In a preferred embodiment, drugs having chemotherapeutic properties which are useful against certain neoplastic disorders with wide safety margins as evidenced by their low toxicity, and molecular actions. Such drugs include as active ingredient(s) one or more N-substituted 2-(1H) pyridone(s) and/or N-substituted 3-(1H) pyridone(s). The compositions of this invention are novel as anti-neoplastic drugs, namely as an agent for treating leukemias, lymphomas, and leiomyomas as agent(s) for treating benign and malignant tumors, including lymphomas, leukemias, and leiomyomas.
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1. A method of treating lymphomas, leukemias, and/or leiomyomas one or more malignant tumors and/or one or more benign tumors in a laboratory animal or a human, comprising: administering to said laboratory animal or said human an effective dose of a composition including one or more pharmaceutical substances selected from the group consisting of N-substituted 2-(1H) pyridones, N-substituted 3-(1) pyridones, and pharmaceutically acceptable salts thereof, wherein said 2-(1H) pyridones have the following general structural formula:
##STR00003##
where: R1 is selected from the group consisting of (1) an alkyl group, with R3 hydrogen, and (2) hydrogen, with R3 consisting of an alkyl group; A is an aryl group; and R2 and R4 are hydrogen; and wherein said 3-(1H) pyridones have the following general structural formula:
##STR00004##
where: R2 is selected from the group consisting of (1) an alkyl group, with R3 hydrogen, and (2) hydrogen, with R3 consisting of an alkyl group; A is an aryl group; and R1 and R4 are hydrogen.
4. A method of treating lymphomas, leukemias, and/or leiomyomas one or more malignant tumors and/or one or more benign tumors in a laboratory animal or a human, comprising: administering to said laboratory animal or said human an effective dose of a composition including one or more pharmaceutical substances selected from the group consisting of N-substituted 2-(1H) pyridones, N-substituted 3-(1H) pyridones, and pharmaceutically acceptable salts thereof, said N-substituted 2-(1H) pyridones and said N-substituted 3-(1H) pyridones being selected from the group consisting of: 5-Methyl-1-phenyl-2-(1H) pyridone, 5-Methyl-1-(3-nitrophenyl-2)-(1H) pyridone, 5-Methyl-1-(4′-methoxyphenyl)-2-(1H) pyridone, 5-Methyl-1-p-tolyl-2-(1H) pyridone, 5-Methyl-1-(3′-trifluoromethylphenyl)-2-(1H) pyridone, 1-(4′Chlorophenyl)-5-methyl-2-(1H) pyridone, 5-Methyl-1-(2′-naphthyl)-2-(1H) pyridone, 5-Methyl-1-(1′-naphthyl)-2-(1H) pyridone, 3-Methyl-1-phenyl-2-(1H) pyridone, 6-Methyl-1-phenyl-2-(1H) pyridone, 3,6-Dimethyl-1-phenyl-2-(1H) pyridone, 5-Methyl-1-(2′thienyl)-2-(1H) pyridone, 1-(2′-Furyl)-5-methyl-2-(1H) pyridone, 5-Methyl-1-(5′-quinolyl)-2-(1H) pyridone, 5-Methyl-1-(4′-pyridyl)-2-(1H) pyridone, 5-Methyl-1-(3′-pyridyl)-2-(1H) pyridone, 5-Methyl-1-(2′-pyridyl)-2-(1H) pyridone, 5-Methyl-1-(2′-quinolyl)-2-(1H) pyridone, 5-Methyl-1-(4′-quinolyl)-2-(1H) pyridone, 5-Methyl-1-(2′-thiazolyl)-2-(1H) pyridone, 1-(2′-Imidazolyl-5-methyl-2-(1H) pyridone, 5-Ethyl-1-phenyl-2-(1H) pyridone, 3-Ethyl-1-phenyl-2-(1H) pyridone, 1-Phenyl-2-(1H) pyridone, 1-(4′-Nitrophenyl)-2-(1H) pyridone, 5-Methyl-3-phenyl-1-(2′-thienyl)-2-(1H) pyridone, 5-Methyl-1-phenyl-3-(1H) pyridone, 5-Methyl-1-(4′-methoxyphenyl)-3-(1H) pyridone, 5-Methyl-1-p-tolyl-3-(1H) pyridone, 1-(4′-Chlorophenyl)-5-methyl-3-(1H) pyridone, 5-Methyl-1-(2′-naphthyl)-3-(1H) pyridone, 4-Methyl-1-phenyl-3-(1H) pyridone, 6-Methyl-1-phenyl-3-(1H) pyridone, 5-Methyl-1-(2′-thienyl)-3-(1H) pyridone, 1-(2′-Furyl)-5-methyl-3-(1H) pyridone, 5-Methyl-1-(5′-quinolyl)-3-(1H) pyridone, 5-Methyl-1-(3′-pyridyl)-3-(1H) pyridone, 5-Methyl-1-(2′-pyridyl)-3-(1H) pyridone, 5-Methyl-1-(2′-quinolyl)-3-(1H) pyridone, 5-Ethyl-1-phenyl-3-(1H) pyridone, and 1-Phenyl-3-(1H) pyridone.
