WO2002007823A2

WO2002007823A2 - Inhibition of mutagenic effects of carcinogens

Info

Publication number: WO2002007823A2
Application number: PCT/US2001/023290
Authority: WO
Inventors: Davide Riccardo Grassetti
Original assignee: Davide Riccardo Grassetti
Priority date: 2000-07-24
Filing date: 2001-07-24
Publication date: 2002-01-31
Also published as: AU2001277143A1; WO2002007823A3

Abstract

A method of treating a human subject to inhibit mutagenic effects of carcinogens by administering to the subject a composition comprising a sufficient amount of diethyl 2,2'-dithiobis-(thiazole-4-carboxylate) to inhibit activity of poly(ADP-ribose) polymerase in said subject to thereby inhibit said effects. Also provided is a method of inhibiting occurrence of mutations and of cancers derived from mutations, which method comprises administration to a subject of a composition comprising a sufficient amount of diethyl 2,2'-dithiobis-(thiazole-4-carboxylate).

Description

INHIBITION OF MUTAGENIC EFFECTS OF CARCINOGENS

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method for inhibiting the mutagenic effects of carcinogens, and in particular, a method for administering to a human the compound diethyl 2,2'-dithiobis-

(thiazole-4-carboxylate) to inhibit the activity of the enzyme poly(ADP-ribose) polymerase to

inhibit the occurrence of mutation-caused cancer.

2. Background of the Invention

Mutations occur in cellular DNA under the effect of ultraviolet light, infrared light, x-rays,

ionizing radiation, and chemicals. It is generally accepted that most mutations lead to cancer. The enzyme poly(ADP-ribose) polymerase (ADPRP) has been suspected to play a

regulatory role in many cellular activities, including DNA repair, differentiation, and malignant

transformation. Studies of inhibitors of ADPRP have provided evidence of the importance of ADPRP in (a) those cellular activities (MJ. Suto et al, 1991, Drugs of the Future, 16:723-739);

and (b) the importance of ADPRP regulation in experimental therapy of various forms of

inflammation and shock, myocardial and intestinal ischaemia-reperfusion, focal cerebral ischaemia,

meningitis-associated intracranial complications, diabetes mellitus, stroke, Alzheimer's disease,

and other forms of brain and spinal cord injury (Szabo, C, Dawson, V.L., Role of poly(ADP-

ribose) synthetase in inflammation and ischaemia-reperfusion, Trends Pharmacol. Sc (1998) Jul;19(7):287-298; Pieper, A. A., Nerma, A., Zhang, J., Snyder, S.H., Poly(ADP-ribose)

polymerase, nitric oxide and cell death, Trends Pharmacol Sci.. (1999) Apr;20(4): 171-81; Nippon

Yakurigaku Zasshi, Hara, H. transl., Involvement of caspase on apoptosis in ischemia-induced

neuronal cell death: usefulness of caspase inhibitors for stroke therapy, (1999) Feb;l 13(2):97-111; Koedel, U., Pfister, H.W., Oxidative stress in bacterial meningitis, Brain Pathol.. (1999)

Jan;9(l):57-67. Many analogs of nicotinamide, a product of ADPRP's action on NAD, have been

studied in the hope of finding a compound that could modulate the activity of ADPRP. It would

be desirable to find a selective inhibitor of ADPRP that could inhibit the malignant transformation

of human cells (Sutro et al).

SUMMARY OF THE INVENTION

It has been discovered that diethyl 2,2'-dithiobis-(thiazole-4-carboxylate) has an antimutagenic effect. The method of the invention is based on the finding that diethyl 2,2'- dithiobis-(thiazole-4-carboxylate) is a useful agent for the prevention of mutations, most of which

lead to cancer.

