U.S. patent number 6,037,376 [Application Number 07/779,744] was granted by the patent office on 2000-03-14 for methods for therapy of cancer.
This patent grant is currently assigned to The United States of America as represented by the Department of Health. Invention is credited to Dvorit Samid.
United States Patent |
6,037,376 |
Samid |
March 14, 2000 |
Methods for therapy of cancer
Abstract
Compositions and methods of treating anemia, cancer, AIDS, or
severs .beta.-chain hemoglobinopathies by administering a
therapeutically effective amount of phenylacetate or
pharmaceutically acceptable derivatives thereof or derivatives
thereof alone or in combination or in conjunction with other
therapeutic agents. Pharmacologically-acceptable salts alone or in
combinations and methods of preventing AIDS and malignant
conditions, and inducing cell differentiation are also aspects of
this invention.
Inventors: |
Samid; Dvorit (Rockville,
MD) |
Assignee: |
The United States of America as
represented by the Department of Health (Washington,
DC)
|
Family
ID: |
25117401 |
Appl.
No.: |
07/779,744 |
Filed: |
October 21, 1991 |
Current U.S.
Class: |
514/568 |
Current CPC
Class: |
A61K
31/20 (20130101); A61K 31/70 (20130101); A61K
31/35 (20130101); A61K 38/212 (20130101); A61K
31/7072 (20130101); A61K 31/19 (20130101); A61P
43/00 (20180101); A61P 31/12 (20180101); A61P
35/00 (20180101); A61P 31/18 (20180101); A61K
31/7076 (20130101); A61K 45/06 (20130101); A61K
31/365 (20130101); A61P 7/06 (20180101); A61K
31/192 (20130101); A61K 31/19 (20130101); A61K
2300/00 (20130101); A61K 31/20 (20130101); A61K
2300/00 (20130101); A61K 31/35 (20130101); A61K
2300/00 (20130101); A61K 31/365 (20130101); A61K
2300/00 (20130101); A61K 31/70 (20130101); A61K
2300/00 (20130101); A61K 31/7072 (20130101); A61K
2300/00 (20130101); A61K 31/7076 (20130101); A61K
2300/00 (20130101); A61K 38/212 (20130101); A61K
2300/00 (20130101); A61K 31/7076 (20130101); A61K
31/19 (20130101); A61K 31/7072 (20130101); A61K
31/19 (20130101); A61K 31/70 (20130101); A61K
31/19 (20130101); A61K 31/20 (20130101); A61K
31/19 (20130101); A61K 31/19 (20130101); A61K
31/19 (20130101); A61K 31/19 (20130101); A61K
31/17 (20130101) |
Current International
Class: |
A61K
38/21 (20060101); A61K 31/192 (20060101); A61K
31/20 (20060101); A61K 31/19 (20060101); A61K
31/185 (20060101); A61K 31/7042 (20060101); A61K
31/70 (20060101); A61K 31/7072 (20060101); A61K
45/00 (20060101); A61K 45/06 (20060101); A61K
31/35 (20060101); A61K 38/22 (20060101); A61K
31/365 (20060101); A61K 31/7076 (20060101); A61K
031/19 () |
Field of
Search: |
;514/568,569,570,564 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Burzynski, S.R. et al., "Preclinical Studies on Antineoplaston
AS2-1 and Antineoplaston AS2-5," Drugs Exptl. Clin. Res.,
Supplemental 1, XII:11-16 (1986). .
Shechter, Y. et al., "Hydroxyphenyl Acetate Derivatives Inhibit
Protein Tyrosine Activity and Proliferation in Nb2 Rat Lymphoma
Cells and Insulin-Induced Lipogenesis in Rat Adipocytes," Molecular
and Cellular Endrocrinology, vol. 80, pp. 183-192 (1991). .
Samid, D. et al., "Interferon in Combination with Antitumourigenic
Phenyl Derivatives: Potentiation of IFN .alpha. Activity In-Vitro,"
British J. Haematology, vol. 79, Suppl. 1, pp. 81-83 (Oct. 10,
1991). .
Timothy J. Levy, et al., "5-Azacytidine Selectively Increases
.gamma.-globin Synthesis in a Patient with .beta..sup.+
Thalassemia", New England Journal of Medicine, vol. 307:1469-1475
(Dec. 9, 1982). .
Michael B. Sporn, et al., "Chemoprevention of Cancer with
Retinoids", Federation Proceedings, vol. 38:2528-2534 (Oct. 1979).
.
Richard L. Momparler, et al., "Clinical Trial on
5-AZA-2'-Deoxycytidine in Patients with Acute Leukemia", Pharmac.
Ther., vol. 30:277-286 (1985). .
Gary J. Kelloff, et al., "Chemoprevention Clinical Trials",
Mutation Research, vol. 267:291-295 (1992). .
I. Bernard Weinstein, "Cancer Prevention: Recent Progress and
Future Opportunities", Cancer Research, vol. 51:5080s-5085s (1991).
.
Olli Simell, et al, "Waste Nitrogen Excretion Via Amino Acid
Acylation: Benzoate and Phenylacetate in Lysinuric Protein
Intolerance", Pediatr. Res., vol. 20:1117-1121 (1986). .
Neish, et al., "Phenylacetic Acid as a Potential Therapeutic Agent
for the Treatment of Human Cancer", Experentia, vol. 27:860-861
(1971). .
J.A. Stamatoyannopoulos, et al., "Therapeutic Approaches to
Hemoglobin Switching in Treatment of Hemoglobinopathies", Annu.
Rev. Med., vol. 43:497-521 (1992). .
Jones, G.L., Anti sickling effects of Beta Di
Ethy1aminoethylidiphenylpropyl acetate SFK-525-A, Phamracologist,
20(3):204 (1978). .
Ross, Philip D. and Subramanian, S., Inhibition of sickle cell
hemoglobin gelation by some aromatic compounds, Biochem. Biophys.
Res. Commun., 77:1217-1223 (1977). .
Erhum, Wilson O., Acetonyl esters of hydroxybenzoic acids as
potential antisickling agents, Niger. J. Pharm., 12:285-287 (1981).
.
Chemical Abstracts 83:53278s (1975)..
|
Primary Examiner: Goldberg; Jerome D.
Attorney, Agent or Firm: Needle & Rosenberg, P.C.
Government Interests
I. GOVERNMENT INTEREST
The invention described herein may be manufactured, used and
licensed by or for the Government for governmental purposes without
the payment to us of any royalties thereon.
Claims
What is claimed:
1. A method of inhibiting the growth of a rapidly proliferating
nonmalignant or malignant mammalian tumor cell sensitive to a
compound recited below in a host in need of such inhibition,
comprising administering to the host an amount effective to attain
said inhibition of said compound having the formula (I): ##STR9##
wherein R and R.sup.1 are independently H, lower alkoxy, or lower
alkyl;
R.sup.2 is phenyl, unsubstituted or substituted with halogen,
hydroxy, or lower alkyl;
R.sup.3 and R.sup.4 are H; and
n is 0 or 2;
or a pharmaceutically acceptable salt thereof.
2. The method of claim 1, wherein the pharmaceutically acceptable
salt is an alkali metal salt or alkaline earth metal salt.
3. The method of claim 1, wherein R is H and R.sup.1 is H, CH.sub.3
O, CH.sub.3, C.sub.2 H.sub.5, or C.sub.3 H.sub.7.
4. The method of claim 1, wherein R is H and R.sup.1 is H.
5. The method of claim 1, wherein R.sup.2 is phenyl or phenyl
substituted with from 1 to 4 halogens, from 1 to 4 hydroxy
moieties, or from 1 to 2 methyl moieties.
6. The method of claim 1, wherein R.sup.2 is phenyl or phenyl
substituted with from 1 to 4 halogens of Cl or F, from 1 to 4
hydroxy moieties, or from 1 to 2 methyl moieties.
7. The method of claim 1, wherein R.sup.2 is phenyl,
3-methylphenyl, 4-methylphenyl, 2,6-dimethylphenyl, 3-chlorophenyl,
4-chlorophenyl, 2,6-dichlorophenyl or 4-fluorophenyl.
8. The method of claim 1, wherein R.sup.2 is phenyl.
9. The method of claim 1, wherein the compound is the sodium salt
of phenylacetate, phenylbutyrate, 3-chlorophenylacetate,
4-chlorophenylacetate, 2,6-dichlorophenylacetate, or
4-fluorophenylacetate.
10. The method of claim 1, wherein the compound is the sodium salt
of formula I and R is H, R.sup.1 is H, and R.sup.2 is phenyl.
11. The method of claim 1, wherein the compound is sodium
phenylacetate.
12. The method of claim 1, wherein the compound is sodium
phenylbutyrate.
13. The method of claim 1, wherein n is 0.
14. The method of claim 1, comprising administering a
pharmacologically-effective amount of the compound to the host to
suppress the growth of the nonmalignant or malignant tumor
cell.
15. The method of claim 1, wherein the compound is administered
intravenously at a dosage level of from about 50 mg/kg/day to about
1,000 mg/kg/day.
16. The method of claim 1, wherein the compound is administered
subcutaneously at a dosage level of from about 50 mg/kg/day to
about 1,000 mg/kg/day.
17. The method of claim 1, wherein the compound is administered
orally at a dosage level of from about 50 mg/kg/day to about 1,000
mg/kg/day.
18. The method of claim 1, wherein the compound is applied
topically at a dosage concentration of from about 1 to about 10
mg/ml.
19. The method of claim 1, wherein the tumor is a prostatic
carcinoma tumor, melanoma tumor, glial brain tumor, Kaposi's
sarcoma tumor or lymphoma tumor, leukemic tumor, lung
adenocarcinoma tumor, breast cancer tumor, osteosarcoma tumor,
fibrosarcoma tumor, or squamous cancer tumor.
20. The method of claim 1, wherein the host is a human.
21. A method of inhibiting the growth of a rapidly proliferating
nonmalignant or malignant mammalian tumor cell sensitive to a
compound recited below in a host in need of such inhibition,
comprising administering to the host an amount effective to attain
said inhibition of said compound of phenylacetic acid or
phenylbutyric acid or a pharmaceutically acceptable salt
thereof.
22. The method of claim 21, wherein the tumor is a prostatic
carcinoma tumor, melanoma tumor, glial brain tumor, Kaposi's
sarcoma tumor or lymphoma tumor, leukemic tumor, lung
adenocarcinoma tumor, breast cancer tumor, osteosarcoma tumor,
fibrosarcoma tumor, or squamous cancer tumor.
23. A method of inhibiting the growth of a rapidly proliferating
nonmalignant or malignant mammalian tumor sensitive to a compound
recited below in a host in need of said inhibition, consisting
essentially of administering to the host an amount effective to
attain said inhibition of said compound having the formula (I):
##STR10## wherein R and R.sup.1 are independently H, lower alkoxy,
or lower alkyl;
R.sup.2 is phenyl, unsubstituted or substituted with halogen,
hydroxy, or lower alkyl;
R.sup.3 and R.sup.4 are H; and
n is 0 or 2;
or a pharmaceutically-acceptable salt thereof.
24. The method of claim 23, wherein the compound is phenylacetic
acid or phenylbutyric acid or a pharmaceutically acceptable salt
thereof.
