SMITH KIELLAND INGRID (NO)
| GB1233312A | 1971-05-26 |
TETRAHEDRON LETTERS, Volume 30, No. 41, 1989, M. NISHIZAWA et al.: "Isolation of a New Cycloartanoid Triterpene from Leaves of Lansium Domesticum Novel Skin-Tumor Promotion Inhibitors", page 5615 - page 5618.
CHEMICAL SOCIETY LONDON JOURNAL, 1962, page 4034-4039, C. DJERASSI et al.: "789. Terpenoids. Part LI. The Isolation of Some New Cyclopropane-Containing Triterpenes from Spanish Moss (Tillandsia Usneoides, L.)".
TETRAHEDRON, Volume 26, 1970, J.L. FOURREY et al.: "Sur Quelques Oxydations de Type Biogenetique", page 3839 - page 3847.
| 1. | Pharmaceutical mixture , c h a r a c t e r i z e d by containing a compound having the general formula (I) R. or stereoisomers thereof, where R, is one of the groups OR', SR' , NHR' , N(R')2 or N+(R')3; R' is defined as hydrogen, branched or unbranched alkyl, branched or unbranched mono or polyalkenyl, branched or unbranched mono or polyalkynyl, formyl, branched or unbranched alkylcarbonyl, branched or unbranched mono or polyalkenylcarbonyl, branched or unbranched mono or polyalkynylcarbonyl or glycosyl, and when R, is a amino group substituted with two or three R' substituents the R' substituents can be identical or different; and R2 is an alkenyl group which is branched or unbranched and in which at least two carbon atoms are bound together by a double bond, and the said alkenyl group is substituted with one or more organic groups defined similarly as the substituent R,. |
| 2. | Pharmaceutical mixture according to claim 1, c h a r a c t e r i z e d by containing a compound having the formula or stereoisomers thereof. |
| 3. | Pharmaceutical mixture according to claim 1, c h a r a c t e r i z e d by containing a compound having the formula OH or stereoisomers thereof. se of a compound having formula (I) R. or stereoisomers thereof, where R. is one of the groups OR', SR' , NHR' , N(R')2 or N+(R')3; R' is defined as hydrogen, branched or unbranched alkyl, branched or unbranched mono or polyalkenyl, branched or unbranched mono or polyalkynyl, formyl, branched or unbranched alkylcarbonyl, branched or unbranched mono or polyalkenylcarbonyl, branched or unbranched mono or polyalkynylcarbonyl or glycosyl, and wheirRj is a amino group substituted with two or three R' substituents the R' substituents can be identical or different; and R2 is an alkenyl group which is branched or unbranched and in which at least two carbon atoms are bound together by a double bond, and the said alkenyl group is substituted with one or more organic groups defined similarly as the substituent R,, for preparation of a remedy useful for treatment of cancerous diseases. |
This invention relates to a pharmaceutical mixture containing derivatives of cycloartenol.
As known, lots of resources are continuously used in research concerning therapeuticals against cancerous diseases. But still a solution to the entire cancer problem seems to be far away. The present invention relates to a pharmaceutical mixture never used in treatment of cancerous diseases, having good cytotoxic, cell inactivating effect on diverse human tumour cells and no signs of side effects.
Cycloartenol derivatives are members of the group of chemical natural products called triterpenes. Triterpenes are built up from six C 5 - (isoprene) -units and contain 30 carbon atoms. (A C 5 - (isoprene) -unit equals a 2-methyl-1,3-butadiene group.) Triterpenes are present in many plants. The biological function for triterpenes in the plants seems generally to be protecting, and they are for instance present in resins and waxes.
The structural formula of cycloartenol is:
During the latest 20 years triterpenes of the cycloartenol type are reported to reduce the cholesterol level, have anti- inflammatorical and a ti-oxidizing effect, and to be useful in cosmetics. Several articles and patents on these subjects are published. Among these patents are GB-1233312, GB-1233313 and US- 3.625.194.
Only very limited information is available on the effects of cycloartenol derivatives on tumours, and none of this information can be regarded as prior art concerning the present invention.