2. A method, as defined in
3. A method, as defined in
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This application is a CIP of Ser. No. 09/162,011 filed Sep. 28, 1998 which is a CIP of Ser. No. 08/913,202 filed Sep. 3, 1997 ABN., which is a CIP of PCT/US96/02737 filed Mar. 4, 1996 which is a CIP of Ser. No. 08/397,962 filed Mar. 3, 1995 ABN.
1. Field of the Invention
The present invention relates to medical compositions and methods for the chemotherapeutic treatment of lymphomas, leukemias, and leiomyomas generally and, more particularly, to compositions comprising one or more N-substituted 2(1H) pyridones and/or one or more N-substituted 3(1H) pyridones as active ingredient(s). The selected compounds nay be used alone or as an adjunct to other forms of neoplastic therapy including surgery, other chemotherapeutic compounds, radiation therapy, and immunotherapeutic agents.
2. Background Art
The causes of leukemias and leiomyomas are poorly understood, are complex, and involve interplay between the basic genetic material in the nucleus of cells. An abnormal reaction of cellular genetic DNA to internal or external factors can create a new deviation in the cell genetic code, or in the genetic DNA generated communication proteins which creates neoplastic perturbations in the transcription process governing the specific cell cycle stages of otherwise normal cell division, and proliferation.
Cell proliferation is defined as the increase in number of cells resulting from completion of the cell cycle, as contrast to growth, which is the increase in the individual cell mass.
Extracellular or intracellular factors can determine whether a quiescent cell will begin to proliferate and also whether a normal proliferating cell in phase G1 will begin to cycle or will revert to quiescence. After cells enter into the S Phase, cell-cycle events become largely independent of prior extracellular factors., while they go on to divide and produce two daughter cells.
Among the carcinogenic factors of external origin, acting internally, are physical carcinogens such as ionizing or ultraviolet radiation, and the presence of foreign substances such as asbestos. Carcinogenic substances acting internally include various chemicals, natural or man-made, which can effect directly or indirectly cell DNA to elicit intracellular oncogenic events. In addition biological substances such as bacteria, viruses, parasites, hormones and cytokines have been implicated in mammalian carcinogenesis.
In malignant neoplasms (such as leukemiasbenign and malignant tumors including lymphatic or hymelogenous leukemias, lymphomas, and leiomyomas.
Methods of preparation of some N-substituted 2(1-H) pyridones useful in the present invention are described in U.S. Pat. No. 3,839,346, issued Oct. 1, 1974, to Gadekar, and titled N-SUBSTITUTED PYRIDONE AND GENERAL METHOD FOR PREPARING PYRIDONES, the disclosure of which is incorporated by reference hereto.
Inhibition of proliferation by pirfenidone of human promyelocytic leukemia (HL-60) cells in vitro.
Pirfenidone was suspended in RPMI, 2.0% FBS, at 100 micrograms to 900 micrograms per ml, and one other culture served as a control. Five ml of each pirfenidone concentration, and of the control were placed into a T25 tissue culture flask along with 1×105 cells. Every day for 4 days an aliquote from each flask was taken out, then stained with Trypan Blue, and counts were repeated three times on each aliquote.
The inhibition of proliferation of HL-60 cells by pirfenidone was directly related to the graded concentrations of pirfenidone in the respective culture flasks. After 3 and 4 days at a concentration of 900 micrograms/ml, a 63% pharmacologic inhibition was maintained without causing a toxic death of the cells (see FIG. 1). Inhibition of proliferation as well as deaths of almost all cells occur with pirfenidone concentrations at 1400 micrograms/ml.
Inhibition of proliferation by pirfenidone of human T-cell leukemia (JURKAT) cells in vitro.
To determine the inhibitory effect of pirfenidone concentrations on human T-cell leukemia (JURKAT), a MTS assay was used which incorporates a tetrazolium dye that measures the cellular conversion of the dye into a formazan product by the activation of NADH-generating dehydrogenases found in metabolically active cells.