25. The method of claim 23, wherein n is 0.
26. The method of claim 23, wherein the tumor is a prostatic
carcinoma tumor, melanoma tumor, glial brain tumor, Kaposi's
sarcoma tumor or lymphoma tumor, leukemic tumor, lung
adenocarcinoma tumor, breast cancer tumor, osteosarcoma tumor,
fibrosarcoma tumor, or squamous cancer tumor.
27. A method of treating a host afflicted with a condition of
cancer sensitive to a compound recited below, comprising
administering to said host a pharmacological amount effective to
ameliorate said condition of said compound having the formula (I):
##STR11## wherein R and R.sup.1 are independently H, lower alkoxy,
or lower alkyl;
R.sup.2 is phenyl, unsubstituted or substituted with halogen,
hydroxy, or lower alkyl;
R.sup.3 and R.sup.4 are H; and
n is 0 or 2;
or a pharmaceutically-acceptable salt thereof.
28. The method of claim 27, wherein the compound is phenylacetic
acid or phenylbutyric acid or a pharmaceutically acceptable salt
thereof.
29. The method of claim 27, wherein n is 0.
30. The method of claim 27, wherein the cancer is a prostatic
cancer, melanoma, glial brain tumor, Kaposi's sarcoma or lymphoma,
leukemia, lung adenocarcinoma, breast cancer, osteosarcoma,
fibrosarcoma, or squamous cancer.
31. A method of inducing differentiation of a tumor cell sensitive
to a compound recited below in a host in need of such treatment
comprising administering to said host a therapeutically effective
amount of said compound having the formula (I): ##STR12## wherein R
and R.sup.1 are independently H, lower alkoxy, or lower alkyl;
R.sup.2 is phenyl, unsubstituted or substituted with halogen,
hydroxy, or lower alkyl;
R.sup.3 and R.sup.4 are H; and
n is 0 or 2;
or a pharmaceutically-acceptable salt thereof.
32. The method of claim 31, wherein the compound is phenylacetic
acid or phenylbutyric acid or a pharmaceutically acceptable salt
thereof.
33. The method of claim 31, wherein n is 0.
34. The method of claim 31, wherein the tumor is a prostatic
carcinoma tumor, melanoma tumor, glial brain tumor, Kaposi's
sarcoma tumor or lymphoma tumor, leukemic tumor, lung
adenocarcinoma tumor, breast cancer tumor, osteosarcoma tumor,
fibrosarcoma tumor, or squamous cancer tumor.
35. A method of treating a malignant condition sensitive to a
compound recited below in a host in need of such treatment
comprising administering to the host a therapeutically effective
amount of said compound having the formula (I): ##STR13## wherein R
and R.sup.1 are independently H, lower alkoxy, or lower alkyl;
R.sup.2 is phenyl, unsubstituted or substituted with halogen,
hydroxy, or lower alkyl;
R.sup.3 and R.sup.4 are H; and
n is 0 or 2;
or a pharmaceutically-acceptable salt thereof.
36. The method of claim 35, wherein the compound is phenylacetic
acid or phenylbutyric acid or a pharmaceutically acceptable salt
thereof.
37. The method of claim 35, wherein n is 0.
38. The method of claim 35, wherein the malignant condition to be
treated is a prostatic cancer, melanoma, glial brain tumor,
Kaposi's sarcoma or lymphoma, leukemia, lung adenocarcinoma, breast
cancer, osteosarcoma, fibrosarcoma, or squamous cancer.
39. The method of claim 35, wherein the malignant condition to be
treated is prostatic cancer.
40. The method of claim 35, wherein the malignant condition to be
treated is melanoma.
41. The method of claim 35, wherein the malignant condition to be
treated is a glial brain tumor or Kaposi's sarcoma or lymphoma.
Description
II. FIELD OF THE INVENTION
This invention relates to methods of using phenylacetic acid and
its pharmaceutically acceptable derivatives as antitumor, and
antiviral agents, and treatment of severe beta-chain
hemoglobinopathies.
III. BACKGROUND OF THE INVENTION
Some of the most dreadful epidemics, inherited diseases and
cancerous infections in the history of mankind are afflicting the
world's people at a rapid and discomforting rate. These maladies
are being caused in many instances by the increasing cases of
cancer, of viral infections, such as human immunodeficiency viruses
(HIV) or HTLV and of severe beta-chain hemoglobinopathies. When one
pauses to reflect upon the devastating pain, suffering and
ultimately death experienced by persons afflicted, these moments of
reflection underscore the tremendous importance which must be
accorded medical research. In response to the need to alleviate
suffering and add comfort to human life, the scientific community
throught the world is searching for effective treatments to prevent
or ameliorate diseases.
In order to present the enormous scope of this unitary invention in
a comprehensive form while preserving the essential need for
clarity in presentment, this invention focusing on phenylacetate
and its derivatives will be described in the following three (3)
subsections, designated herein as A. Phenylacetate In Cancer
prevention and maintenance therapy; B. Phenylacetate and its
derivatives in the Treatment and Prevention of AIDS; and C.
Induction of fetal hemoglobin synthesis in .beta.-chain
hemoglobinopathies by phenylacetate and its derivatives.
DESCRIPTION OF RELATED DISCLOSURES
Phenylacetic acid (PAA) is a protein decomposition product found
throughout the phylogenetic spectrum, ranging from bacteria to man.
Highly conserved in evolution, PAA may play a fundamental role in
growth control and differentiation. In plants, PAA serves as a
growth hormone (auxin) promoting cell proliferation and enlargement
at low doses (10-5-10-7M), while inhibiting growth at higher
concentrations. The effect on animal and human cells is less well
characterized. In humans, PAA acid is known to conjugate glutamine
with subsequent renal excretion of phenylacetylglutamine (PAG). The
latter, leading to waste nitrogen excretion, has been the basis for
using PAA or preferably its salt sodium phenylacetate (NaPA) in the
treatment of hyperammonemia associated with inborn errors of
ureagenesis. Clinical experience indicates that acute or long-term
treatment with high NaPa doses is well tolerated, essentially free
of adverse effects, and effective in removing excess glutamine.
[Brusilow, S. W., Horwich, A. L. Urea cycle enzymes. Metabolic
Basis of Inherited Diseases, Vol. 6:629-633 (1989)]. These
characteristics should be of value in cancer intervention,
treatments to inhibit virus replication and treatment of severe
beta-chain hemoglobinopathies.
Glutamine is the major nitrogen source for nucleic acid and protein
synthesis, and substrate for energy in rapidly dividing normal and
tumor cells. Compared with normal tissues, most tumors, due to
decreased synthesis of glutamine along with accelerated utilization
and catabolism, operate at limiting levels of glutamine
availability, and consequently are sensitive to further glutamine
depletion. Considering the imbalance in glutamine metabolism in
tumor cells and the ability of PAA to remove glutamine, PAA has
been proposed as a potential antitumor agent, however, no data was
provided to substantiate this proposal. [Neish, W. J. P.
"Phenylacetic Acid as a Potential Therapeutic Agent for the
Treatment of Human Cancer", Experentia, Vol. 27, pp. 860-861
(1971)].
Despite efforts to fight cancer, many malignant diseases that are
of interest in this application still present a major challenge to
clinical oncology. Prostate cancer, for example, is the second most
common cause of cancer deaths in men. Current treatment protocols
rely primarily on hormonal manipulations, however, in spite of
initial high response rates, patients often develop
hormone-refractory tumors, leading to rapid disease progression
with poor prognosis. Overall, the results of cytotoxic chemotherapy
have been disappointing, indicating a long felt need for new
approaches to treatment of advanced prostatic cancer. Other
diseases resulting from abnormal cell replication for example,
metastatic melanomas, brain tumors of glial origin (e.g.,
astrocytomas), and lung adenocarcinoma, are all highly aggressive
malignancies with poor prognosis. The incidence of melanoma and
lung adenocarcinoma has been increasing significantly in recent
years. Surgical treatment of brain tumors often fails to remove all
tumor tissues, resulting in recurrences. Systemic chemotherapy is
hindered by blood barriers. Therefore there is an urgent need for
new approaches to the treatment of human malignancies such as
advanced prostatic cancer, melanoma, brain tumors, and others.
The development of the methods of the present invention was guided
by the hypothesis that metabolic traits that distinguish tumors
from normal cells could potentially serve as targets for
therapeutic intervention. Tumor cells show unique requirements for
specific amino acids, of which glutamine would be the desired
choice because of its major contribution to energy metabolism and
to synthesis of purines, pyrimidines, and proteins. Along this
line, promising antineoplastic activities have been demonstrated
with glutamine-depleting enzymes such as glutaminase, and various
glutamine antimetabolites, unfortunately, the clinical usefulness
of these drugs has been limited by unacceptable toxicities.
Consequently, the present invention focuses on PAA, a plasma
component known to conjugate glutamine in vivo.
In addition to its effect on glutamine phenylacetate can induce
tumor cells to undergo differentiation. (See examples 1-5, 8 and 9
herein) Differentiation therapy is a known desirable approach to
cancer intervention. The underlying hypothesis is that neoplastic
transformation results from defects in cellular differentiation.
Inducing tumor cells to differentiate would prevent tumor
progression and bring about reversal of malignancy. Several
differentiation agents are known, but their clinical applications
have been hindered by unacceptable toxicities and/or deleterious
side effects.
Accordingly, a major object of the present invention is to provide
a method for treating various cancerous conditions with PAA and its
pharmaceutically acceptable salts and derivatives.
Another object of the present invention is to provide a method for
the prevention of tumor progression and the development of
malignant conditions in high risk individuals by administering
prophylactically effective amounts of nontoxic agents such as
phenylacetate and its pharmaceutically acceptable derivatives.
Another object of the present invention is to provide a method for
the amelioration of and prophlactic treatment against viral
infections. Still another object of the present invention is to
provide a method for the amelioration of and prophylactic treatment
against severe anemia associated with beta-chain
hemoglobinopathies.
It is yet a further object to provide a method of treating or
preventing the onset of malignancies, viral infections associated
with AIDS or severe beta-chain hemoglobinopathies with a
combination of Phenylacetate (or its pharmaceutically acceptable
derivatives) and various other therapeutic or preventive agents
alone or in conjunction with conventional therapies.
A further object of the invention is to provide effective
pharmaceutical formulations of PAA and its pharmaceutically
acceptable derivatives for carrying out the above methods.
IV. BRIEF SUMMARY OF THE INVENTION
The present invention is directed to methods for therapy of cancer.
The methods relate to administering a compound or salt of a
compound having the formula (I).
The present invention provides a method of (1) suppressing the
growth of tumor cells in a host in need of such suppression
comprising administering an amount of PAA or a pharmaceutically
acceptable derivative thereof effective to suppress the growth of
said tumor cells and (2) preventing the onset of or ameliorating
the effects of viral infections or severe beta-chain
hemoglobinopathies.
For the purpose of the present application, the PAA derivatives
include its pharmacological acceptable salts, preferably sodium;
analogs containing halogen substitutions, preferably chlorine or
fluorine; analogs containing alkyl substitutions, preferably methyl
or methoxy; precursors of phenylacetate, preferably phenylbutyrate;
and natural analogs such as naphtylacetate.