The method of the invention comprises treating a human subject to inhibit the mutagenic

effects of carcinogens. The method involves administering to the subject a composition

comprising a sufficient amount of diethyl 2,2'-dithiobis-(thiazole-4-carboxylate) to inhibit the activity of poly(ADP-ribose) polymerase to thereby inhibit the mutagenic effects, and in

particular, their carcinogenic effects. In another aspect, the invention involves a method of inhibiting the occurrence of mutations and of cancers derived from mutations, and involves administering to a subject a composition comprising a sufficient amount of diethyl 2,2'-dithiobis-

(thiazole-4-carboxylate).

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwise indicated, conventional

molecular and cell biology, cell culture, biochemistry, and organic and medicinal chemical

synthesis within the skill of the art. Such techniques are explained fully in the literature. See

Cancer Chemotherapy: Principles and Practice, ed. B.A. Chabner, J.M. Collins, Phil., Lippincott Publ., 1990; Cancer: Principles and Practice of Oncology, ed. de Vita, Jr., V.T., Hellman, S., and

Rosenberg, S.A., Lippincott Co., Philadelphia, 1993; The Chemotherapy Source Book, ed. Perry,

M.C., Williams and Wilkins Publ., Baltimore, 1991; Silverman, Richard B., The Organic

Chemistry of Drug Design and Drug Action, Academic Press, Inc., New York, 1992; Smith,

Michael B., Organic Synthesis. McGraw Hill, Inc., New York, 1994.

Methods are well known in the art for determining therapeutically effective amounts of the compounds used in the method of the invention. Such methods involve analysis of the pharmaceutical/pharmacokinetic parameters in anti-cancer or antitumor therapy, i.e for inhibiting

the growth of cancerous tumors (Wedge. S.R., Porteus, J.K., Newlands, E.S., Cancer Chemother.

Pharmacol., (1997) 40:266-272; Legha, S.S., Seminar in Oncology. (1997) 24.S4-24-31; Motzer,

R. J., Vogelzang, N.J., Chemotherapy for Renal Cell Carcinoma, in: Raghaven, D., Scher, H.I.,

Leibel, S.A., et al: eds. Principles and Practice of Genitourinary Oncology. Lippincott-Raven

Publ, Philadelphia, pp. 885-96, 1997; Bloom, H.J., Medroxyprogesterone acetate (Provera) in the treatment of metastatic renal cancer, Br. J. Cancer, (1971) 25:250-65). The present invention shows that in the presence of diethyl 2,2'-dithiobis-(thiazole-4- carboxylate) the mutagenic effect of carcinogenic chemicals, exemplified by benzo-[a]-pyrene, 2-

aminofluorene, N-acetyl-2-aminofluorene, and N-methyl-N'-nitro-N-nitrosoguanidine, is greatly diminished.

Diethyl 2,2'-dithiobis-(thiazole-4-carboxylate) is known to prevent metastases in mice

(D.R. Grassetti, 1986, Cancer Letters, 31:187-195).

Examples 1, 2, 3 and 4 report experiments in which the antimutagenic effects of diethyl 2,2'-dithiobis-(thiazole-4-carboxylate) were observed. These experiments were performed with a mutant strain of Salmonella typhimurium bacteria T-100. When the cells were exposed to varying amounts of benzo-[a]-pyrene, 2-aminofluorene, N-acetyl-2-aminofluorene, and N-methyl-

N'-nitro-N-nitrosoguanidine, a number of colonies (mutant) were formed. When diethyl 2,2- dithiobis-(thiazole-4-carboxylate) was present in the culture prior to the addition of carcinogen, the number of mutants decreased by as little as 8% and as much as 89%.