Procedure
1. 100 microl. of cells was seeded into 96 well plates at 1×105 cells/ml in media containing 100 mcg/ml, 300 mcg/ml and 900 mcg/ml of pirfenidone, as well as a pirfenidone-free media control.
2. Cells were incubated at 37 degrees C., at 5.0% CO2 for 72 hours.
3. After incubation a standard curve was made of 100 microliters of cells added to the plate at 5×105 cells/ml making 1:2 dilutions all the way down to a pre-set degree of dilution.
4. 2.0 mls of the MTS solution was mixed with a 100 microliter of the PMS solution, and 20 microliters of this combined dye solution was added to each well and incubated at 37 degrees C., 5.0% CO2 for 3 hours.
5. Subsequently the absorbance was read at 490 nm.
Exposure for only 72 hours of human T-cell leukemia (JURKAT) cells, at 900 mcg/ml of pirfenidone caused a statistically significant 75% reduction in number of cell which were active (75% no longer were able to proliferate; FIG. 2).
Inhibition of proliferation by pirfenidone of human Burkitt's lymphoma (RAJI) cells in vitro.
To determine the inhibitory effect of pirfenidone concentrations on Burkitt's lymphoma (RAJI) cells, a MTS assay was used which incorporates a tetrazolium dye that measures the cellular conversion of the dye into a formazan product by the activation of NADH-generating dehydrogenases found in metabolically active cells.
Procedure
1. 100 microl. of cells was seeded into 96 well plates at 1×105 cells/ml in media containing 100 mcg/ml, 300 mcg/ml and 900 mcg/ml of pirfenidone, as well as a pirfenidone-free media control.
2. Cells were incubated at 37 degrees C., at 5.0% CO2 for 72 hours.
3. After incubation a standard curve was made of 100 microliters of cells added to the plate at 5×105 cells/ml making 1:2 dilutions all the way down to a pre-set degree of dilution.
4. 2.0 mls of the MTS solution was mixed with a 100 microliter of the PMS solution, and 20 microliters of this combined dye solution was added to each well and incubated at 37 degrees C., 5.0% CO2 for 3 hours.
5. Subsequently the absorbance was read at 490 nm.
Exposure for only 72 hours of these human Burkitt's lymphoma (RAJI) cells, at 900 mcg/ml of pirfenidone caused a statistically significant 45% reduction in number of cells which were active (45% no longer were able to proliferate; FIG. 3).
Leiomyoma tissues were obtained from several premenopausal women with symptomatic uterine fibroids at elective hysterectomy, and who were not receiving any hormonal or other drug therapy. The fibroid tissues were minced into 1-2 mm# explants and placed in suitable DMEM supplemented with 10% bovine serum. The fibroid tissue was digested for 14-18 hours at 37 degrees C. in an incubator. After centrifugation, a resulting cell pellet was resuspended in DMEM and then placed in culture flasks. The cultures were maintained at 37 degrees centigrade in a humidified atmosphere of 5% CO2 and 95% air.
For determining cell proliferation utilizing tritiated thymidine incorporation as an assay, leiomyoma cells were cultured in 96-well plates (15,000 cells/well) for 48 hours in DMEM plus 10.0% FBS serum. Cells were then made quiescent by culturing in DMEM plus 0.5% FBS serum for 48 hours. These quiescent cells were washed, and then placed in DMEM plus 10.0% FBS serum containing graded concentrations of pirfenidone (10.0, 100, 300 and 1000 micrograms/ml). After 18 hours, the cells received 0.2 uCi/well of [3H]-thymidine, and the incubation was continued for 6 more hours. Subsequently, cells were harvested and counted in a beta-counter to the rate of incorporation of [3H]-thymidine. Results are summarized in Table 1.
TABLE 1
INHIBITION BY PIRFENIDONE
OF PROLIFERATION OF HUMAN LEIOMYOMA CELLS
PIRFENIDONE
RECENT DEAD
TREATMENT
CELL COUNTS (DAY 7)
CELLS (DAY 7)
(CONC/ML)
LEIOMYOMA
LEIOMYOMA
0.0
MG
380,000
9.0
0.01
MG
315,000
8.0
0.1
MG
205,000
10.0
0.3
MG
170,000
6.0
1.0
MG
69,000
16.0
Leiomyoma cells were plated in 100 mm dishes (100,000 each dish) and allowed to attach overnight in DMEM plus 10% fetal bovine serum until they reached 80-90% confluence. The following day all cells received DMEM plus 10% fbs containing various concentrations of pirfenidone (0.0 micrograms, 100 micrograms, 300 micrograms or 1000 microgram/ml for seven (7) days. The medium was changed with addition of fresh treatments on days 3 and 5. On day 7, cells were harvested and counted. Cell viability was assessed using the Trypan Blue exclusive stain. Results are displayed in FIG. 4.