The compounds of the present invention can be administered
intravenously, enterally, parentally, intramuscularly,
intranasally, subcutaneously, topically or orally. The dosage
amounts are based on the effective inhibitory concentrations
observed in vitro and in vivo in antitumorigenicity studies. The
varied and efficacious utility of the compounds of the present
invention is further illustrated by the finds that they may also be
administered concomitantly or in combination with other antitumor
agents such as hydroxyurea, 5-azacytidin, 5-aza-2' deoxycytidine,
suramin; retinoids; hormones; biological response modifiers, such
as interferon and hematopoetic growth factors; and conventional
chemo- and radiation therapy or various combinations thereof.
The present invention also provides methods of inducing tumor cell
differentiation in a host comprising administering to the host a
therapeutically effective amount of PAA or a pharmaceutically
acceptable derivative thereof.
The present invention also provides methods of preventing the
formation of malignancies by administering to a host a
prophylactically effective amount of PAA or a pharmaceutically
acceptable derivative thereof.
The present invention also provides methods of treating malignant
conditions, such as prostatic cancer, melanoma, adult and pediatric
tumors, e.g. brain tumors of glial origin, astrocytoma, Kaposi's
sarcoma, lung adenocarcinoma and leukemias, as well as hyperplastic
lesions, e.g. benign hyperplastic prostate and papillomas by
administering a therapeutically effective amount of PAA or a
pharmaceutically acceptable derivative thereof.
V. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the inhibition of HL-60 leukemia and permalignant
10T1/2 cell proliferation by NaPA.
FIG. 2 shows the induction of HL-60 cell differentiation. The
number of NBT positive cells was determined after 4 or 7 days of
treatment. NaPA (h), 1.6 mg/ml; NaPA (1), 0.8 mg/ml. 4-hydroxy PA
and PAG were used at 1.6 mg/ml. Potentiation by RA 10 nM was
comparable to that by IFN gamma 300 IU/ml, and the effect of
acivicin 3 ug/ml similar to DON 30 ug/ml. Glutamine Starvation
(Gln, <0.06 mM) was as described (18). Cell viability was over
95% in all cases, except for DON and acivicin (75% and 63%,
respectively).
FIG. 3 shows adipocyte conversion in 10T1/2 cultures. Lipids
stained with Oil-Red O were extracted with butanol, and the optical
density at 510 nm determined. Increased lipid accumulation was
evident with NaPA concentrations as low as 0.024 mg/ml.
FIG. 4 shows the effect of NaPA on cell proliferation. PC3; DU145;
LNCap; and, FS4 cultures were treated with NaPA or PAG for four
days.
FIG. 5 shows the inhibition of tumor cell invasion by NaPA cells
treated in culture for 7 days were harvested and assayed for their
invasive properties using a modified Boyder Chamber with a
matrigel-coated filter. Results scored 6-24 hours later.
VI. DETAILED DESCRIPTION OF THE INVENTION
According to the present invention, PAA, in particular its sodium
salt NaPA has been found to be an excellent inhibitor of the growth
of specific tumor cells, affecting the proliferation of the
malignant cells while sparing normal tissues. Also, according to
the present invention, NaPA has been found to induce tumor cell
differentiation, thus offering a most desirable approach to cancer
prevention and therapy. Additionally, NaPA has been found to be of
potential value for the treatment of AIDS and severe beta-chain
hemoglobino-pathies. The exact mechanisms by which the compounds
used in the method of this invention exert their effect is
uncertain, one mechanism may involve depletion of plasma glutamine.
Based on the data reported herein, it is believed that glutamine
depletion alone cannot explain the molecular and phenotypic changes
observed in vitro following exposure to NaPA. It will be
understood, however, that the present invention is not to be
limited by any theoretical basis for the observed results. Most
significantly, it has now been discovered for the first time:
1. A pharmacetical composition for inhibiting (1) abnormal cell
growth and inducing differentiation in nonmalignant or malignant
mammalian tumor cells; (2) altering gene expression and inducing
differentiation in nonmalignant mammalian cells; or (3) viral
replication and spread, comprising a pharmacologically-effective
amount of a compound of the formula ##STR1## wherein
R and R.sup.1 is H, lower alkoxy, or lower alkyl; R.sup.2 =aryl and
substituted aryl; stereoisomers thereof,
pharmaceutically-acceptable derivatives or salts thereof; and
mixtures thereof.
2. The composition of claim 1, wherein the
pharmaceutically-acceptable salts are selected from the group
consisting of a alkali and alkaline earth metal.
3. The composition of claim 1, wherein the
pharmaceutically-acceptable salt is an alkali metal.
4. The composition of claim 3, wherein the alkalimetal is
sodium.
5. The composition of claim 1, wherein R is H and R.sup.1 is H,
CH.sub.3, CH.sub.3 --O--, C.sub.2 H.sub.5, or C.sub.3 H.sub.7.
6. The composition of claim 5 wherein R.sup.2 is ##STR2## X is
halogen or hydroxy; and n is 0 to 4.
7. The composition of claim 6, wherein X is Cl, F, or hydroxy.
8. The composition of claim 7, wherein X is Cl.
9. The composition of claim 8, wherein R.sup.2 is ##STR3##
10. The composition of claim 9, wherein R is H or C.sub.3 H.sub.7
and R.sup.1 is H.
11. The composition of claim 10, wherein R.sup.2 is ##STR4##
12. The composition of claim 11, wherein R.sup.2 is ##STR5## and R
is hydrogen.
13. The composition of claim 11 wherein R.sup.2 is ##STR6## and R
is C.sub.3 H.sub.7.
14. The composition of claim 11, wherein R.sup.2 is ##STR7##
15. The composition of claim 11, wherein R.sup.2 is ##STR8##
16. The composition of claim 1, comprising about 0.010 to 99.990
weight percent (%) of said compound.
17. A method of inhibiting the growth of rapidly proliferating
nonmalignant and malignant mammalian tumor cells, comprising
administering to a host in need of said inhibition an amount of the
composition of claim 1 effective to attain said inhibition.
18. A method of inhibiting the altering gene expression and
inducing differentiating in nonmalignant or malignant mammalian
tumor cells in red blood derived from a patient afflicted anemia
resulting from abnormal hemoglobin production, comprising
administering to host in need of inhibition an inhibitory amount of
the composition of claim 1 effective to attain said inhibition.
19. The method of inhibiting viral replication and spread of
virus-infected abnormal mammalian cells, comprising administering
to a host in need of said inhibition an amount of the composition
of claim 1 effective to attain said inhibition.
20. The method of claim 17, wherein said abnormal mammlian cells
are red blood derived from a patient afflicted with anemia
resulting from abnormal production adult-form hemoglobin.
21. The method of claim 18, wherein the anemic cells are selected
from the group comprising sickle cell anemia or
beta-thalassemia.
22. The method of claim 19, wherein the virus-infected cell is
infected with a retrovirus.
23. The method of claim 22, wherein the retrovirus is a Human
Immunodeficiency Virus or HTLV.
24. The method of claim 17, wherein the composition is
prophylactically administered prior to the onset of malignancy.
25. The method of claim 24, wherein the composition is
prophylactically administered prior to the onset of AID or
AIDS-Associated disorders.
26. A method of treating a host afflicted with a condition of
cancer, anemia, HIV or HTLV comprising administering to said host a
pharmacological amount of the composition of claim 1 effective to
ameliorate said conditions.
27. The method in accordance with claim 17 comprising administering
to said host an amount of phenylacetic acid or a pharmaceutically
acceptable derivative or salt thereof a pharmacologically-effective
to suppress the growth of beneign or malignant tumor cells.
28. The method in accordance with claim 18 comprising administering
to said host an amount of phenylacetate derivative or salt thereof
a pharmalogically-effective to induce the production of the
fetal-form hemoglobin.
29. The method according to claim 26, wherein phenylacetic acid or
a pharmaceutically acceptable derivative thereof is administered
intravenously at a dosage level of from about 50 mg/kg/day to about
100 mg/kg/day.
30. The method according to claim 26, wherein phenylacetic acid or
a pharmaceutically acceptable derivative thereof is administered
subcutaneously at a dosage level of from about 50 mg/kg/day to
about 1000 mg/kg/day.
31. The method according to claim 26, wherein, phenylacetic acid or
a pharmaceutically acceptable derivative thereof is administered
orally at a dosage level of from about 50 mg/kg/day to about 1000
mg/kg/day.
32. The method according to claim 26, wherein phenylacetic acid or
a pharmaceutically acceptable derivative thereof is applied
topically at a dosage concentration to about 1 to 10 mg/ml.
33. The method according to claim 26, wherein phenylacetic acid or
a pharmaceutically acceptable derivative thereof is administered
concomitantly or in combination with an antitumor or antiviral
agent.
34. The method according to claim 27, wherein phenylacetic acid or
a pharmaceutically acceptable derivative thereof is administered
concomitantly or in combination with a biological response
modifier.
35. The method according to claim 33, wherein the antitumor agent
is selected from the group consisting of hydroxyurea, suramin,
retinoids 5-azacytidine and 5-aza-2-deoxcytidine.
36. The method according to claim 33, wherein the the antiviral
agent is AZT or DDI.
37. The method according to claim 34, wherein the biological
response modifier is selected from the group consisting of
interferons, hormones, hormone-antagonists.
38. The method according to claim 26, wherein phenylacetic acid or
a pharmaceutically acceptable derivative therof is administered
concomitantly or in combination with conventional biotherapy,
chemotherapy, hormone manipulation, or radiation therapy.
39. The method according to claim 26, wherein the pharmaceutically
acceptable derivative is sodium phenylacetate.
40. The method according to claim 26, wherein the pharmaceutically
acceptable derivative is sodium phenylbutyrate.
41. A method of inducing tumor cell differentiation in a host in
need of such treatment comprising administering to said host a
therapeutically effective amount of phenylacetic acid or a
pharmaceutically acceptable derivative thereof.
42. The method according to claim 41, wherein the anemia is sickle
cell or beta-thalassemia.
43. A method of treating malignant conditions comprising
administering to a host in need of such treatment a therapeutically
effective amount of phenylacetic acid or a pharmaceutically
acceptable derivative thereof.
44. A method according to claim 43, wherein the malignant condition
to be treated comprises prostatic, melanoma, glial brain tumor,
AIDS-Associated Kaposi's Sarcoma and Lymphomas, leukemia, lung
adenocarcenoma, brest cancer, osteosarcoma, fibrosarcoma, and
squamous cancers.
45. The method of claim 44 provided that R.sup.11 cannot be phenyl
when the condition being treated is brest cancer.
46. The method according to claim 43, wherein the malignant
condition to be treated is prostatic cancer.
47. The method according to claim 43, wherein the malignant
condition to be treated is melanoma.
48. The method of claim 43 wherein the malignant condition being
treated is selected from the group consisting essential of glial
brain tumors, AIDS and AIDS associated klaposi's sarcoma and
lyphomas.2
VII. EXAMPLES
The herein offered examples, including experiments, provide methods
for illustrating, without any implied limitation, the practice of
this invention focusing on phenylacetate and its derivatives
directed to A. Cancer therapy and prevention; B. Treatment and
prevention of AIDS; and C. Induction of fetal hemoglobin synthesis
in .beta.-chain hemoglobinopathies.
SECTION A.
PHENYLACETATE IN CANCER PREVENTION AND MAINTENANCE THERAPY
Recent advances in molecular techniques allow the detection of
genetic disorders associated with predisposition to cancer.
