Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Hormones – e.g. – prolactin – thymosin – growth factors – etc.
Patent
1994-06-27
1996-03-12
Sayala, Chhaya D.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Hormones, e.g., prolactin, thymosin, growth factors, etc.
530350, C07K 1447, C07K 14475
Patent
active
054986982
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a megakaryocyte potentiator (abbreviated as Meg-POT) of human origin that acts on megakaryocyte colony-Forming units (CFU-Meg) differentiated From pluripotent blood stem cells, and promotes maturation of megakaryocytes in the presence of a substance having a megakaryocyte colony-stimulating factor (abbreviated as Meg-CSF) activity, such as interleukin 3 (IL-3).
BACKGROUND ART
Platelets are one of the corporeal components of the blood having important significance with respect to hemostasis and thrombus formation in the body. Platelets are released into the blood from mature megakaryocytes formed from hematopoietic stem cells in bone marrow via megakaryocytic precursor cells and megakaryoblasts.
Factors having two different types of action are considered to be required to form megakaryocyte colonies from myeloid cells (Williams, N. et al., "J. Cell Physiol.", 110, 101 (1982)). More specifically, these factors consist of Meg-CSF, which by itself result in the formation of megakaryocyte colonies, and Meg-POT, which, although does not result in the formation of megakaryocyte colonies by itself, increases the number of megakaryocyte colonies and promotes their maturation when added together with Meg-CSF.
In human, IL-3 (Teramura, M. et al., "Exp. Hematol.", 16, 84 (1988)) and granulocyte-macrophage colony stimulating factor (Teramura, M. et al., "Exp. Hematol.", 17, 1011 (1989)) have Meg-CSF activity. In addition, examples of substances having Meg-POT activity in human include interleukin 6 (Teramura M. and Mizoguchi, H. "Int. J. Cell Cloning", 8, 245 (1990)), interleukin 11 (Teramura, M. et al., "Blood", 79, 827 (1992)), and erythropoietin (Bruno, E. et al., "Blood", 78, 671 (1989)).
However, it is also known that these substances are not specific factors for the megakaryocyte-platelet lineage, but rather also act on other blood cells as well as on cells outside the blood cell system. Thus, in the case of administration of these substances as pharmaceuticals in anticipation of their action on the megakaryocyte and platelet systems, there is also the risk of the manifestation of other actions different from that which is expected. As such, there is a need for a physiologically active substance having a high degree of usefulness as a pharmaceutical product that specifically acts on the megakaryocyte-platelet system.
DISCLOSURE OF THE INVENTION
Thus, the present invention provides a novel human megakaryocyte potentiator.
After culturing cloned cells (HPC-Y5) derived from human pancreatic cancer cells (Nozomi Yamaguchi et al., "CANCER RESEARCH", 50, 7008, (1990)) (deposited as an international deposition FERM BP-3703 under the Budapest Treaty with Fermentation Research Institute, Agency of Industrial Science and Technology on Dec. 27, (1991)), the inventors of the present invention succeeded in purifying megakaryocyte potentiator (Meg-POT) from a supernatant of that culture, and clarified the characteristics of that substance, thus leading to the completion of the present invention. Namely, the present invention provides a megakaryocyte potentiator having the following properties: presence of IL-3 in vitro; measured by SDS-polyacrylamide gel electrophoresis; 40-45% in 0.1% trifluoroacetic acid (TFA) in reverse phase high-performance liquid chromatography (HPLC) (column: Vydac Protein C4, 4.6.times.250 mm, particle size 5 .mu.m, Vydac); and,
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 represents a result of reverse phase HPLC (III) of Step 7 in Example 3;
FIG. 2 represents a result of molecular weight measurement by SDS-PAGE of the Meg-POT in Example 4; and
FIG. 3 represents a dose-response curve of Meg-POT activity of the megakaryocyte potentiator in Example 7. Each value is the mean of duplicate cultures. Furthermore, the Meg-POT activity on the axis of ordinate is indicated as the difference in the number of megakaryocyte colonies where the test sample was added and where the test sample was not added (recombinant mouse IL-3 alone). The number of megakaryoc
REFERENCES:
Kawa Kita, Medical Immunology, vol. 14, No. 4, pp. 463-470, Oct. 1987.
Ishibashi et al, Proc. Natl. Acad. Sci. USA, vol. 86, pp. 5953-5957 (Aug. 1989).
Yonemura et al, Exp. Hematol., 20:1011-1016 (1992).
Tayrien et al, The Journal of Biological Chemistry, vol. 262, No. 7, pp. 3262-3268 (Mar. 5, 1987).
Yamaguchi et al, The Journal of Biological Chemistry, vol. 269, No. 2, pp. 805-808 (Jan. 14, 1994).
Hattori Kunihiro
Oh-Eda Masayoshi
Yamaguchi Nozomi
Chugai Seiyaku Kabushiki Kaisha
Sayala Chhaya D.
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