Abstract:
A method of promoting regeneration of hemopoietic cells in a subject undergoing chemotherapy or radiotherapy, which method includes the steps of (i) administering to the subject a first amount of AcSDKP or an agonist thereof, the first amount being effective to reduce the proliferation of hemopoietic cells during the chemotherapy or radiotherapy; (ii) administering to the subject a second amount of an angiotensin-converting enzyme (ACE) inhibitor, said second amount being effective to reduce the degradation of said AcSDKP or an agonist thereof by angiotensin-converting enzyme; and (iii) after the chemotherapy or radiotherapy, administering to the subject a second amount of a hemopoiesis growth factor, the second amount being effective to stimulate the proliferation of hemopoietic cells.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation-in-part of U.S. patent application Ser. No. 08/304, filed Sep. 12, 1994, now U.S. Pat. No. 5,595,973. 
    
    
     BACKGROUND OF THE INVENTION 
     Acute and chronic bone marrow toxicities are the major limiting factors in the treatment of cancer. They are both related to two causes. The first is a decrease in the number of hemopoietic cells (e.g., pluripotent stem cells and other progenitor cells) caused by both a lethal effect of cytotoxic agents or radiation on these cells and by differentiation of stem cells provoked by a feed-back mechanism induced by the depletion of more mature marrow compartments. The second cause is a reduction in self-renewal capacity of stem cells, which is also related to both direct (mutation) and indirect (aging of stem cell population) effects. Stimulators and inhibitors of bone marrow kinetics play a prominent role in the induction of damage and recovery patterns (Tubiana, M., et al., Radiotherapy and Oncology 29:1, 1993). 
     Acute myelosuppression as a consequence of cytotoxic chemotherapy is well recognized as a dose-limiting factor in cancer treatment. Although other normal tissues may be adversely affected, bone marrow is particularly sensitive to the proliferation-specific treatment such as chemotherapy or radiotherapy. 
     Self-renewing, pluripotent hemopoietic stem cells, as measured by spleen colony-forming units (CFU-S), are responsible for generation of the hemopoietic system (Till, J. E., et al., Radiat. Res. 14:213, 1961). Such stem cells are mainly quiescent, and renewal of mature cells is provided by the partially differentiated, lineage-committed hemopoietic progenitors. At this stage, the stem cells are relatively invulnerable to chemotherapy and radiotherapy that affects only those cells that are already in proliferation or about to begin proliferation. The stem cell compartment of the bone marrow that guarantees a sufficient supply of cells bound for differentiation, though mainly quiescent, still has approximately 10% of its cellular component in some state of mitosis (Lajtha, L. G., In Stem Cells, Ed: C. S. Potten, Churchill Livingstone, Edinburgh, 1-11, 1983). During chemotherapy or radiotherapy, the first treatment kills proliferating malignant or infected cells as well as proliferating hemopoietic cells in the bone marrow, but has no effect on the quiescent CFU-S. Therefore, the given situation before treatment is favorable for therapy as the stem cell population is relatively resistant. Subsequent treatment, however, will cause severe damage to the stem cell population, since they will have begun compensatory proliferation. 
     SUMMARY OF THE INVENTION 
     The present invention relates to a method of promoting regeneration of hemopoietic cells in a subject undergoing chemotherapy or radiotherapy. 
     More specifically, the method of this invention includes the steps of (i) administering to the subject a first amount of AcSDKP (CH 3  --CO-Ser-Asp-Lys-Pro-OH) or an agonist thereof, the first amount being effective to reduce the proliferation of hemopoietic cells during the chemotherapy or radiotherapy; (ii) administering to the subject a second amount of an angiotensin-converting enzyme (ACE) inhibitor, said second amount being effective to reduce the degradation of said AcSDKP or an agonist thereof by angiotensin-converting enzyme; and (iii) after the chemotherapy or radiotherapy, administering to the subject a second amount of a hemopoiesis growth factor, the second amount being effective to stimulate the proliferation of hemopoietic cells. As will be further discussed below, by &#34;chemotherapy&#34; is meant a process of killing proliferating cells using a cytotoxic agent. The phrase &#34;during the chemotherapy&#34; above refers to the period in which the effect of the administered cytotoxic agent lasts. On the other hand, the phrase &#34;after the chemotherapy&#34; above is meant to cover all situations in which a hemopoiesis growth factor is administered after the administration of a cytotoxic agent regardless of any prior administration of the same or another hemopoiesis growth factor and also regardless of the persistence of the effect of the administered cytotoxic agent. 