Consequently, it is now possible to identify high-risk individuals
as well as patients in remission with residual disease. Despite
such remarkable capabilities, there is no acceptable preventive
treatment. Chemopreventive drugs are needed also for adjuvant
therapy, to minimize the carcinogenic effects of the prevailing
anticancer agents and maintain tumor responses.
To qualify for use in chemoprevention, a drug should have antitumor
activities, be nontoxic and well tolerated by humans, easy to
administer (orally), and inexpensive. We suggest that NaPA may
possess all of the above characteristics.
Experimental Data
1. Prevention of Neoplastic transformation--Oncogene transfer
studies.
NIH 3T3 cells carrying activated EJras oncogene (originally
isolated from human bladder carcinoma) were used as a model to
study the potential benefit of NaPA treatment to high risk
individuals, in which predisposition is associated with oncogene
activation. Cell treatment with NaPA was initiated 24-48 hr after
oncogene transfer. Results, scored 14-21 days later, showed
dose-dependent reduction in the formation of ras-transformed foci
in cultures treated with NaPA. Molecular analyses indicated that
the drug did not interfere with oncogene uptake and transcription,
but rather prevented the process of neoplastic transformation. The
effect was reversible upon cessation of treatment. In treated
humans, however, the fate of the premalignant cells may be
substantially different due to involvement of humoral and cellular
immunity (see below).
2. Prevention of tumor progression by genotoxic chemotherapy
Current approaches to combat cancer rely primarily on the use of
chemicals and radiation, which are themselves carcinogenic and may
promote recurrences and the development of metastatic disease. One
example is the chemotherapeutic drug 5-aza-2' deoxycytidine
(5Azadc). While this drug shows promise in treatment of some
leukemias and severe inborn anemias, the clinical applications have
been hindered by concerns regarding toxicity and carcinogenic
effects. Our data indicate that NaPA can prevent tumor progression
induced by 5azadC.
The experimental model involved non malignant 4C8a10 cells
(revertants of Ha-ras-transformed NIH3T3 fibroblasts). Transient
treatment of the premalignant cells with 5AzadC resulted in
malignant conversion evident within 2 days, as determined by cell
morphology, loss of contact inhibition and anchorage dependent
growth in culture, acquired invasive properties and tumorigenicity
in recipient athymic mice. Remarkably, NaPA prevented the
development of the malignant phenotype in the 5AzadC treated
cultures.
TABLE 1 ______________________________________ Tumor
Formation.sup.a Growth Treatment Incidence Size (mm) on
matrigel.sup.b ______________________________________ None 3/8 1
(0.5-2) - 5Azadc (0.1 uM) 8/8 11.5 (4-19) + NaPA (1.5 mg/ml) 0/8 -
5Azadc + NaPA (0.1 uM) (1.5 mg/ml) 0/8 0 -
______________________________________ .sup.a Cells pretreated in
culture were injected s.c. (5x10.sup.5 cells per site) into 3 month
old female athymic nude mice (Division of Cancer Treatment, NCI
animal Program, Frederick Cancer Research Facility). Results
indicate the incidence (tumor bearing/injected animals), as well as
tumor size as mean (range), determined after 3 weeks. .sup.b Cells
were plated on top of matrigel (reconstituted basement membrane)
and observed for malignant growth pattern, i.e., active
replication, development of characteristic processes, and
invasion.
Anticipated Activity in Humans.
In terms of cancer prevention, the beneficial effect of NaPA humans
may be even more dramatic than that observed with the experimental
models. In humans, NaPA is known to deplete circulating glutamine,
an aminoacid critical for the development and progression of
cancer. The enzymatic reaction leading to glutamine depletion takes
place in the liver and kidney; it is not clear whether or not
glutamine depletion occurs in the cultured tumor cells. Moreover,
molecular analysis revealed that NaPA can induce the expression of
histocompatibility class I antigens, which are localized on the
surface of tumor cells and affect the immune responses of the host.
While the therapeutic benefit of NaPA observed in cultures is in
some cases reversible upon cessation of treatment, in patients the
tumor cells might eventually be eliminated by the immune system.
Even if chemoprevention will require continuous treatment with
NaPA, this would be acceptable considering the lack of
toxicity.
Pharmaceutical compositions containing phenylacelate have been
shown to cause reversal of malignancy and to induce differenciation
of tumor cells. To demonstrate the capacity of drugs to induce
differentiation of tumor cells, three in vitro differentiation
model systems were used. (See sections A and D herein) The first
system used a human promyelocytic leukemia cell line HL-60. This
cell line represents uncommitted precursor cells that can be
induced to terminally differentiate along the myeloid or monocytic
lineage. In the second system, immortalized embryonic mesenchymal
C3H 10T1/2 cells were used which have the capabilities of
differentiating into myocytes, adipocytes, or chondrocytes. In the
third system, human erythroleukemia cells (K562) were used which
can be induced to produce hemoglobin.
EXAMPLE 1
Referring now to the data obtained using the first system, the
results of which are illustrated in FIG. 1, logarithmically growing
HL-60 [--.largecircle.--] and 10T1/2 [--.largecircle.--] cells were
treated for four days with NaPA [++] or phenylacetylglutamate (PAG)
[- - -]. The adherent cells were detached with trypsin/EDTA and the
cell number determined using a hemocytometer. Data points indicate
the mean.+-.S.D. of duplicates from two independent experiments.
The cell lines were obtained from the American Type Culture
Collection and maintained in RPMI 1640 (HL-60) or Dulbecco's
Modified Eagle's Medium (10T1/2) supplemented with 10% heat
inactivated fetal calf serum (Gibco Laboratories), 2 mM
L-Glutamine, and antibiotics. PAA (Sigma, St. Louis Mo.) and PAG
were each dissolved in distilled water, brought to pH 7.0 by the
addition of NaOH, and stored in -20.degree. C. until used. As
demonstrated in FIG. 1, NaPA treatment of the HL-60 and 10T1/2
cultures was associated with dose dependent inhibition of cell
proliferation.
EXAMPLE 2
To further evaluate the effectiveness of NaPA as an inducer of
tumor cell differentiation, the ability of NaPA to induce
granulocyte differentiation in HL-60 was investigated. The ability
of cells to reduce nitroblue tetrazolium (NBT) is indicative of
oxidase activity which is characteristic of the more mature forms
of human bone marrow granulocytes. NBT reduction thus serves as an
indicator of granulocyte differentiation. In FIG. 2, the number of
NBT positive cells was determined after 4 days [solid bars] or 7
days [hatched bar] of treatment. NaPA (h), 1.6 mg/ml; NaPA (1), 0.8
mg/ml. 4-hydroxyphenylacetate (4HPA) and PAG were used at 1.6
mg/ml. Potentiation by retinoic acid (RA) 10 nM was comparable to
that by interferon gamma 300 IU/ml. The direction of
differentiation towards granulocytes in cultures treated with NaPA,
whether used alone or in combination with RA, was confirmed by
microscopic evaluation of cells stained with Wright Stain and the
lack of nonspecific esterase activity. The effect of acivicin (ACV)
1 ug/ml was similar to 6-diazo-5-oxo-L-norleucine (DON) 25 ug/ml.
Glutamine starvation (Gln, <0.06 mM) was as described. Cell
viability determined by trypan blue exclusion was over 95% in all
cases, except for DON and ACV which were 75% and 63%, respectively.
DON, ACV and HPA are glutamine antagonists. As illustrated in FIG.
2, it is clear that NaPA is capable of inducing granulocyte
differentiation in HL-60. As further illustrated in FIG. 2,
differentiation of HL-60, assessed morphologically and
functionally, was sequential and could be further enhanced by the
addition of low doses of retinoic acid (RA, 10 nM) or interferon
gamma (300 IU/ml). After seven days of NaPA treatment, or four
days, when combined with RA, the HL-60 cultures were composed of
early stage myelocytes and metamyelocytes (30-50%), as well as
banded and segmented neutrophils (30-40%) capable of NBT.
Pharmacokinetics studies in children with urea cycle disorders
indicate that infusion of NaPA 300-500 mg/kg/day, a well tolerated
treatment, results in plasma levels of approximately 800 ug/ml.
Brusilow, S. W. et al. Treatment of episodic hyperammonemia in
children with inborn errors of urea synthesis. The New England
Journal of Medicine. 310:1630-1634 (1984). This same concentration
was shown to effectively induce tumor cell differentiation in the
present experimental system.
EXAMPLE 3
That NaPA is capable of inducing adipocyte conversion in 10T1/2
cultures is illustrated in FIG. 3. The results in FIG. 3 show that
differentiation was dose and time-dependent, and apparently
irreversible upon cessation of treatment. NaPA at 800 ug/ml was
quite efficient and totally free of cytotoxic effect. In the 10T1/2
model, adipocyte conversion involved over 80% of the cell
population. It was noted that higher drug concentrations further
increased the efficiency of differentiation as well as the size of
lipid droplets in each cell.
It is known that glutamine conjugation by NaPA is limited to humans
and higher primates and that in rodents NaPA binds glycine. [James,
M. O. et al. The conjugation of phenylacetic acid in man, sub-human
primates and some nonprimate species. Proc. R. Soc. Lond. B.
182:25-35 (1972). Consequently, the effect of NaPA on the mouse
10T1/2 cell line could not be explained by an effect on glutamine.
In agreement, neither glutamine starvation nor by treatment with
glutamine antagonists such as DON and ACV resulted in adipocyte
conversion.
EXAMPLE 4
TABLE 2 ______________________________________ Phenylacetate and
Derivatives: Induction of cellular differentiation in premalignant
10T1/2 cells Compounds DC50* Differentiation (sodium salts) at 1 mM
(mM) (%) ______________________________________ Phenylacetate 65
0.7 1-naphthylacetate >95 <0.1 3-chlorophenylacetate 80 0.5
4-chlorophenylacetate 50 1.0 2,6-dichlorophenylacetate 75 0.5
4-fluorophenylaceatae 65 0.7 ______________________________________
*DC50, concentration of compound causing 50% differentiation
Potential clinical use of phenylacetate and derivatives
As shown in the table, phenylacetate and its derivatives
efficiently induced lipid accumulation and adipocyte (fat cell)
differentiation in premalignant cells. This and other results
indicate that the tested compounds might be of value in:
1. Cancer prevention. Non replicating, differentiated tumor cells
are not likely to progress to malignancy.
2. Differentiation therapy of malignant and phathological
nonmalignant conditions.
3. Treatment of lipid disorders, in which patients would benifit
from increased lipid accumulation.
4. Wound healing. This is indicated by the ability of phenylacetate
to induce collagen synthesis in fibroblasts (shown in FIG. 13).
It is known that studies in plants reveal that NaPA can interact
with intracellular regulatory proteins and modulate cellular RNA
levels. In an attempt to explore the possible mechanism of action,
Northern blot analysis of HL-60 and 10T1/2 cells was performed
according to conventional methods. As shown in FIG. 4a, cytoplasmic
RNA was extracted, separated and analyzed (20 .ltoreq.g/lane) from
confluent cultures treated for 72 hrs with NaPA or PAG (mg/ml); C
is the untreated control. The aP2 cDNA probe was labeled with
[32P]dCTP (NEN) using a commercially available random primed DNA
labeling kit. Ethidium bromide-stained 28S rRNA indicates the
relative amounts of total RNA in each lane.
The results of the Northern blot analysis of HL-60 and 10T1/2
cells, showed marked changes in gene expression shortly after NaPA
treatment. Expression of the adipocyte-specific aP2 gene was
induced within 24 hrs in treated 10T1/2 confluent cultures reaching
maximal mRNA levels by 72 hrs.