A significant inhibitory effect on proliferation was seen for leiomyoma cells exposed to graded concentrations of pirfenidone in the culture media (FIG. 4). A significant increase in the percentage (16%) of dead leiomyoma cells was found at the 1000 micrograms/ml concentration of pirfenidone (Table 1).
The data demonstrates the anti-tumor effect of on leiomyomas indicate indicates that pirfenidone will be useful in eliminating the most severe symptoms associated with leiomyoma tumors (excessive abnormal arterial bleeding, pelvic pain, infertility and increased urinating frequency).
These compounds also can be employed in combination to enhance other types of therapy (surgery, radiation, immunotherapy or other chemotherapeutic compounds). Another facet of the present invention is absence of any severely debilitating adverse effects. The absence of such severe toxic reactions reduces or eliminates the patient discomfort inherent in conventional treatments for neoplasias. The delivery method to patients being treated may consist of oral, intramuscular or intravenous administration.
The N-substituted pyridones of the present invention pharmacologically arrest the proliferation of leukemias, Llymphomas, or leiomyoma cells or tissue, at concentrations that are ⅓rd- 1/10th that which is toxic or killing to these neoplastic cells. Accordingly, this invention characterizes a group of chemotherapeutic compounds which provide a means of (1) arresting the proliferation and (2) then destroying the malignant cells by raising the pirfenidone concentration of the cell of tissue to the cytotoxic level or by adding another known cytotoxic anti-neoplastic agent, the neoplastic cells can be destroyed or eradicated without serious or fatal injury to healthy normal cells and tissues.
These compounds also can be employed in combination to enhance other types of therapy (surgery, radiation, immunotherapy or other chemotherapeutic compounds).
Another facet of the present invention is absence of any severely debilitating generalized adverse effects. The absence of such severe toxic reactions reduces or eliminates the patient discomfort inherent in conventional neoplasia treatments.
The differences in the concentrations of pirfenidone which pharmacologically arrest the proliferation of neoplasia neoplastic cells and the pirfenidone concentration which kills neoplasia cells affords a larger margin of safety for patients, (Raghu, G., et al., Amer. J. Resp. and Critical Care Med., 1997, Vol. 155:A741), and thereby distinctly reduces the incidence of serious adverse effects experienced by patients during treatment, as compared to treatment with currently conventional anti-neoplasia anti-neoplastic agents.
The intracellular action of pirfenidone in (1) arresting the proliferation and (2) subsequent destruction of the abnormal or neoplastic cells takes place in the cell nucleus and directly involves the signaling via the specific gene activated proteins (for example, p53, Rb, WT1, etc.) and ameliorating or blocking the impact of such gene proteins on the cell transcription apparatus and cyclins. These specific gene proteins act on the check points of the cell cycle to prevent or correct the aberrant gene protein signals impacting on the cyclins and check points.
It is estimated that the effective human dosage of one or more N-substituted 2-(1H) pyridone(s) and/or N-substituted 3-(1H) pyridone(s) in practicing the present invention is from about 250 to about 750 mg/kg of body weight per day, which dosage may be taken in the diet.
The general structural formula of N-substituted 2-(1H) pyridones is:
##STR00001##
where: R1 is selected from the group consisting of (1) an alkyl group, with R3 hydrogen, and (2) hydrogen, with R3 consisting of an alkyl group; A is an aryl group; and R2 and R4 are hydrogen.
The general structural formula for the N-substituted 3-1(H) pyridones is:
##STR00002##
where: R2 is selected from the group consisting of (1) an alkyl group, with R3 hydrogen, and (2) hydrogen, with R3 consisting of an alkyl group; A is an aryl group; and R1 and R4 are hydrogen.
Examples of the pyridone compounds which have been found or are believed to be effective in practicing the present invention include:
It will thus be seen that the objects set forth above, amoung those elucidated in, or made apparent from, the preceding description, are efficiently attained and, since certain changes may be made in the above compositions and methods without departing from the scope of the invention, it is intended that all matter contained in the foregoing disclosure shall be interpreted as illustrative only and not in a limiting sense.
It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween.
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