U.S. patent number RE40,155 [Application Number 09/724,378] was granted by the patent office on 2008-03-18 for methods for treatment of benign and malignant tumors, including lymphomas, leukemias, and leiomyomas.
Invention is credited to Solomon B Margolin.
United States Patent |
RE40,155 |
Margolin |
March 18, 2008 |
Methods for treatment of benign and malignant tumors, including
lymphomas, leukemias, and leiomyomas
Abstract
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.]. .Iadd.as agent(s) for
treating benign and malignant tumors, including lymphomas,
leukemias, and leiomyomas.Iaddend..
Inventors: |
Margolin; Solomon B (Dallas,
TX) |
Family
ID: |
22901121 |
Appl.
No.: |
09/724,378 |
Filed: |
November 27, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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09162011 |
Sep 28, 1998 |
6090822 |
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08913202 |
Sep 3, 1997 |
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PCT/US96/02737 |
Mar 4, 1996 |
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08397962 |
Mar 3, 1995 |
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Reissue of: |
09239211 |
Jan 28, 1999 |
06114353 |
Sep 5, 2000 |
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Current U.S.
Class: |
514/313; 514/336;
514/334 |
Current CPC
Class: |
A61K
31/47 (20130101); A61K 31/44 (20130101) |
Current International
Class: |
A61K
31/47 (20060101) |
Field of
Search: |
;514/313,334,336,341,342,345 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Ross, Russell, PhD. Medical Progress; The Pathogenesis of
Athersclerosis--An Update; The New England Journal of Medicine vol.
314, No. 8, pp. 488, 493, 494; Department of Patholology,
University of Washington School of Medicine, Seattle, Washington;
Feb. 20, 1986. cited by examiner .
Saven, Alan, M.D., et al.; Brief Communications,
2-Chlorodeoxyadenosine-induced Complete Remissions in
Langerhans-Cell Histiocytosis, pp. 430-432; Reprint from Annals of
Internal Medicine, vol. 121, No. 6, Sep. 15, 1994. cited by
examiner .
Schwartz, Stephen M., et al; Remodeling: How Vessels Narrow,
Clinical Trials and Remoldeling; pp. 128-131; University of
Washington, Seattle, WA; Massachusetts General Hospital, Boston,
MA; and ZymoGenetics, Inc., Seattle WA. 1996. cited by examiner
.
Waltenberger, Johannes, M.D., Current Perspectives, Modulation of
Growth Factor Action, Implications for the Treatment of
Cardiovascular Diseases; Circulation, pp. 4083-4094; vol. 96, No.
11, Department of Internal Medicine II (Cardiology); Ulm University
Medical Center, Ulm Germany; Dec. 2, 1997. cited by examiner .
Copy of Brochure for Leustatin.RTM. (cladribine) Injection; Ortho
Biotech, Raritan, New Jersey, Dec. 1996. cited by examiner .
Lee et al., Pirfenidone: A Novel Pharmacological Agent That
Inhibits Leimomyoma Cell Proliferation and Collegen Production,
1998, US, J. Clin. Endocrinal Metab 83: 219-223. cited by
other.
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Primary Examiner: Low; Christopher S. F.
Assistant Examiner: Gembeh; Shirley V.
Attorney, Agent or Firm: Crozier; John H.
Parent Case Text
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.
Claims
I claim:
1. A method of treating .[.lymphomas, leukemias, and/or
leiomyomas.]. .Iadd.one or more malignant tumors and/or one or more
benign tumors .Iaddend.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.
2. A method, as defined in claim 1, wherein: said composition is
administered orally or parenterally to said laboratory animal at a
rate of from about 250 to about 750 mg/kg of body weight per
day.
3. A method, as defined in claim 1, wherein: said composition is
administered orally or parenterally to .[.a.]. .Iadd.said
.Iaddend.human at a rate of from about 20 to about 60 mg/kg of body
weight per day.
4. A method of treating .[.lymphomas, leukemias, and/or
leiomyomas.]. .Iadd.one or more malignant tumors and/or one or more
benign tumors .Iaddend.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.
Description
BACKGROUND OF THE INVENTION
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 .Iadd.benign and malignant tumors including .Iaddend.lymphomas,
leukemias and leiomyomas, the control of proliferation is deranged.
After induction of altered proliferation control, deranged cell
differentiation is initiated in phase G1, and is a hallmark of
neoplastic cells (Pardee. A. B., Science, Nov. 3, 1989, p.603).
Neoplasms are manifest when the normal progression of the orderly
relationship between cell division and cell differentiation
malfunctions. With the usual cell division sequence in normal
cells, the proliferation of cells is restricted to
non-differentiated stem cells which ordinarily differentiate and
reproduce to provide a replacement for aged dying cells.
Neoplasms arising from lympho-hematopoietic origin may be
identified as leukemias, lymphomas, leiomyomas, etc.