EXAMPLE 5
In HL-60, cell transformation has been linked to myc amplification
and overexpression, and differentiation would typically require
down regulation of myc expression. [Collins, S. J. The HL-60
promyelocytic leukemia cell line: Proliferation, differentiation,
and cellular oncogene expression. Blood. 70:1233-1244 (1987)]. To
demonstrate the kinetics of myc inhibition and HLA-A induction,
Northern blot analysis of cytoplasmic RNA (20 ug/lane) was carried
out on cells treated with NaPA and PAG for specified durations of
time and untreated controls (-). Two concentrations of NaPA, 1.6
mg/ml (++) and 0.8 mg/ml (+), and PAG at 1.6 mg/ml was
investigated. The 32P-labeled probes used were myc 3rd exon (Oncor)
and HLA-A3 Hind III/EcoRI fragment. NaPA caused a rapid decline in
the amounts of myc mRNA. This occurred within 4 hours of treatment,
preceding the phenotypic changes detectable by 48 hrs,
approximately two cell cycles, after treatment. Similar kinetics of
myc inhibition have been reported for other differentiation agents
such as dimethyl sulfoxide, sodium butyrate, bromodeoxyuridine,
retinoids, and 1,25-dihydroxyvitamin D3. The results suggest that
down regulation of oncogene expression by NaPA may be responsible
in part for the growth arrest and induction of terminal
differentiation. In addition, NaPA treatment of the leukemic cells
was associated with time- and dose-dependent accumulation of HLA-A
mRNA coding for class I major histocompatibility antigens. It is
believed that this may enhance the immunogenicity of tumors in
vivo.
EXAMPLE 6
Further support for the use of NaPA as a non-toxic inducer of tumor
cell differentiation was found in the ability of NaPA to promote
hemoglobin biosynthesis in erythroleukemia cells. It is known that
K562 leukemic cells have a nonfunctional betta globin gene and,
therefore, do not normally make much hemoglobin. When K562 human
erythroleukemia cells were grown in the presence of NaPA at 0.8 and
1.6 mg/ml concentrations, hemoglobin accumulation, a marker of
differentiation, was found to increase 4-9 fold over the control
cells grown in the absence of NaPA. Hemoglobin accumulation was
determined by Benzidine staining of cells for hemoglobin and direct
quantitation of the protein.
It has been shown that high concentrations of NaPA inhibit DNA
methylation in plants. [Vanjusin, B. J. et al. Biochemia 1,46:47-53
(1981)]. Alterations in DNA methylation can promote oncogenesis in
the evolution of cells with metastatic capabilities. [Rimoldi, D.
et al. Cancer Research. 51:1-7 (1991)]. These observations raised
some concerns regarding potential long-term adverse effects with
the use of NaPA. To determine the potential tumorigenicity of NaPA,
a comparative analysis was performed using NaPA and a known
hypomethylating agent 5-aza-2'-deoxycytidine (5AzadC).
Premalignant cells (3-4.times.105) were plated in 75 cm2 dishes and
5AzadC 0.1 uM was added to the growth medium at 20 and 48 hrs after
plating. The cells were then subcultured in the absence of the
nucleoside analog for an additional seven weeks. Cells treated with
NaPA at 1.6 mg/ml were subcultured in the continuous presence of
the drug. For the tumorigenicity assay, 4-5 week-old female athymic
nude mice were inoculated s.c. with 1.times.106 cells and observed
for tumor growth at the site of injection.
The results set forth in Table 1 show that NaPA, unlike the
cytosine analog, did not cause tumor progression.
TABLE 3 ______________________________________ Tumorigenicity of
C3H 10T1/2 Cells in Athymic Mice Tumors Treatment Incidence Time
(positive/ Diameter (weeks) injected mice) (mm .+-. - S.D.)
______________________________________ None 0/8 0 5 AzadC 8/8 5.5
.+-. 2.5 NaPA 0/8 0 ______________________________________
The transient treatment of actively growing 10T1/2 cells with
5AzadC resulted in the development of foci of neoplastically
transformed cells with a frequency of about 7.times.10-4. These
foci eventually became capable of tumor formation in athymic mice.
By contrast, actively replicating 10T1/2 cultures treated for seven
weeks with NaPA, 800-1600 ug/ml, differentiated solely into
adipocytes, forming neither neoplastic foci in vitro nor tumors in
recipient mice.
Furthermore, experiments have demonstrated that NaPA can prevent
spontaneous or 5AzadC-induced neoplastic transformation, thus
suggesting a potential role in cancer prevention. It is known that
the treatment of premalignant 4C8 and 10T1/2 cells with carcinogens
such as 5AzadC produces malignant conversion of the respective
cells. When 4C8 [Remold: et al., Cancer Research, 51:1-7 (1990)]
and 10T1/2 cells were exposed to 5AzadC, malignant conversion was
evident in two days and two weeks, respectively. NaPA (0.8-1.6
mg/ml) prevented the appearance of the malignant phenotype, as
determined by cell morphology, contact inhibition and anchorage
dependent growth in culture. Additionally, see section B,
herein.
EXAMPLE 7
The K562 erythroleukemia line serves as a model for inherited
anemias that are associated with a genetic defect in the beta
globin gene leading to severe B-chain hemoglobinopathies.
The results reported in Table 3 also show that there is a
synergistic affect when leukemia cells are exposed NaPA in
combinaiton with interferon alpha, a known biological response
modifier or with the chemotherapeutic drug hydroxyurea (HU).
TABLE 4 ______________________________________ Induction of
Hemoglobin Synthesis in Erythroleukemia K562 cells BENZIDINE CELL
POSITIVE VIABILITY TREATMENT CELLS* (%) (%)
______________________________________ Control 1.8 >95 NaPA 0.8
mg/ 6.0 1.6 mg/ml 17.1 Interferon 500 IU/ml 13.5 HU 100 uM 17.2
NaPA (0.8 mg/ml) + HU or IFN 40-42
______________________________________ *Results at seven days of
treatment.
Analysis of gene transcripts showed accumulation of mRNA coding for
gamma globin, the fetal form of globin. This was confirmed at the
protein level.
Using the erythroleukemia K562 cell line described above it was
found that 4 hydroxyphenylacetate was as effective as NaPA in
inducing fetal hemoglobin accumulation, but was less inhibitory to
cell proliferation. In contrast, some other analogs such as 2,4- or
3,5-dihydroxyphenylacetate were found to be highly toxic (Please
see section III, herein for further discussion).
EXAMPLE 8
The effectiveness of NaPA as an antitumor agent was further
evaluated in a variety of experimental models. Studies of depth
were performed with two androgen- independent human prostate
adenocarcinoma cell lines, PC3 and DU145, established from bone and
brain metastases, respectively. NaPA treatment of the prostatic
cells resulted in concentration-dependent growth arrest,
accompanied by cellular swelling and accumulation of lipid. The
results of this study are shown in FIG. 4. As illustrated therein,
an IC50 for NaPA occurred at 600-800 ug/ml. Significantly higher
doses were needed to affect the growth of actively replicating
normal human FS4 skin fibroblasts, indicating a selective
cytostatic effect of the drug.
EXAMPLE 9
It is known that PC3 cells are invasive in vitro and metastatic in
recipient athymic mice. [Albini, A. et al. A rapid in vitro assay
for quantitating the invasive potential of tumor cells. Cancer Res.
47:3239-3245 (1987)]. The invasiveness of PC3 cells which is
indicative of their malignant phenotype can be assessed by their
ability to degrade and cross tissue barriers such as matrigel, a
reconstituted basement membrane. Untreated PC3 cells and PC3 cells
treated with NaPA for 4 days in culture were quantitatively
analyzed in a modified Boyden chamber containing a matrigel-coated
filter with FS4 conditioned medium as a chemoattractant. After 4
days of treatment with 800 ug/ml of NaPA in T.C. plastic dishes,
5.times.10.sup.4 cells were replated onto 16 mm Costar dishes
coated with 250.ltoreq.1 of matrigel. Pictures of controls taken
after 1 and 8 days, show the characteristic growth pattern of
untreated cells, i.e., formation of net-like structures composed of
actively replicating cells which eventually degraded the matrigel
and formed monolayers on the plastic surface beneath. In contrast
to the controls, the NaPA treated cells formed isolated small
colonies which resembled normal human FS4 cells as shown in C and D
taken 8 days after plating. The NaPA treated cells failed to
degrade the matrigel barrier. The formation of small noninvasive
colonies on top of the matrigel is indicative of loss of malignant
properties following treatment. Results of the in vitro invasion
assays correlate highly with the biological behavior of cells in
vivo.
EXAMPLE 10
Indeed, PC3 cells treated with NaPA for one week in culture, in
contrast to untreated cells or those treated with PAG, failed to
form tumors when transplanted s.c. into athymic mice. These results
are shown in Table 5.
TABLE 5 ______________________________________ Tumorigenicity of
Prostatic PC3 Cells in Nude Mice. TUMORS TREATMENT Incidence
Diameter Weight (mg/ml) (mm .+-. S.D.) (mg)
______________________________________ None 7/7 9 .+-. 3 285 .+-.
60 NaPA 0.8 1/7 2 50 PAG 0.8 3/4 8 .+-. 2 245 .+-. 35
______________________________________
PC3 cells were pretreated for 1 week in culture and then injected
(2.times.10.sup.5 cells/animal) s.c. into 4-5 week-old female
athymic nude mice. The results in Table 5 indicate the incidence of
tumor bearing animals/injected animals as well as tumor size
measured as mean diameter .+-.S.D. 8 weeks later. The data in Table
4 are a summary of two independent experiments.
EXAMPLE 11
To further substantiate the phenotypic changes observed in the NaPA
treated prostatic PC3 cells, Northern blot analysis revealed that
NaPA inhibited the expression of collagenase type IV, one of the
major metalloproteases implicated in degradation of basement
membrane components, tumor cell invasion, and metastasis.
Furthermore, it was found that NaPA treated prostatic PC3 cells
showed an increase in the level of HLA-A mRNA which codes for major
histocompatibility class I antigen known to affect tumor
immunogenicity in vivo.
NaPA in Combination with Suramin
TABLE 6 ______________________________________ Malignant Melanoma
A375 Treatment Growth Viability (ug/ml) (% of control) (%)
______________________________________ None 100 >95 NaPA 400
63.3 >95 Suramin 38 78.3 >95 75 56.8 >95 150 38.6 92 300
26.6 82 NaPA (400) + Suramin (38) 45.5 >95 + Suramin (75) 30.1
94 + Suramin (150) 21.8 92
______________________________________
TABLE 7 ______________________________________ Prostate
Adenocarcinoma PC3 Treatment Growth Viability (ug/ml) (% of
control) (%) ______________________________________ None 100 >95
NaPA 800 59.6 >95 Suramin 75 58.5 nd 150 46.5 nd 300 31.0 nd
NaPA (800) + Suramin (75) 24.2 90 + Suramin (150) 10.9 64
______________________________________
NaPA was found to significantly potentiate the therapeutic effect
of suramine, the only experimental drug known to be active against
prostrate cancer.
