above that can be expressed in a host cell. Synthetic DNA containing. 8. The following stages: 1) Proliferate and inhibit myeloid-derived inhibitor-producing macrophage cell line in growth medium 2) contacting the supernatant with an anion exchanger; and The anion exchanger was eluted with 0.25-0.30M NaCl to remove the first phase containing the inhibitor. obtaining an eluent; 3) Contact the first eluent with Sepharose-heparin to The phosphorus is eluted with a solution of at least 1M molar NaCl and the obtaining a second eluent; 4) contacting the second eluent with Sepharose blue; and Sepharose blue in a solution of MgC12 with a molar concentration of at least 3M. The inhibition according to 1 above, comprising: separating the eluent to obtain a third eluent containing the inhibitor. Methods of isolating and purifying agents. 9. 1 or 2 above for use in a method of treating a human or animal body for therapy. Inhibitors as described. 10. 1 above to protect a patient's hematopoietic stem cells from damage caused by cytotoxic drugs. or receiving a cell cycle-specific cytotoxic drug, including administering an inhibitor according to 2. how to treat patients who