As shown in Example 5 below, a method of the present invention is based on the finding that diethyl 2,2'-dithiobis-(thiazole-4-carboxylate) inhibited the activity of poly(ADP-ribose) polymerase (ADPRP). Inhibition of ADPRP is useful for blocking the mutagenic effects of carcinogens (Suto, M.J., et al, (1991), Drugs of the Future, 16(8):723-739). It is understood that diethyl 2,2'-dithiobis-(thiazole-4-carboxylate) includes derivatives thereof, including, but not

limited to alkaline metal salts and alkyl esters. The method of the present invention includes administering a pharmaceutical composition comprising an effective amount of diethyl 2,2'-dithiobis-(thiazole-4-carboxylate) in pure form or as a pharmaceutically acceptable crude concentrate in association with a pharmaceutical carrier or diluent. By way of example and not limitation, such compositions conveniently contain, in capsule form, less than 1% by weight, and preferably about 0.2% by weight, of diethyl 2,2'- dithiobis-(thiazole-4-carboxylate), and involve administration of at least about 0.1 mg/kilo of body weight of diethyl 2,2'-dithiobis-(thiazole-4-carboxylate). A typical dosage is about 100 mg per day administered in 1 capsule per day. The compositions may be prepared by conventional techniques to be efficacious and to be in conventional forms, for example, capsules, tablets, suppositories, dispersible powders, syrups, elixirs, suspensions or solutions for enteral, parenteral, intravenous or transdermal administration (Genaro, A.R., ed., The Science and Practice of Pharmacy, Mack Publ. Co., Eston, PA, 1995). Suitable pharmaceutical diluents or carriers include, for example, water, alcohols, natural or hardened oils and waxes, calcium and sodium carbonates, calcium phosphate, kaolin, talc and lactose as well as suitable preserving agents, such as ethyl-p-hydroxybenzoate, suspending agents such as methyl cellulose, tragacanth and sodium alginate, wetting agents such as lecithin, polyoxyethylene stearate and polyoxyethylene sorbitan mono-oleate, granulating and disintegrating agents such as starch and alginic acid, binding agents such as starch, gelatin and acacia and lubricating agents such as magnesium stearate, stearic acid and talc, in order to provide an elegant and palatable pharmaceutical preparation. Compositions in tablet form may be coated by conventional techniques to delay disintegration of the tablet and absorption of the active ingredient in the gastrointestinal tract and thereby provide sustained action over a long period. Other compounds and methods known in the art for delaying disintegration or for time-delayed or time-measured delivery of the active ingredients also find use in formulating the active ingredients for use in the methods of the invention. For example, diethyl 2,2'-dithiobis-(thiazole-4-carboxylate) may also be combined with liposomes or other delayed- release carrier means to protect the compounds from degradation until they reach their targets and/or facilitate movement of the compounds across tissue barriers.

The preferred compositions from the standpoint of ease of administration are solid compositions, particularly solid-filled gelatin capsules or tablets.

The carcinogens tested here are representative of the carcinogens which form a major part of air pollutants caused by tobacco smoke, the exhaust fumes of internal combustion engines, among other sources.

It has been observed that diethyl 2,2'-dithiobis-(thiazole-4-carboxylate) is not a toxic compound. It can be taken in doses of at least approximately 0.1 gram per day for indefinite periods. This fills the requirements for a non-toxic, low-cost chemopreventive agent, which could be administered orally.

The effect of diethyl 2,2'-dithiobis-(thiazole-4-carboxylate) (0.3 mg plate with dimethyl sulfoxide) added to each plate in the Ames test, which is a reverse mutation assay (Ames, B.N. et al, 1975, Mutation Research 31 :347) with Salmonella typhimurium bacteria T-100, was studied. See Tables 1, 2, 3 and 4. The diethyl 2,2'-dithiobis-(thiazole-4-carboxylate) was added to each plate one hour before the addition of the carcinogen. The results reported in the tables represent the average values (number of colonies per plate) of three individual determinations.