It is known that a disease state characterized by the presence of
benign hyperplastic lesions of the prostate exists as a separate
disease entity and has been identified in many patients that
progress to a diagnosis of prostatic cancer. Based on the above, it
is anticipated that NaPA, in addition to being effective in the
treatment of prostatic cancer, would be effective in treating
patients having benign hyperplastic prostatic lesions.
Further experiments demonstrated that NaPA appears to have broad
antitumor activity affecting a wide spectrum of malignancies. The
experimental data indicate presented in Table 5 that NaPA 0.4-0.8
mg/ml caused about 50% inhibition of growth in treated
adenocarcinoma of the prostate cell lines PC3 and DU145, melanoma
A375 and SK MEL 28, lung adenocarcinoma H596 and H661, and
astrocytoma U87, U373, and 343. Somewhat higher concentrations
(1.0-1.5 mg/ml) were needed to cause a similar inhibition of
squamous cell carcinoma A431, breast tumor MCS-7, osteosarcoma
KRIB, and fibrosarcoma V7T. Typically, NaPA treatment was
associated with growth arrest, induction of differentiation
markers, reduced invasiveness in vitro, and loss of tumorigenicity
in nude mice.
TABLE 8 ______________________________________ RESPONSES OF
DIFFERENT TUMOR CELL LINE TO NaPA TREATMENT % Inhibition by # Tumor
Cell Line NaPA 0.8 mg/ml a ______________________________________ 1
Melanoma A375 .gtoreq.70 SK MEL 28 >50 2 Prostatic Ca b PC3
.gtoreq.50 Du145 .gtoreq.50 LaNCoP >50 3 Astrocytoma U87
.gtoreq.50 U343 .gtoreq.50 U373 .gtoreq.50 4 Kaposi's Sarcoma KS
.ltoreq.40 5 Leukemia HL-60 .ltoreq.40 6 Leukemia K562 .ltoreq.30 7
Breast Ca. MCF-7 .ltoreq.30 8 Osteosarcoma KRIB .ltoreq.30 HOS
<20 9 Fibrosarcoma V7T .ltoreq.30 RS485 .ltoreq.30 10 Squamous
Ca. of Head & Neck A431 <30
______________________________________ a Pharmacologically
attainable concentration b Carcinoma
Of major interest in Table 4 are the following:
#1-3 Tumor cells show significant response i.e., .gtoreq.50
inhibition of proliferation within one week of treatment.
#4 KS, an HIV-associated disorder, may be more dramatically
affected by NaPA in humans, due to inhibition of HIV expression and
of essential growth factors released by infected lymphocytes.
#5,6 The treated HL-60 promeyelocytic leukemic cells undergo
terminal differentiation, a desirable outcome of chemotherapy. In
the K562 erythroleukemia, NaPA induced reversible erythroid
diferentation with no significant growth arrest (<30); thus the
K562 data is of interest with respect to treatment of certain
anemias, not cancer.
Less attractive:
#7-10 For effective responses, the tumors may require much higher
drug concentrations if used alone.
Although some of the malignant cell lines seem more sensitive than
others, all were significantly more affected by NaPA when compared
to normal or benign cells. For example, NaPA inhibited the growth
of malignant osteosarcoma (KRIB) cells more so than benign
osteosarcoma-derived HOS cells. A differential effect was seen also
in ras-transformed fibrosarcoma V7T, when compared to the parental
non-tumorigenic N1H3T3 cells. As to normal human cells, as much as
2-4 mg/ml of NaPA were needed to cause a significant inhibition of
growth to primary human skin FS4 fibroblasts. It should be noted
that the treatment was not toxic to either the malignant or the
normal cells.
The concentration range found to selectively suppress malignant
growth can be readily obtained in the clinical setting without
causing significant side effects. Intravenous infusion of humans
with NaPA at 250-500 mg/kg/day which results in plasma levels of
600-800 ug/ml has been found to be a well tolerated treatment.
Cytotoxicity in tissue culture was observed when the NaPA
concentration was >3 mg/ml.
SECTION B
PHENYLACETATE AND ITS DERIVATIVES IN THE TREATMENT AND PREVENTION
OF AIDS
The etiology of human acquired immunodeficiency syndrome (AIDS) has
been linked to the human immunodeficiency virus (HIV), which is
capable of selective infection and suppression of the host immune
system. The immune defect renders the human body susceptible to
opportunistic infections and cancer development, which are
ultimately fatal. The spread of HIV throughout the world is rapid,
with no effective therapeutics on hand. It is suggested that NaPA,
a nontoxic natural compound capable of glutamine depletion in vivo,
could potentially be used in the treatment and prevention of
AIDS.
HIV is a retrovirus. The production of retroviruses is dependent on
transcriptional activation by the long terminal repeat (LTR)
element, and the availability of glutamine (Gln) for translational
control. Experimental data obtained with chronically infected
cultured cells and animal models indicate that virus replication is
inhibited specifically in cells starved for glutamine, but not for
other amino acids (Gloger and Panet (1986); (J. Gen. Virol.
67:2207-2213) Roberts and McGregor, (1991), (J. Gen. Virol
72:2199-305). The results could not be attributed to either an
effect on cell cycle or a general inhibition of protein
synthesis.
The reason why glutamine depletion leads to virus suppression cab
be explained as follows. Replication competent murine retroviruses
contain an amber termination codon at the junction of gag and pol
genes, which can be recognized by amber suppressor tRNA.sup.Gln.
Glutamine is thus essential for the readthrough of viral mRNA
transcripts (Yoshinaka et al (1985)); PNAS 82:1618-1622 reduction
in glutamine concentrations disrupts viral mRNA translational
readthrough and protein synthesis, with subsequent inhibition of
viral assembly and secondary spread. Although human retroviruses
are somewhat different from the murine viruses studied, it has been
shown that reduction in the levels of amber suppressor tRNA.sup.Gln
in human cells infected with HIV causes a significant reduction in
the synthesis of viral proteins [(Muller et al Air Research and
Human Retroviruses 4:279-286 1988)]. Such data suggest that agents
which can lower glutamine levels in humans, are likely to benefit
patients infected with HIV. NaPA may be such as agent, since it is
known to conjugate to glutamine in humans with subsequent renal
excretion of phenylacetylglutamine. Since NaPA possesses also
antitumor activities, the drug is likely to affect Kaposi's
sarcomas, the tumors found in as many as 30% of all AIDS patients,
as well as lymphoma associated with AIDS.
EXAMPLE 13
Evidence from experimental model systems in support the above
hypotheses include: (a) Our preliminary findings with cultured
cells indicate that NaPA can inhibit expression of genes controlled
by the retroviral LTR; (b) While animal studies have been hindered
by the fact that glutamine depletion by NaPA is limited to humans
and high primates, an acceptable animal model (other than primates)
involves rodents treated with glutaminase. Glutaminase is a
bacterial enzyme that causes reduction of extracellular (and
presumably intracellular) glutamine concentrations. Glutaminase
treatment of viremic mice infected with Rouscher murine leukemia
virus (RLV) inhibited retroviral replication and the development of
splenomegaly, and significantly increased animal survival [Roberts
and McGregor J. Gen. Virology 72:299-305 (1991)]. The efficacy of
glutaminase therapy compared favorably with AZT, the drug currently
used for treatment of AIDS. The results are of particular interest
since the RLV serves as a model in the search for anti-HIV drugs
(Ruprecht et al, 1986). Unfortunately, however, glutamine depletion
by glutaminase in vivo is only transient due to development of
neutralizing antibodies to the enzyme; once this occurs, viral
replication can resume, eventually killing the host. NaPA, unlike
the bacterial glutaminase, is a natural component of the human
body, and thus is less likely to induce the production of
neutralizing antibodies; (c) There is clinical evidence for
sustained reduction by NaPA of plasma glutamine concentrations.
NaPA is currently being used for treatment of hyperammonemia
associated with inborn disorders of urea metabolism. The clinical
experience indicate that long-term treatment with NaPA effectively
reduces glutamine levels. Such treatment is nontoxic and well
tolerated even by newborns. In conclusion, we propose that NaPA
might benefit patients with HIV infection. NaPA could inhibit viral
replication through (among other mechanisms) inhibition of LTR and
depletion of glutamine, the aminoacid required for appropriate
processing of viral proteins. If NaPA proves to have anti-HIV
activities in humans, it could be used to prevent disease
progression in asymptomatic HIV-positive individuals. The lack of
toxicity, easy oral administration and relatively low cost,
uniquely qualify NaPA as a chemopreventive drug. In fact, the drug
is so well tolerated by humans that treatment can start just a few
hours after birth. In addition, NaPA could be used (alone or in
combination with other drugs) in treatment of AIDS-associated
disorders including opportunistic infections, HIV encephalopathy,
and neoplasia.
SECTION C
INDUCTION OF FETAL HEMOGLOBIN SYNTHESIS IN .beta.-CHAIN
HEMOGLOBINOPATHY BY PHENYLACETATE AND ITS DERIVATIVES
There is considerable interest in identifying nontoxic therapeutic
agents for treatment of severe .beta.-chain hemoglobinopathies.
Employing the human leukemic K562 cell line as a model, we have
explored the cellular responses to NaPA, an amino acid derivative
essentially nontoxic to humans. Treatment of cultures with
pharmacologically attainable concentrations of NaPA resulted in
time- and dose dependent inhibition of cell proliferation and
caused an increase in hemoglobin production. Molecular analysis
revealed accumulation of the fetal form of hemoglobin (HbF), which
was associated with elevated steady-state levels of gamma globin
mRNA. All NaPA effects reversed upon cessation of treatment.
Interestingly, addition of NaPA to other antitumor agents of
clinical interest, i.e., 5-azacytidine and hydroxyurea, resulted in
superinduction of HbF biosynthesis. The results suggest that NaPA,
an agent known to be well tolerated by newborns, could be used
alone or in combination with other drugs for long-term treatment of
some inborn blood disorders. The pathophysiology of inherited blood
disorders such as sickle cell anemia and severe .beta.-thalassemias
is based on genetic abnormalities in the .beta.-globin gene which
result in deficient or absent .beta.-globin synthesis. The latter
prevents the production of hemoglobin and results in ineffective
red blood cell production and circulation. Recent data indicate
that pharmacological manipulation of the kinetics of cell growth
and differentiation might have beneficial effect in patients with
the .beta.-chain hemoglobinopathies, due to the induction of fetal
hemoglobin (HbF) synthesis. To date, several antitumor drugs
including 5-azacytidine (5AzaC), 5-aza-2'-deoxycytidine (5AzadC),
hydroxyurea (HU), vinblastine, and arabinosylcytosine (ara-C) have
been shown to increase the production of HbF in experimental models
[Dover, Ann NY Acad Sci 612:184-190 (1990)]. Moreover, there is
clinical evidence for 5AzaC and HU activity in sever
.beta.-thalassemia and sickle cell anemia, respectively. However,
concerns regarding toxic and potential carcinogenic effects of the
prevailing antitumor drugs raise the need to identify safe
alternatives for long-term treatment of the inborn nonmalignant
diseases. The accumulation of fetal hemoglobin in adults is thought
to be due to changes in the kinetics of erythroid differentiation
rather than a direct effect on the fetal globin genes. According to
this hypothesis, other agents that can induce differentiation would
also be expected to affect HbF production. The focus here is on the
efficacy of a novel nontoxic differentiating agent, sodium
phenylacetate (NaPA).