The term "anti-neoplastic" or "anti-tumor" refers herein to the (a)
chemotherapeutic inhibition of arrest of the growth, and (b) the
destruction of mammalian benign .[.,.]. .Iadd.(for example
leiomyomas) and/.Iaddend.or malignant tissues .Iadd.(.Iaddend.such
as leukemias.[.,.]. .Iadd.or .Iaddend.lymphomas), .[.and
leiomyomas.]. found in various organs and tissues of the body.
Although most tissues and organs of the human body may become
neoplastic, the basic processes leading to diverse tumors appear to
be quite similar. Normal cells proliferate or reproduce in rigorous
compliance with programmed guidance from parental or adjacent
cells. Such unceasing, disciplined instruction ensures that each
tissue maintains a size, architecture and function appropriate to
the body's needs.
Neoplastic cells, in distinct contrast, become unresponsive to the
usual controls of parental or adjacent cells with respect to
proliferation, architecture and/or function. These neoplastic cells
frequently (a) migrate from the site where they began, (b) invade
nearby tissues, and (c) travel through the blood and lymphatic
circulatory systems to form neoplastic lesions at distant sites in
the body. These lesions become lethal when they disrupt the normal
function of other tissues or organs essential for the patient
survival.
Multiple genetic changes occur during the transformation of normal
cells into neoplastic cells. This is facilitated in neoplastic
cells by loss of fidelity in the processes that replicate, repair,
and segregate the genome structure. Advances in our understanding
of the cell cycle reveal how fidelity is normally achieved by the
coordinated activity of cyclin-dependent kinases, checkpoint
controls, and repair pathways, and how this fidelity can be
abrogated by specific genetic changes. The recognition of molecular
mechanisms for cellular transformation may help identify the
mechanisms by which chemotherapeutic compounds are useful in the
treatment of neoplastic diseases (Hartwell, L. and Kasten, M.,
1994, Science. 266:1821-1828).
Control systems enforcing interdependency in the cell cycle are
called "checkpoints." Elimination of checkpoints can result in cell
death, infidelity in the distribution chromosomes or other cellular
organelles, or increased susceptibility to external perturbations
such as DNA damaging agents. Such perturbations can result in
neoplastic transformation of cells and tissues (Hartwell, L. and
Weinert, T., 1989, Science, 246:629-634).
The cell-type-specific expression of most genes is determined at
the transcription level. Transcription factors are involved in the
control of the process. To understand the basis of this regulation,
it has become important to analyze the control of transcription
factors themselves. A variety of transcriptional, translational,
and post-translational mechanisms have been described. The most
direct way for a cell to regulate the abundance of a factor is to
adjust the production of the mRNA encoding it. Thus far, the
control of many cell-type and tissue-specific transcription factors
has been found to occur at the transcription level (Falvey, E., and
Schibler, U., FASEB J., 1991, 5:309-314).
Recent studies indicate that extracellular signals often effect
cell proliferation and differentiation by modulating intracellular
transcription factor activity via protein phosphorylation cascades,
which involves the transduction systems used to transmit
information (signals) from the cell surface to the transcription
machinery of the cell nucleus (Karin, M., 1992, FASEB J.
6:2581-2590).
Cell nuclear transcription factors regulate tissue and
stimulus-specific gene expression through their ability to
integrate extracellular signals at the nucleus. Several human
diseases, including neoplasias, cardiovascular disease, and
neurological and autoimmune disorders, result from aberrations in
the expression of genes regulated by these transcription factors
(Manning, A. Gonzales, R., and Bennett, B., Expert Opin. Ther.
Pat., 1997, 7:225-231).
Normally, the body's tissues prevent excessive proliferation of
cells by depriving them of excessive amounts of growth-stimulating
factors, or by flooding the cells with antiproliferative factors
derived from adjacent or parental cells which block the actions of
the growth stimulating factors.
Certain cellular proteins, through their intrinsic ability to
regulate a host of other genes involved in the control of cell
proliferation, can reorganize and redirect a cell's normal or
abnormal fate. Thus, the loss of these growth controlling genes by
deletion or mutation is a common occurrence in neoplasias (Lozano,
G. and Hulboy, D. L., Methods (San Diego) 1995, 8:215-224.)
Some cell cycle derangements stem from extracellular influences.
Many neoplasia causing oncogenes, for example, turn out to encode
components of the pathways through which various growth factor
signals feed into the cell cycle to stimulate cell division. This
is an important demonstration that the protein encoded by the p53
tumor suppresser gene inhibits cell growth by turning on the
production of a specialized protein that blocks the cell cycle. The
intracellular gene encoding one component of the cell cycle
machinery, a protein called cyclin D1, as well as several others,
are oncogene candidates. A significant amount of experimental
evidence indicates that excess cyclin D1 causes neoplasias, but
additional data suggest that several gene changes are implicated in
the transformation of normal cells into a neoplastic configuration.