     By &#34;agonist&#34; is meant an analog (with one or more modifications) or a fragment of the compound with the same desired activity, i.e., inhibiting or stimulating the proliferation of hemopoietic cells. By &#34;ACE inhibitors&#34; is meant a compound which is capable of inhibiting the binding of ACE to its substrates. Examples of ACE inhibitors include captopril, enalapril, and fusinopril. 
     Examples of a hemopoiesis growth factor which can be used to practice the above method include, but are not limited to, cytokines or agonists thereof, such as natural, synthetic or modified interleukins (e.g., IL-1, IL-3, IL-6, IL-11, or its agonist), G-CSF (i.e., granulocyte colony-stimulating factor), GM-CSF (i.e., granulocyte/macrophage-colony stimulating factor), erythropoietin, stem cell factor, and leukemia inhibitory factor. 
     In one embodiment of this method, the subject is undergoing chemotherapy with a cytotoxic agent. By &#34;cytotoxic agent&#34; is meant an agent which kills proliferating cells, e.g., tumor cells, virally infected cells, or hemopoietic cells. Examples of a cytotoxic agent which can be used to practice the above method include, but are not limited to, cyclophosphamide, taxol, 5-fluorouracil, adriamycin, cisplatinum, methotrexate, cytosine arabinoside, mitomycin C, prednisone, vindesine, carboplatinum, vincristine, and an agonist of any of the above compounds. A cytotoxic agent can also be an antiviral agent, e.g., AZT (i.e., 3&#39;-azido-3&#39;-deoxythymidine). In another embodiment of this method, the subject is undergoing radiotherapy. Note that the terms &#34;chemotherapy&#34; and &#34;radiotherapy&#34; used herein refer to the process of killing proliferating cells by administration of a cytotoxic agent or by irradiation. 
     When the method of this invention is applied to chemotherapy, a hemopoiesis inhibitory factor can be administered prior to, during, or subsequent to the chemotherapy (i.e., prior to, during, or subsequent to the administration of a cytotoxic agent). In any event, it is preferred that a hemopoiesis growth factor be administered subsequent to the administration of a hemopoiesis inhibitory factor. When the method of this invention is applied to radiotherapy, a hemopoiesis inhibitory factor can be administered prior to or during the radiotherapy (i.e., prior to or during the irradiation). Note that the timing of when to administer a hemopoiesis inhibitory factor depends on the half life of that factor, the duration of its inhibitory activity, the administration route, etc., as well as the conditions of the chemotherapy or radiotherapy (e.g., the half life of the cytotoxic agent which is used in chemotherapy). 
     The ACE inhibitor is preferably administered in concurrence with hemopoiesis inhibitory factor. 
     The effective amount of the hemopoiesis inhibitory factor or growth factor used to practice the present invention varies depending upon the manner of administration, the age and the body weight of the subject, and the condition of the subject to be treated. Ultimately, it will be decided by the attending veterinarian or physician. Any such amount of the inhibitory factor, ACE inhibitor, or the growth factor as determined by the attending veterinarian or physician is referred to herein as &#34;effective amount&#34;. 
     Also note that the hemopoiesis inhibitory factor, the ACE inhibitor, and the hemopoiesis growth factor may be administered by any route appropriate to the condition being treated. Preferably, it is injected into the bloodstream of the subject being treated. However, it will be readily appreciated by those skilled in the art that the route, such as intravenous, subcutaneous, intramuscular, intraperitoneal, nasal, oral, etc., will vary with the condition being treated and the activity of the compound being used. Note that continuous administration using a subcutaneous infusion pump may be desirable when the compound to be used has a rather short half life or lacks long-lasting activity. Conversely, single or intermittent administration is acceptable or even preferable when the factor to be used has a long half life or long-lasting activity. 