As discussed in Section A Applicant's laboratory has found that
NaPA can also affect the maturation (i.e., differentiated state) of
various animal and human cell types. The drug caused growth arrest
and reversal of malignant properties in a variety of in vitro tumor
models including cell lines established from adenocarcinomas of the
prostate and lung, malignant melanomas, and astrocytomas. Moreover,
NaPA treatment was associated with adipocyte conversion in
premalignant mesenchymal C3H 10T1/2 cells, and granulocyte
differentiation in promyelocytic leukemia HL-60 cultures. Studies
indicated that NaPA, in contrast to the chemotherapeutic
differentiating drugs 5AzaC and 5AzadC, may be free of adverse
effects such as cytotoxicity and tumor progression.
Indeed, NaPA is well tolerated by humans as indicated by the vast
clinical experience with NaPA is in the treatment of hyperammonemia
in infants with inborn errors of ureagenesis. The clinical
experience indicates that acute or long-term treatment with high
doses of NaPA is essentially free of adverse effects. The lack of
toxicity and the ability to induce cellular differentiation
prompted Applicant to examine the effect of NaPA on HbF
expression.
EXAMPLE 14
The experimental system involved the human leukemic K562 cells,
which carry a nonfunctional .beta.-globin gene, but produce low
levels of the fetal gamma globin and of HbF. The K562 cell line was
originally established from a patient with chronic myelogenous
leukemia in the blast cells transformation, and has since been
extensively utilized as a model in studies of erythroid
differentiation and regulation of the gamma globin gene expression.
We show here for the first time that pharmacologically attainable
concentrations of NaPA can promote HbF biosynthesis in the human
leukemic cells, and can cause superinduction when combined with the
other chemotherapeutic agents of interest, 5AzaC and HU.
MATERIALS AND METHODS
Cell Culture and reagents. The human leukemia K562 cells were
maintained in RPMI 1640 medium supplemented with 10%
heat-inactivated fetal calf serum (Gibco), 50 U/ml penicillin, 50
ug/ml streptomycin, and 2 mM L-glutamine unless otherwise
indicated. The suspension cultures were kept in exponential growth
phase by diluting every 3-5 days with fresh medium, and cell
viability was determined by trypan blue exclusion. Phenylacetic
acid, 4-hydroxyphenyl acetic acid, 3,4-dihydroxyphenyl acetic acid,
2,5-dihyroxyphenyl acetic acid (Sigma, St. Louis Mo.) and PAG (a
gift from L. Trombetta, Houston Tex.) were dissolved in distilled
water, and brought to pH 7.0 by the addition of NaOH, DON,
acivicin, 5AzadC, 5AzaC, and HU (Sigma) were also dissolved in
distilled. All drug stock solutions were stored in aliquots at
-20.degree. C. until used.
Determination of Hemoglobin Production. K562 cells were seeded at
1.times.10.sup.5 cells/ml and treated with the drugs for four to
seven days prior to assay. Qualitative estimation of hemoglobin
production was determined by benzidine staining of intact cells in
suspension. The hemoglobin concentration within cells was
determined by the protein absorption at 414 nm (). Briefly,
1.times.10.sup.7 cells were lysed in 1 ml of lysing buffer (0.12%
Tris pH 7.4, 0.8% NaCl, 0.03% Mg-acetate, and 0.5% Np-40), vortexed
and incubated on ice gor 15 minutes. The lysate were then
centrifuged for 15 minutes at 1500 rpm at 4.degree. C., and the
absorption of the supernatant monitored between 350 nm and 650 nm
using Beckman Du-7 scanning spectrophotometer. The hemoglobin was
quantitated using the relationship of 1.0 optical density (OD) at
414 nm corresponding to 0.13 mg/ml hemoglobin as described
before.
Northern Blot Analysis and DNA probes. Cytoplasmic RNA was prepared
from cultures at logarithmic phase of growth and separated on 1%
agarose-formaldehyde gels. Gel electrophoresis, transfer of RNA
onto nytran membranes (Schleicher & Schuell), hybridization
with radiolabeled DNA probes, and autoradiography (Kodak X-ray film
XAR5) were according to established procedures. The probe for gamma
globin was a 0.6 Kb Eco RI/Hind III fragment of the human gamma
globin gene. Probes were labeled with [.sup.32 P]dCTP (NEN) using
random primed DNA labeling kit (Boehringer Mannheim, West
Germany).
Analysis of HbF Protein Synthesis. Newly synthesized proteins were
labeled with .sup.35 S-methionine and the HbF immunoprecipitated
and analyzed as previously described. Briefly, cells
(1.times.10.sup.6 per point in 1 ml) were first subjected to 1 hr
starvation in methionine-free medium, then incubated in the
presence of 100 uCi/ml of .sup.35 S-methionine for 2 hrs. The
labeled cells were harvested, washed and lysed in a lysing buffer
containing 10 mM phosphate buffer pH 7.4, 1% Triton X100, 0.1% SDS,
0.5% deoxychilate, 100 mM NaCl, 0>1% NaN3, 2 mM PMSF, and 10
ug/ml lenpeptin. 1.times.10.sup.7 cpm of TCA precipitable count of
cytoextract was incubated with rabbit anti-human HbF (Pharmacia)
and protein A Sepharose at 4.degree. C., and the immunoprecipitates
were separated by electrophoresis on 12% SDS-polyacrylamide
gels.
RESULTS
The Effect of NaPA and Analogues on Cell Growth and
Differentiation. Treatment of the K562 cultures with NaPA resulted
in dose dependent inhibition of cell proliferation, with 1.4 mg/ml
causing 50% reduction in cell number after four days of treatment.
No toxicity was observed with doses as high as 2.0 mg/ml. In
addition to the cytostatic effect, NaPA also induced erythroid
differentiation, as indicated by an increase in the number of
benzidine-positive cells (FIG. 5) and confirmed by quantitative
analysis of hemoglobin production (Table 9). Similar treatment with
PAG, which is the glutamine conjugated form of NaPA, had no
significant effect on either cell proliferation or hemoglobin
accumulation suggesting that the changes associated with NaPA
treatment are specific and not due to alterations in culture
conditions.
The effect of NaPA on cell growth and differentiation could be
mimicked by the use of 4-hydroxyphenylacetate (Table 10). This was
in marked contrast to the analogues 3,4-dihydroxyphenylacetate and
2,5-dihyroxyphenylacetate, which were highly toxic to the cells
(LD50 of 60 and 100 ug/ml, respectively), and did not induce
differentiation.
Regulation of Fetal Hemoglobin Production by NaPA. K562 cells
normally express low but detectable levels of HbF. Protein analysis
employing anti-HbF antibodies revealed significantly increased
amounts of HbF in cells treated with NaPA compared to untreated
controls; this was associated with elevated steady-state levels of
the fetal gamma globin mRNA. The effect of NaPA on HbF production
was time and dose dependent, and apparently reversible upon
cessation of treatment.
Glutamine Starvation and HbF Production. NaPA treatment of humans
can lead to depletion of circulating glutamine due to conjugation
to glutamine and formation of PAG, an enzymatic reaction known to
take place in the liver and kidney. The in vivo reduction in plasma
glutamine was mimicked in vitro by culturing the K562 cells in the
presence of lowered glutamine concentrations. Results presented in
Table 9 show, in agreement with previous reports, that glutamine
starvation alone can affect the growth rate as well as HbF
production in the K562 cells. Addition of NaPA to the
glutamine-depleted growth medium further augmented the cytostatic
and differentiating effects observed. We speculate therefore that
the effect of NaPA on erythroid differentiation and HbF production
in humans may be even more dramatic than that observed with the in
vitro model, due to depletion of circulating glutamine and a direct
effect on the erythroid progenitor cells.
Potentiation by NaPA of Erythroid Differentiation induced by Other
Chemotherapeutic Drugs. There is considerable interest in the use
of 5AzaC, 5AzadC and HU for treatment of sickle cell anemia and
.beta.-thalassemia; however, the clinical use of these drugs is
often limited by unacceptable toxicities. Combination treatments
with nontoxic differentiating agents like NaPA could enhance
hemoglobin production while minimizing the adverse effects. We
tested therefore the efficacy of various combinations of NaPA with
the other drugs of clinical interest. Results, summarized in Table
10, show that addition of NaPA 800 ug/ml, to low doses of 5AzadC or
HU act synergitically to further augment HbF production with no
toxic effect to cells. The concentration of HU used in these
experiments is comparable to the plasma HU levels measured in
sickle cell anemia patients following an oral administration of 25
mg/kg [(Goldberg et al. New England J Med 323:366-372 (1990)]. As
to NaPA, pharmacokinetics studies in children with urea cycle
disorders indicate that plasma levels of approximately 800 ug/ml
can be obtained by infusion with 300-500 mg/kg/day, a treatment
well tolerated even by newborns.
DISCUSSION
Chemotherapeutic agents selected for their low cytotoxic/mutagenic
potential could be used for induction of fetal hemoglobin in
patients with congenital sever anemias such as sickle cell and
.beta.-thalassemia. Drug toxicity is an important consideration in
view of overall health condition and the variable life-span of
patients with these nonmalignant blood disorders. Unfortunately,
recombinant human erythropoietin, which has proved to be both
nontoxic and effective therapy for anemia associated with chronic
renal disease, is apparently ineffective in the treatment of sickle
cell anemia. The application of other active drugs such as 5AzadC,
HU, vinblastine and ara-C has been hindered by concerns regarding
their carcinogenic effects. HU is also difficult to use because of
the narrow margin between toxicity and the desired effect on
increased HbF production [(Dover, et al., Blood 67:735-738 (1986)].
By contrast, NaPA, shown here to affect HbF production, is so well
tolerated by humans that treatment can be initiated just a few
hours after birth.
Using an in vitro model involving human leukemic K562 cells, we
have demonstrated that NaPA can promote the maturation of early
erythroid progenitor cells that have an active HbF program.
Addition of NaPA to other therapeutic agents currently in clinical
use, i.e., 5AzaC, 5AzadC, or HU resulted in superinduction of HbF
synthesis. 5AzaC has been shown to be less toxic and more effective
than HU in stimulating HbF production. Moreover, 5AzaC, unlike HU,
is effective in treatment of both sickle cell anemia and
.beta.-thalassemia. Such data are consistent with the
interpretation that 5AzaC acts by both perturbation of
erythropoiesis and by its effect on DNA methylation. However, while
hypomethylation can lead to gene activation and cell
differentiation, it can also promote oncogenesis and the evolution
of cells with metastatic capabilities. Our results obtained with
the K562 erythroid progenitor cells indicate that the therapeutic
effects of NaPA compare favorably with those of 5AzadC, yet NaPA
(unlike the cytosine analog) did not cause tumor progression.
Moreover, NaPA was shown to prevent tumor progression induced by
5AzadC.
The data presented here suggest that NaPA, used alone or in
combination with other drugs, might be of value in treatment of
leukemias and .beta.-chain hemoglobinopathies. In addition to
promoting the production of red blood cells expressing HbF through
nontoxic mechanisms, NaPA may also minimize the adverse effects of
other antitumor drugs currently in clinical use.
TABLE 9 ______________________________________ HbF Accumulation in
Treated K562 Cells Treatment (mg/ml) Benzidine Positive Cells HbF
production increase (%) fold increase (pg/cell) fold
______________________________________ None 2.2 .+-. 0.8 1 0.35
.+-. 0.06 1 NaPA 0.4 2.7 .+-. 0.2 1.2 0.49 .+-. 0.02 1.4 0.8 7.0
.+-. 0.3 3.2 1.15 .+-. 0.20 3.3 1.6 14.6 .+-. 0.2 6.6 2.40 .+-.
