Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-04-07
2001-04-17
Goldberg, Jerome D. (Department: 1614)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C514S452000, C514S456000, C424S649000
Reexamination Certificate
active
06218369
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the use of flavanolignanes, alone or combined with known chemotherapeutic agents, for the preparation of medicaments for the therapy and prophylaxis of uterus, ovary and breast tumors.
BACKGROUND OF THE INVENTION
Recently, some flavonoids have been found to have antitumoral activity (Verna, Cancer Research 48, 5754, 1988) and chemoprophylactic activity in some tumors (Cassady, J. Nat. Prod. 53, 23, 1990). Particularly quercetin, a flavonoid which is almost ubiquitous in plants, has proved some inhibiting activity on the proliferation of human leukemia cells (Larocca, Br. J. of Haematology 75, 489, 1990) and on other cell lines (Scambia, Br. J. Cancer 62, 942, 1990—Int. J. Cancer 46, 1112, 1990—Cancer Chemother. Pharmacol. 28, 255, 1991—Gynecologic Oncology 45, 13, 1992) besides having a synergistic activity with the usual chemotherapeuticals. Though the mechanism of such an inhibiting action on proliferation is unknown, it seems to be connected with the interaction of this flavonoid with the estrogen receptors of type II (Markaverich, J. steroid Biochem. 30, 71, 1988). These receptors, first described by Clark (J. Biol. Chem. 253, 7630, 1978) in the rat uterus, are different from the real estrogen receptors (ER) since these are present in a higher concentration and have a dissociation affinity constant (K
D
: 10-20 nM) for estradiol lower than that of the estrogen receptors (K
D
: 0.2-1 nM).
SUMMARY OF THE INVENTION
The invention relates to methods for preventing, inhibiting, or suppressing tumors by administering to a person in need of such treatment a therapeutically effective amount of a flavanolignane selected from the group of silymarin, silybin, silidianin, silicristin, dehydrosilybin, and mixtures thereof. The flavanolignane exhibits antagonistic activity on type II estrogen receptors and antiproliferative activity. In one embodiment, the flavanolignane is provided in a concentration from 0.01 &mgr;M to 20 &mgr;M. In a preferred embodiment, the flavanolignane is provided in an amount from 50 to 1,500 mg/day.
In a preferred embodiment, the method also includes administering a therapeutically amount of a different antitumor agent with the flavanolignane. In a more preferred embodiment, the different antitumor agent is administered concurrently or sequentially with the flavanolignane. In another preferred embodiment, the different antitumor agent includes cisplatin or adriamycin.
In one embodiment, the flavanolignane is administered together with a pharmaceutically acceptable carrier or excipient. In a preferred embodiment, the carrier or excipient includes a glyceride or a phospholipid. In a more preferred embodiment, the glyceride includes a liquid semi-synthetic glyceride of one or more medium-chain fatty acids. In a different embodiment, the administration is selected to be oral.
The invention also relates to antitumor pharmaceutical compositions including a therapeutically effective amount of a flavanolignane selected from the group of silymarin, silybin, silidianin, silicristin, dehydrosilybin, and mixtures thereof, in combination with a different antitumor agent. In one embodiment, the flavanolignane is present in a concentration from 0.01 &mgr;M to 20 &mgr;M. In a preferred embodiment, the composition includes an amount from 50 to 1,500 mg/day of the flavanolignane.
In a preferred embodiment, the different antitumor agent is concurrently administered with the flavanolignane. In a more preferred embodiment, the different antitumor agent includes cisplatin or adriamycin.
In another embodiment, the composition includes a pharmaceutically acceptable carrier or excipient. In a preferred embodiment, the carrier includes a phospholipid or a liquid semi-synthetic glyceride of one or more medium-chain fatty acids. In a different embodiment, the composition is suitable for oral administration.
The invention also relates to a pharmaceutical composition includes a therapeutically effective amount of a flavanolignane selected from the group of silidianin, silicristin, and mixtures thereof, with a pharmaceutically acceptable carrier or excipient.
Now it has surprisingly been found that flavanolignanes, among which silymarin, already widely used in therapy for the treatment of hepatopathias of various origin, the three main components thereof being known under the names silybin, silidianin, silicristin and dehydrosilybin and having the structures reported below:
have a high affinity to the estrogen receptors of type II and a very marked antiproliferative activity on uterus, ovary and breast tumoral cell lines resistant to Cis-platin and adriamycin. In order to verify the antiproliferative effect of flavanolignanes, the growth curves of different stabilized cell lines deriving from various human tumors have been examined in the presence of the compounds and their capability to interact with the Type II EBS in ovary carcinoma samples has been evaluated.
DETAILED DESCRIPTION OF THE INVENTION
The determination of the binding to the estrogen receptor has been carried out on cells of ovary tumor or of other organ tumors, cultured in monolayers using as medium the Dulbecco Modified Medium added with calf serum and with 200 unities/ml of penicillin. The cells used for the tests have been trypsinated every week and placed on a plate at a 8×10
−4
/ml density and incubated at 37° C. under air atmosphere containing 5% CO
2
and humidity. For the control of the antiproliferative activity of the products, the cells have been placed into wells (Falcon 3046, Becton Dickinson N.Y.). at a concentration of 4×10
4
/ml. After 24 hours the medium is substituted with fresh medium and the flavanolignanes dissolved in absolute ethanol are added. The controls are treated in the same experimental conditions only with the vehicle in the absence of the active ingredient. The treatment described above is repeated at 24 hour intervals during the 72 hours of the test time. The inhibition of the cell proliferation is evaluated by direct count of the cells, comparing the growth of the controls versus that of the treated samples.
For the dosage of the receptors, the cells after 24 hours are incubated with scalar amounts of labelled estradiol (
3
H-E2 40Ci/mmol, Amershan UK) alone or in the presence of a 100-fold amount of diethylstilbestrol at 4° C. for 2.5 hours.
At the end of the incubation time, the cells are quickly washed with fresh substrate and incubated for 30 minutes with 1M NaOH. Radioactivity is measured by means of a scintiller and binding specificity is calculated from the difference between the preparations containing or not diethylstilbestrol. Results are expressed as the number of binding sites per cell, according to conventional methods of the literature (Raneletti, 1988).
The inhibition on cell proliferation is evaluated by direct count of the cells, comparing the growth of controls versus that of the treated ones.
The results on different cell lines are reported in FIG.
1
and in Tables I and V.
REFERENCES:
Carter et al., Chemotherapy of Cancer, 2nd.,John Wiley & sons, N.Y.,N.Y., pp. 107-108, Aug. 13, 1981.
Bombardelli Ezio
Morazzoni Paolo
Goldberg Jerome D.
Indena S.p.A.
Pennie & Edmonds LLP
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