     The inhibitory factor, the ACE inhibitor, or the growth factor may be conveniently be presented as an ingredient of a pharmaceutical composition in unit dosage form according to any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient(s) into association with the carrier which constitutes one or more accessory ingredients. In general, the formulations for tablets or powders are prepared by uniformly and intimately blending the active ingredient with finely divided solid carriers, and then, if necessary as in the case of tablets, forming the product into the desired shape and size. 
     Other features and advantages of the present invention will be apparent from the following drawings and description of the preferred embodiments, and also from the appending claims. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     It is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference. 
     AcSDKP or an agonist thereof is used as the hemopoiesis inhibitory factor to practice this invention to inhibit the proliferation of hemopoietic cells. In other words, by the term &#34;hemopoiesis inhibitory factor&#34; is meant herein AcSDKP or an agonist thereof. Suitable agonists of AcSDKP are described in Thierry et al., J. Med. Chem. 33:2122, 1990 and Robinson et al., Stem Cells, 11:422, 1993. 
     On the other hand, the hemopoiesis growth factor which can be used to practice this invention is a compound capable of stimulating the proliferation of hemopoietic cells, such as cytokines. Preferred cytokines include interleukins, GM-CSF, and G-CSF. E.g., see R. Van Furth (ed.), Hemopoietic Growth Factors and Mononuclear Phagocytes (Karges 1993) and Moore, M. article cited in the preceding paragraph. 
     Interleukin-1 (IL-1) has shown dramatic hemopoietic protective and restorative effects against lethal doses of irradiation either alone (Neta, R. et al., J. Immunol. 136:2483, 1986) or in concert with bone marrow transfer (Oppenheim, J. J., et al., Blood 74:2257, 1989), and against chemotherapeutic drugs such as cyclophosphamide (Castelli, M. P., et al., J. Immunol. 140:3830, 1988; Futami, H., et al., J. Immunol. 145:4121, 1990; Fibbe, W. E., et al., Exp. Hematol. 17:805, 1989). GM-CSF and G-CSF have also been found to have marked restorative effects after irradiation (Tanikawa, S., et al., Exp. Hematol. 17:883, 1989; Schuening, F. G., et al., Blood 74:1308, 1989) or treatment with chemotherapeutic drugs both in preclinical models (Moore, M. A. S., et al., Proc. Natl. Acad. Sci. USA 84:7134, 1987; Welte, K., et al., J. Exp. Med. 165:941, 1987), as well as in human trials (Steward, W. P., et al., Cancer Treat. Rev. 17:77, 1990; Gianni, A. M., et al., J. Clin. Oncol. 8:768, 1990). 
     Examples of angiotensin-converting enzyme (ACE) inhibitors include sulfhydryl-containing inhibitors (e.g., captopril, fentiapril, pivalpril, zefenopril, ano-alacepril), dicarboxyl-containing ACE inhibitors (e.g., enalapril, lisinopril, benzepril, indolapril, pentopril, indalapril, and cliazapril), and phosphorus-containing ACE inhibitors (e.g., fusinopril). Jackson, et al., Renin and Angiotensin in Goodman &amp; Gilman&#39;s The Pharmacological Basis of Therapeutics, 9th ed., eds. Hardman, et al. (McGraw Hill, 1996). 
     When the method of this invention is applied to chemotherapy, the cytotoxic agent which can be used include cyclophosphamide, taxol, daunorubicine, 5-fluorouracil, adriamycin, cisplatinum, methotrexate, cytosine arabinoside, mitomycin C, prednisone, vindesine, carbaplatinum, and vincristine. The cytotoxic agent can also be an antiviral compound which is capable of destroying proliferating cells. For a general discussion of cytotoxic agents used in chemotherapy, see Sathe, M. et al., Cancer Chemotherapeutic Agents: Handbook of Clinical Data (1978). 