0.16 6.8 4HP 1.6 14.2 .+-. 0.5 6.45 ND PAG 2.6 2.1 .+-. 0.5 0.95
0.37 .+-. 0.03 1.06 ______________________________________
TABLE 10 ______________________________________ Glutamine
Starvation and HbF Production HbF (pg/cell) Gln starvation Gln (mM)
alone plus NaPA (0.8 mg/ml) ______________________________________
2.0 0.39 .+-. 0.04 1.0 .+-. 0.06 0.5 0.56 .+-. 0.01 1.15 .+-. 0.01
0.2.sup.a 1.17 .+-. 0.12 1.75 .+-. 0.22 0.1.sup.a 1.86 .+-. 0.40
2.22 .+-. 0.20 ______________________________________ .sup.a The
concentration of NaPA used in this study (0.8 mg/ml) is
pharmacologically attainable without toxicity. In children such a
treatment is expected to cause a drop in circulating glutamine
plasma levels to 0.1-0.2 mM. The results presented above indicate
that under suc conditions HbF production increases 4.5-5.7 fold
compared to controls. We propose therefore that the effect of NaPA
in children might be more dramatic then that seen under routine
culture conditions # (i.e., cell growth in medium with 2 mM
Gln).
TABLE 11 ______________________________________ Potentiation by
NaPA of HU's Therapeutic Effect Treatment HbF (pg/cell)
______________________________________ none 0.39 .+-. 0.04 NaPA
(0.8 mg/ml) 1.64 .+-. 0.07 HU (50 uM) 1.00 .+-. 0.03 HU (50 uM) +
NaPA 5.91 .+-. 0.6.sup.b HU (100 uM) 2.12 .+-. 0.04 HU (100 uM) +
NaPA 6.71 .+-. 0.05.sup.b ______________________________________
.sup.a To mimic the effect of NaPA in vivo, treatments involving
NaPA wer performed in medium supplemented with 0.2 mM Gln (see
explanation to tabl 2). Control untreated cells and those treated
with HU or 5AzadC alone wer maintained in growth medium with 2 mM
gln. .sup.b The results indicate that NaPA and HU act
synergistically to induc HbF Production in the erytroid progenitor
cells Note: Similar results have been obtained for the combination
NaPA 0.8 mg/ml and 5AzadC 0.3 uM.
Contemplated Models of Drug Administration
NaPA may be administered locally or systemically. Systemic
administration means any or route of administration which results
in effective levels of active ingredient appearing in the blood or
at a site remote from the site of administration of said active
ingredient.
The pharmaceutical formulation for systemic administration
according to the invention may be formulated for intravenous,
intramuscular, sub-cutaneous, oral, nasal, enteral, parenteral or
topical administration. In some cases, combination of types of
formulations may be used simultaneously to achieve systemic
administration of the active ingredient.
Suitable formulations for oral administration include hard or soft
gelatin capsules, dragees, pills, tablets, including coated
tablets, elixirs, suspensions, and syrups or inhalations.
Solid dosage forms in addition to those formulated for oral
administration include rectal suppositories.
The compounds of the present invention may also be administered in
the form of an implant.
Suitable formulations for topical administration include creams,
gels, jellies, mucilages, pastes and ointments.
Suitable injectable solutions include intravenous, subcutaneous,
and intramuscular injectable solutions. The compounds of the
present invention may also be administered in the form of an
infusion solution or as a nasal inhalation or spray.
The compounds of the present invention may also be used
concomitantly or in combination with selected biological response
modifiers, e.g., interferons, interleukins, tumor necrosis factor,
glutamine antagonists, hormones, vitamins, as well as anti-tumor
agents and hematopoetic growth factors, discussed above.
It has been observed that NaPA is somewhat malodorous. Therefore,
it may be preferable to administer this compound in the presence of
any of the pharmaceutically acceptable odor-masking excipients or
as its precursor phenylbutyrate which has no offensive odor.
The PAA and its pharmaceutically acceptable derivatives to be used
as antitumor agents can be prepared easily using pharmaceutical
materials which themselves are available in the art and can be
prepared by established procedures. The following preparations are
illustrative of the preparation of the dosage forms of the present
invention, and are not to be construed as a limitation thereof.
EXAMPLE 14
PARENTERAL SOLUTION
A sterile aqueous solution for parenteral administration containing
200 mg/ml of NaPA for treating a neoplastic disease is prepared by
dissolving 200 g. of sterilized, micronized NaPA in sterilized
Normal Saline Solution, qs to 1000 ml. The resulting sterile
solution is placed into sterile vials and sealed. The above
solution can be used to treat malignant conditions at a dosage
range of from about 100 mg/kg/day to about 1000 mg/kg/day. Infusion
can be continuous over a 24 hour period.
EXAMPLE 15
PARENTERAL SOLUTION
A sterile aqueous solution for parenteral administration containing
50 mg/ml of NaPA is prepared as follows:
______________________________________ Ingredients Amount
______________________________________ NaPA, micronized 50 g.
Benzyl alcohol 0.90% w/v Sodium chloride 0.260% w/v Water for
injection, qs 1000 ml ______________________________________
The above ingredients, except NaPA, are dissolved in water and
sterilized. Sterilized NaPA is then added to the sterile solution
and the resulting solution is placed into sterile vials and sealed.
The above solution can be used to treat a malignant condition by
administering the above solution intravenously at a flow rate to
fall within the dosage range set forth in Example 14.
EXAMPLE 16
PARENTERAL SOLUTION
A sterile aqueous solution for parenteral administration containing
500 mg/ml of sodium phenylbutyrate is prepared as follows:
______________________________________ Ingredients Amount
______________________________________ Sodium phenylbutyrate 500 g.
Dextrose 0.45% w/v Phenylmercuric nitrate 0.002% w/v Water for
injection, qs 1000 ml. ______________________________________
The preparation of the above solution is similar to that described
in Examples 14 and 15.
EXAMPLE 17
TABLET FORMULATION
A tablet for oral administration containing 300 mg of NaPA is
prepared as follows:
______________________________________ Ingredients Amount
______________________________________ NaPA 3000 g.
Polyvinylpyrrolidone 225 g. Lactose 617.5 g. Stearic acid 90 g.
Talc 135 g. Corn starch 432.5 g. Alcohol 45 L
______________________________________
NaPA, polyvinylpyrrolidone and lactose are blended together and
passed through a 40-mesh screen. The alcohol is added slowly and
the granulation is kneaded well. The wet mass is screened through a
4-mesh screen, dried overnight at 50.degree. C. and screened
through a 20-mesh screen. The stearic acid, talc and corn starch is
bolted through 60-mesh screen prior to mixing by tumbling with the
granulation. The resulting granulation is compressed into tablets
using a standard 7/16 inch concave punch.
EXAMPLE 18
TABLET FORMULATION
A tablet for oral administration containing 200 mg of sodium
phenylbutyrate is prepared as follows:
______________________________________ Ingredients Amount
______________________________________ Sodium phenylbutyrate 2240
g. Compressible sugar (Di-Pac) 934 g. Sterotex 78 g. Silica gel
(Syloid) 28 g. ______________________________________
The above ingredients are blended in a twin-shell blender for 15
minutes and compressed on a 13/22 inch concave punch.
EXAMPLE 19
INTRANASAL SUSPENSION
A 500 ml sterile aqueous suspension is prepared for intranasal
installation as follows:
______________________________________ Ingredients Amount
______________________________________ NaPA, micronized 30.0 g.
Polysorbate 80 2.5 g. Methylparaben 1.25 g. Propylparaben 0.09 g.
Deionized water, qs 500 ml
______________________________________
The above ingredients, with the exception of NaPA, are dissolved in
water and sterilized by filtration. Sterilized NaPA is added to the
sterile solution and the final suspensions are aseptically filled
into sterile containers.
EXAMPLE 20
OINTMENT
An ointment is prepared from the following ingredients:
______________________________________ Ingredients Amount
______________________________________ NaPA 10 g. Stearyl alcohol 4
g. White wax 8 g. White petrolatum 78 g.
______________________________________
The stearyl alcohol, white wax and white petrolatum are melted over
a steam bath and allowed to cool. The NaPA is added slowly to the
ointment base with stirring.
EXAMPLE 21
LOTION
______________________________________ Ingredient Amount
______________________________________ Sodium phenylbutyrate 1.00
g. Stearyl methylcellulose (4,500) 25.00 ml. solution (2%)
Benzalkonium chloride 0.03 g. Sterile water 250.00 ml
______________________________________
The benzalkonium chloride is dissolved in about 10 ml. of sterile
water. The sodium phenylbutyrate is dispersed into methylcellulose
solution by means of vigorous stirring. The methylcellulose (4,500)
used is a high viscosity grade. The solution of benzalkonium
chloride is then added slowly while stirring is continued. The
lotion is then brought up to the desired volume with the remaining
water. Preparation of the lotion is carried out under aseptic
conditions.
EXAMPLE 22
DUSTING POWDER
______________________________________ Ingredients Amount
______________________________________ NaPA 25 g. Sterilized
absorbable maize 25 g. starch BP dusting powder
______________________________________
The dusting powder is formulated by gradually adding the sterilized
absorbable dusting powder to NaPA to form a uniform blend. The
powder is then sterilized in conventional manner.
EXAMPLE 23
SUPPOSITORY, RECTAL AND VAGINAL PHARMACEUTICAL PREPARATIONS
Suppositories, each weighing 2.5 g. and containing 100 mg. of NaPA
are prepared as follows:
______________________________________ Ingredients Amount/1000
______________________________________ suppositories NaPA,
micronized 100 g. Propylene glycol 150 g. Polyethylene glycol 4000,
qs 2500 g. ______________________________________
NaPA is finely divided by means of an air micronizer and added to
the propylene glycol and the mixture is passed through a colloid
mill until uniformly dispersed. The polyethylene glycol is melted
and the propylene glycol dispersion added slowly with stirring. The
suspension is poured into unchilled molds at 40.degree. C.
Composition is allowed to cool and solidify and then removed from
the mold and each suppository is foil wrapped.
The foregoing suppositories are inserted rectally or vaginally for
treating neoplastic disease.
It is known that intracellular glutathione plays a major role in
detoxification and repair of cellular injury by chemical and
physical carcinogens. NaPA treatment of normal or tumor cells
markedly induced the activity of intracellular glutathione
approximately 2-10 fold depending on growth conditions. Nontoxic
agents that can induce glutathione are highly desirable since these
are likely to protect cells from damage by a variety of chemical
carcinogens and ionizing radiation.
Taken together, the present invention demonstrates that NaPA has
valuable potential in cancer prevention in cases such as high risk
individuals, for example, heavy smokers with familial history of
lung cancer, inherited disorders of oncogene abnormalities
(Li-Fraumeni syndrome), individuals exposed to radiation, and
patients in remission with residual disease. Furthermore, NaPA can
be used in combination with other therapeutic agents, such as
chemicals and radiation, to enhance tumor responses and minimize
adverse effects such as cytotoxicity and carcinogenesis. The
antitumor activity, lack of toxicity, and easy administration
qualify NaPA as a preferred chemopreventive drug.
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