EXAMPLE 1

EFFECTS OF DIETHYL 2,2'-DITHIOBIS-(THIAZOLE-4-CARBOXYLATE) ON MUT AGENESIS BY BENZO-[a]-PYRENE

TABLE 1

Number of Colonies Per Plate

Benzo-[a]-pyrene No Inhibit or With Inhibitor Decrease (%)

(μg/plate)

0 (control) 96 92 0

2.5 783 663 15

5.0 1177 980 17

10.0 1274 872 32

EXAMPLE 2

EFFECTS OF DIETHYL 2,2'-DITfflOBIS-(TfflAZOLE-4-CARBOXYLATE) ON MUT AGENESIS BY 2-AMINOFLUORENE

TABLE 2

Number of Colonies Per Plate

2-Aminofluorene No Inhibitor With Inhibitor Decrease (%)

(μg/plate)

0 (control) 96 92 0

5.0 767 423 45

15.0 1741 1108 36

25.0 2067 1521 26 EXAMPLE 3

EFFECTS OF DIETHYL 2,2'-DITHIOBIS-(THIAZOLE-4-CARBOXYLATE) ON MUTAGENESIS BY N-ACETYL-2-AMINOFLUORENE

TABLE 3

Number of Colonies Per Plate

N-acetyl-2- No Inhibitor With Inhibitor Decrease (%) aminofluorene

(μg/plate)

0 (control) 96 92 0

20 1038 403 61

50 1666 184 89

100 1818 679 63

EXAMPLE 4

EFFECTS OF DIETHYL 2,2'-DITHIOBIS-(THIAZOLE-4-CARBOXYLATE) ON MUTAGENESIS BY N-METHYL-N'-NITROSOGUANIDLNE

TABLE 4

Number of Colonies Per Plate

N-methyl-N'- No Inhibitor With Inhibitor Decrease (%) nitrosoguanidine

(μg/plate)

0 (control) 96 92 0

0.5 210 193 8

1.0 1000 609 39

1.5 1116 838 25 EXAMPLE 5

INHIBITION OF ADPRP BY DIETHYL 2,2'-DITHIOBIS-(THIAZOLE-4-CARBOXYLATE)

ADPRP was prepared from pig thymus using the method of Khan and Shall (Biochem.

Soc. Transactions 4:778 (1976)) with various modifications. Using a 10 μl enzyme preparation

(approximately 150 ng total protein) and a substrate (NAD) concentration of 25 μM, enzyme

activity was approximately linear up to 15-20 minutes. K_M was approximately 26 μM.

Using a standard assay mix containing 100 mM triethanolamine, 10 mM MgCl₂, 2mM

dithiothreitol (DTT), and 25 μM [¹⁴C] NAD, three experiments were performed with diethyl 2,2'- dithiobis-(thiazole-4-carboxylate) added as a solution in dimethyl sulfoxide (DMSO) (1 μg/μl).

The addition of diethyl 2,2'-dithiobis-(thiazole-4-carboxylate) over a concentration range of 50 μg/ml to 300 μg/ml to the standard incubation mix, which contained 0.1 ml enzyme preparation/ml and the labeled substrate, showed a concentration-dependent inhibitory effect on the enzyme activity, measured as the incorporation of radioactivity into TCA-insoluble material.

The concentration of diethyl 2,2'-dithiobis-(thiazole-4-carboxylate) giving 50% inhibition of

activity was found to be approximately

60 μg/ml.

TABLE 5

INHIBITION OF POLY(ADP-RIBOSE) POLYMERASE ACTIVITY BY DIETHYL 2,2'-DITHIOBIS-(THIAZOLE-4-CARBOXYLATE)

Concn of diethyl 2,2'-dithiobis- Incorporation of ⁴C into TCA-insoluble material

(thiazole-4-carboxylate)