For example, a hallmark of normal healthy cells is their ability to
differentiate, but neoplastic cells cannot differentiate due the
blockade of this response via the combined action of cyclins D2 and
D3 (Marx, J., 1994, Science, 263:319-321).
Other recent data strongly suggest that deregulation of the
restriction checkpoints in the cell-cycle G-1 phase is required for
the transition to neoplastic disarray as seen in neoplasias
(Strauss, M.; Lucas, J.; Bartek, J. Nat. Med. (N.Y.), 1995
1:1245-1246).
Mitogenic stimulation of normal cells initiates a sequence of
events leading to activation of cyclin-dependent kinases,
phosphorylation of Rb, and subsequent entry into of the cell into
the S phase. Many types of neoplasms have lost sensitivity to the
growth-inhibitory actions of TGF-beta-1, and this may derive from
dysregulated expression of cyclin, cdk, and cdk inhibitor genes
(Satterwhite, D. and Moses, H., 1994-1995 (Publ. 1995), Invasion
Metastasis, 14:309-318).
Large T-antigen expression in human fibroblasts selectively
uncouples cyclin D1 from cdk4, and subsequent immortalization of
these cells results in additional changes in the cyclin D-dependent
cell cycle regulatory pathways (Peterson, S. et al., 1995, Cancer
Res., 55:4651-4657).
Representative Neoplasms of the Immune System
Each neoplasm of the immune system exhibits a distinct clinical and
pathologic character, yet these disorders share a number of common
features. Systemic symptoms of fever, night sweats, and weight loss
may be present and are associated with advanced stages of the
disease. These tumors usually appear in one or more organs of the
hematopoietic system (lymph nodes, spleen, liver, bone marrow). If
untreated, fatal dissemination to all of these organs, as well as
other sites occurs.
Chronic myeloid leukemia (CML) usually presents clinically in a
chronic phase of variable duration, after which a fatal condition
similar to acute leukemia (blast crisis) develops. The problem of
initial therapy for chronic phase CML is that current conventional
therapy offers little chance of long-term survival of the patient
with no chance of cure, whereas an agent that offers superior
survival and a chance of cure is very toxic and expensive (Kattan,
M., et al., Ann. Intern. Med., 1996; 125:541-548).
The disease is characterized by overproduction of granulocytic
cells (especially neutrophilic types), leading to marked
splenomegaly, and very high white blood cell counts. Rises in
basophils and thrombocytosis occur frequently.
Inhibition of proliferation by pirfenidone of human uterine
leiomyomas or fibroid cells in vitro. According to the literature,
leiomyomas are the most common pelvic tumors in women with a
reported incidence of 20% or more (Merrill, J., Creasman, W. 1990,
"Danforth's Obstetrics and Gynecology," Scott, J., et al., eds.,
6th edition. Philadelphia. Lippincott, pp. 1023-1039). Most common
symptoms associated with these benign tumors are excessive abnormal
uterine bleeding, pelvic pain, infertility and increased urinary
frequency. Consequently, the presence of leiomyomas is the leading
cause for hysterectomy in the United States (Wilcox, L. et al.,
1994, Hystersectomy in the United States, 1990, Obstet. Gynecol.
83:549-555).
The majority of chemotherapeutic neoplasia agents in current
clinical practice are toxic compounds and exert their greatest
anti-neoplasia effect when employed at the maximum tolerated dose.
With these chemotherapeutic agents, toxic actions to normal tissue
can greatly limit the amount that can be safely administered. To
date, the most commonly utilized agents are only partially
selective in their toxicity. Thus, they are damaging to both normal
and neoplastic cells. These agents disrupt major intracellular
systems such as DNA synthesis and essential enzymes systems.
Nevertheless treatment of neoplastic disease is predicated on
exploiting the small differences between healthy normal cells and
neoplastic cells.
Accordingly, it is a principal object of the present invention to
provide compositions for the inhibition or arrest of the growth or
for the destruction of mammalian benign .Iadd.(for example
leiomyomas) .Iaddend.and.Iadd./or .Iaddend.malignant tumors
.[.lymphomas,.]. .Iadd.(for example leiomyomas.Iaddend..[.,.]. and
leukemias.Iadd., etc.).Iaddend..
It is a further object of the present invention to provide such
compositions that provide a means of (1) arresting the
proliferation of and (2) then killing the abnormal cells of
neoplastic tissue without serious or fatal injury to healthy normal
cells and tissues.
It is an additional object of the invention to provide such
compositions that comprise one or more N-substituted 1-(1H)
pyridone(s) and/or N-substituted 3-(1H) pyridone(s) as active
anti-tumor ingredient(s).
Other objects of the present invention, as well as particular
features and advantages thereof, will be elucidated in, or be
apparent from, the following descriptions.