     The method of this invention can also be applied to radiotherapy, which may comprise of either ionizing waves or particles. Examples of ionizing waves include x-rays and gamma rays. Examples of ionizing particles include alpha rays, beta rays, neutrons, electrons, and protons. Radiotherapy may be administered externally or internally. Examples of external radiotherapy include x-ray units, gamma ray units, electron beams, and neutron beams. Internal radiotherapy includes both sealed and unsealed sources. Examples of sealed sources include cobalt beam units,. caesium beam units, strontium applicators, yttrium rods or pellets, gold grains, or radium, cobalt or caesium needles or tubes. Examples of unsealed sources include iodine, phosphorous, gold, and yttrium. See Walter, J., Cancer and Radiotherapy (Churchill Livingstone 1977); Ed. N. J. McNally, The Scientific Basis of Modern Radiotherapy (British Institute of Radiology 1989); and Franz Buschke and Robert G. Parker, Radiation Therapy in Cancer Management (Grove &amp; Stratton 1972). 
     In preferred embodiments of this invention, the subject undergoes repeated cycles of treatment according to the method of this invention. Preferably, a subsequent cycle commences only after the administration of the hemopoiesis growth factor has been terminated and the subject&#39;s blood cell counts (e.g., white blood cell count) have returned to a therapeutically acceptable level (as determined by the attending veterinarian or physician), permitting the repeated chemotherapy or radiotherapy 
     For obvious reasons, the method of this invention can also be applied to treatment in which radiotherapy and chemotherapy are performed in conjunction. 
     In chemotherapy, it is desirable that the hemopoiesis growth factor be administered only 1-7 days (preferably, 1-5 days; or more preferably, 2-3 days) after the administration of both the hemopoiesis inhibitoryfactor, the ACE inhibitor, and the cytotoxic agent has been terminated. By the same token, in radiotherapy, it is preferred that the hemopoiesis growth factor be administered only after the irradiation has been completed. 
     Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. The following specific example is, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. 
     ASSAY 
     An assay was designed to show the effect of combining the hemopoiesis inhibitory factor, an ACE inhibitor, and hemopoiesis growth factor on the proliferation of hemopoietic cells during chemotherapy. The study was designed to determine whether the progenitor cell compartment of the bone marrow can be adequately protected from the cytotoxic effects of chemotherapy so that intensive therapy (e.g., multiple cycles) could be delivered and duration of the negative effects of a leukopenic nadir abbreviated without the need for marrow rescue. 
     Patients treated as follows (i.e., with Ara-C, the abbreviation of cytosine arabinoside, and daunorubicine as the cytotoxic agents; AcSDKP as the hemopoiesis inhibitory factor; Captopril as the ACE inhibitor; and G-CSF as the hemopoiesis growth factor): 
     The patients are administered AcSDKP and Captopril for days 1-9. AcSDKP is administered at 25 μg/kg/day by continuous intravenous infusion. Captopril is administered every 12 hours at a concentration of 50-100 mg/kg/day (depending on creatinine clearance). Ara-C is administered at a concentration of 200 mg/m 2  /day for days 3-9 by continuous infusion. Daunorubicine is administered at a concentration of 45 μg/m 2  /day for days 3-6 by continuous infusion for over a period of 3 hours. G-CSF (Lenogastrin™) is administered at a concentration of 5 μg/kg/day by intravenous infusion for a period of 30 minutes from day 12 to day of recovery aplasia (maximum up to day 30). 
     AcSDKP, which was prepared by a process described in PCT Application WO 93/25571 (hereby incorporated by reference), can also be obtained from Sigma Chemical Co., St. Louis, Mo. Ara-C (Aracytine™) was purchased from Laboratoires UpJohn (Paris, France) and Lenogastrin™ was obtained from Immunix Research and Development Corp., Seattle, Wash. Captopril (Captolane™) was purchased from Laboratoires Bellon (Neuilly Sur Seine, France). Daunorubicine (Cerubidine™) was also purchased from Laboratoires Bellon. 
     Other Embodiments 
     The foregoing description has been limited to specific embodiments of this invention. It will be apparent, however, that variations and modifications may be made to the invention, with the attainment of some or all of the advantages of the invention. Such embodiments are also within the scope of the following claims.