(μg/lOO μl) dpm % control

5 6035±420 111±6.6

6 1475±1002 24±14.2

8 1135±731 18±10.8

10 971±971 10±9.9

These are values from individual incubations with activity expressed as a percentage of the corresponding control (DMSO only) value. These results indicate that diethyl 2,2'-dithiobis- (thiazole-4-carboxylate) is an effective inhibitor of ADPRP, forming the basis of the method of the invention for treating a human subject to inhibit mutagenic effects of carcinogens by administering to a human subject a composition comprising a sufficient amount of diethyl 2,2'- dithiobis-(thiazole-4-carboxylate) (or derivatives thereof) to inhibit activity of ADPRP in the subjects in order to inhibit the mutagenic effects of the carcinogens. Alternatively, the invention also directs itself to a method of inhibiting ADPRP in a human subject in order to inhibit the mutagenic effects of carcinogens, the method involving administering to a human subject a composition comprising a sufficient amount of diethyl 2,2'-dithiobis-(thiazole-4-carboxylate) (or derivatives thereof) to specifically inhibit activity of ADPRP in the subjects in order to inhibit the mutagenic effects of the carcinogens. It should be understood that the method of the invention includes a method of treating a human subject with diethyl 2,2'-dithiobis-(thiazole-4-carboxylate) to inhibit or regulate ADPRP to

achieve therapeutic effects in various forms of inflammation and shock, myocardial and intestinal

ischaemia-reperfusion, focal cerebral ischaemia, meningitis-associated intracranial complications,

diabetes mellitus, stroke, Alzheimer's disease, and other forms of brain and spinal cord injury. It

is known in the art that ADPRP inhibition may play an active role in the experimental therapy of

various forms of inflammation and shock, myocardial and intestinal ischaemia-reperfusion, focal cerebral ischaemia, meningitis-associated intracranial complications, diabetes mellitus, stroke, Alzheimer's disease, and other forms of brain and spinal cord injury (Szabo, C, Dawson, V.L., Role of poly(ADP-ribose) synthetase in inflammation and ischaemia-reperfusion, Trends

Pharmacol. Sci.. (1998) Jul;19(7):287-298; Pieper, AA., Verma, A., Zhang, L, Snyder, S.H.,

Poly(ADP-ribose) polymerase, nitric oxide and cell death, Trends Pharmacol Sci., (1999) Apr;20(4): 171-81; Nippon Yakurigaku Zasshi, Hara, H. transl., Involvement of caspase on

apoptosis in ischemia-induced neuronal cell death: usefulness of caspase inhibitors for stroke therapy, (1999) Feb;l 13(2):97-111; Koedel, U., Pfister, H.W., Oxidative stress in bacterial

meningitis, Brain Pathol.. (1999) Jan;9(l): 57-67.

Having thus described exemplary embodiments of the present invention, it should be noted

by those skilled in the art that the disclosures herein are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present

invention. Accordingly, the present invention is not limited to the specific embodiments as

illustrated herein, but is only limited by the following claims.

Claims

What is claimed is:

1. A method of treating a human subject to inhibit mutagenic effects of carcinogens in said subject wherein the method comprises administering to said subject a composition comprising a sufficient amount of diethyl 2,2'-dithiobis-(thiazole-4-carboxylate) to specifically inhibit activity of poly(ADP-ribose) polymerase in said subject to thereby inhibit said effects.

2. The method of claim 1 wherein a sufficient amount of diethyl 2,2'-dithiobis- (thiazole-4-carboxylate) is at least 0.1 mg/kilo of body weight.

3. A method of specifically inhibiting poly(ADP-ribose) polymerase in a human subject to inhibit the mutagenic effects of carcinogens on said subject, said method comprising administering to said subject a composition comprising a sufficient amount of diethyl 2,2'- dithiobis-(thiazole-4-carboxylate) to said human to thereby inhibit said effects.

4. The method of claim 3 wherein a sufficient amount of diethyl 2,2'-dithiobis-

(thiazole-4-carboxylate) is at least 0.1 mg/kilo of body weight.

5. A method of inhibiting occurrence of mutations and of cancers derived from mutations, which method comprises administration to a subject of a composition comprising a sufficient amount of diethyl 2,2'-dithiobis-(thiazole-4-carboxylate).

6. The method of claim 5 wherein a sufficient amount of diethyl 2,2'-dithiobis- (thiazole-4-carboxylate) is at least 0.1 mg/kilo of body weight.