SUMMARY OF THE INVENTION
The present invention achieves the above objects, among others, and
overcomes the limitations of the prior art by providing, an 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
.Iadd.malignant neoplasms (such as leukemias.Iaddend. .[.,.].
.Iadd.and lymphomas, and/or benign neoplasms (such as
leiomyomas).Iaddend..
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a bar graph showing the inhibition by pirfenidone of the
proliferation of human pro-myelocytic leukemia cells.
FIG. 2 is a bar graph showing the inhibition by prifenidone of
human T-cell leukemia cells.
FIG. 3 is a bar graph showing the inhibition by pirfenidone of
proliferation of human Burkitt's lymphoma cells.
FIG. 4 is a bar graph showing the inhibition by pirfenidone of
human leiomyoma cells.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Methods for Evaluating Anti-Cancer Agents in the Present
Invention
All-trans-retinoic acid is known to specifically induce fresh human
promyelocytic leukemia cells (AML3) to differentiate in vitro to
mature functional gramulocytes which loose their self-renewal
potency and spontaneously die. These results were confirmed in
vivo: AML-3 patients treated with oral all-trans retinoic acid
alone achieve complete remission (Chomienne, Ch., "Retinoids: Basic
Sci. Clin. Appl.," 1994, p.233-241. Edited by Livrea and Vidali,
Birkhaeuser, Basel, Switzerland).
Evidence that in vitro prednisolone resistance is of prognostic
value in childhood acute lymphoblastic leukemia (ALL) was explored
further in adult ALL leukemia. Blast cells from 30 patients were
exposed to prednisolone (PDN) (0.1) microMol to 35 microMol), and
cytotoxicity was assayed calorimetrically with soluble tetrazolium
formazan (2,3-bis
(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbamyl]-2H-tetrazoliu-
m hydroxide, XTT). The IC50 varied greatly among the samples from
0.3 microMol to >35 microMol. 15 microMol was subsequently
chosen as IC50 cutoff between in vitro resistant and sensitive
cases. By regression analysis, PDN-induced cytotoxicity was found
to be significantly related to apoptosis. However, correlation with
clinical symptoms was not demonstrated. Nevertheless, disease free
survival was significantly better in sensitive patients (Tosi, P.
et al., Eur. J. Haematology, 1996, 52:134-141.)
Chronic lymphocytic leukemia (CLL) is characterized by delayed
senescence and a slow accumulation of small lymphocytes. Elevated
cytokine levels have been detected in urine from patients with a
variety of neoplastic diseases including various leukemias. The
source of the elevated levels of cytokine has not been determined.
In the present study, the intracellular cytokine level in
lymphocytes from 36 patients with B-CLL and 15 normal donors was
determined using an enzyme-lined immunoassay. In cells derived from
patients with high risk disease, the median level of cytokine was
382 pg/200,000 cells compared to 91 pg/200,000 cells in patients
with intermediate disease. In patients with low-risk disease,
median cytokine level was 4.9 pg/200,000 cells, and in normal
controls, it was 6.0 pg/200,000 cells. The respective differences
were statistically significant (Menzel et al., Blood, 1996,
87:1056-1063).
In 1996, approximately 3,500 new cases (mostly children) of acute
lymphoblastic leukemia (ALL) were diagnosed in the US. Current
chemotherapy induces complete remission in many patients, but 20%
suffer a critical relapse.
It has been discovered by the present inventor that pirfenidone and
other N-substituted 2(1-H) pyridone compounds and N-substituted
3(1H) pyridone compounds have anti-tumor activity. Heretofore,
before the discovery of the invention disclosed herein, no
effective N-substituted pyridone agent or composition has been
available for the inhibition and arrest of the growth, or for the
destruction of mammalian benign or malignant tissues such as found
in leukemias, lymphomas or leiomyomas.
The "anti-tumor" activity discovered by the present inventor and as
used herein refers to the ability of an active substance to inhibit
or arrest the life-threatening proliferation of neoplastic cells in
.Iadd.benign and malignant tumors including .Iaddend.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.
EXAMPLE I
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.times.10.sup.5
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.
EXAMPLE II
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.times.10.sup.5 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.times.10.sup.5 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).
EXAMPLE III
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.times.10.sup.5 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.times.10.sup.5 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).
EXAMPLE IV
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-US-00001 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
EXAMPLE V
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.]. .Iadd.on
.Iaddend.leiomyomas .[.indicate.]. .Iadd.indicates .Iaddend.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 1/3rd- 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.].
.Iadd.neoplastic .Iaddend.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.]. .Iadd.anti-neoplastic .Iaddend.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 .Iadd.human .Iaddend.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: 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-('-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-methyi-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-pheyl-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 1-Phenyl-3-(1H) pyridone.
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.
* * * * *