Anecdotal evidence reported in the popular press indicated that Euphorbia pulcherrima. the common Poinsettia plant, might contain substances active against tumours. However, there is no reports about cycloartenol derivatives nor any other substances in the Poinsettia plant causing this effect.
Additionally we have shown that cycloartenol (1) itself does not have any cytotoxic properties at all.
In view of the above information the fact that the cytotoxic, cell inactivating effect in the Poinsettia plant is mainly caused by cycloartenol derivatives was a rather surprising discovery.
The pharmaceutical mixture according to this invention is characterized by containing derivatives of cycloartenol having the general formula (I)
or stereoisomers thereof, where
R, is one of the groups -OR', -SR' , HR' , -N(R') 2 .or -N + (R') 3 ; R' is defined as hydrogen, branched or unbranched alkyl, branched or unbranched mono- or polyalkenyl, branched or unbranched mono- or polyalkynyl, formyl, branched or unbranched alkylcarbonyl, branched or unbranched mono- or polyalkenylcarbonyl, branched or unbranched mono or polyalkynylcarbonyl, or glycosyl, and when R, is a amino group substituted with two or three R' -substituents the R' -substituents can be identical or different; and R 2 is an alkenyl group which is branched or unbranched and in which at least two carbon atoms are bound together by a double bond, and the said alkenyl group is substituted with one or more organic groups defined similarly as the substituent Rj.
The expression branched or unbranched alkyl, refers to lower alkyl groups like methyl, ethyl, propyl, isopropyl, n-butyl, 2- butyl and tert.-butyl.
The expression branched or unbranched mono- or polyalkenyl, refers to substituents like vinyl, 1-propenyl, allyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-but-l-enyl, 3- but-1-enyl,- 2-but-2-enyl, 1,3-butadienyl, 2-buta-l,3-dienyl, 1,2- butadienyl, 3-buta-l,2-dienyl and 4-buta-l,2-dienyl.
The expression branched or unbranched mono- or polyalkynyl, refers to substituents like ethynyl, 1-propynyl, 2-propynyl, 1- butyny1, 3-butyny1, 3-but-1-yny1 and 1,3-butadiyny1.
The expressions branched or unbranched alkylcarbonyl, branched or unbranched mono- or polyalkenylcarbonyl, and branched or unbranched mono- or polyalkynylcarbonyl, refer to substituents where the branched and unbranched carbon groups explained in the three sections above, are combined to a carbonyl group.
A preferred feature of the invention is that an allyl alcohol or a derivative thereof is a part of the R 2 -substituent in formula (I).
An even more preferred feature is that the compounds according to formula (I) are 9.19-cycloart-23-ene-3β.25-diol (formula la) or 9.19-cycloart-25-ene-3β.24-diol (formula lb) or stereoisomers thereof.
OH
9.19-cycloart-23-ene-3β.25-diol (la) and 9.19-cycloart-25-ene- 3β.24-diol (lb) were first isolated from "Spanish moss", Tillandsia usneoides (McCrindle, R. & Djerassi, C, Chem. Ind. , (1961), p. 1311-1312, and Djerassi, C. & McCrindle, R.., J. Chem. Soc, (1962), p. 4034-4039). These compounds are since then reported isolated from various plants (i.e. Euphorbia cyparissias. Euphorbia helioscopia, Pachysandra terminalis. Artocarpus altilis. Tricholepis glaberrima.
Poncramia αlabra, Artemisia rubripes. Cassia siamea. Euphorbia tirucalli. Euphorbia broteri. Manσifera indica. Calotropis procera) . However, it is not reported anything about the applications of these compounds (la and lb) .
Fourrey, J.L. et al. (Tetrahedron, (1970), vol 26, p. 3839-3847) did synthesize the 3-monoacetate of compound (la) and (lb) . Nor for these compounds were any applications mentioned in this article.
This invention relates to a pharmaceutical mixture containing derivatives of cycloartenol, useful for treatment of cancerous diseases, either alone or in combination with other compatible components, used in the treatment of such diseases.
The therapeutical active compounds defined as in formula (I) , may be given to the patient as a mixture containing cycloartenol derivatives dissolved in an oil or fat, such as cod liver oil or olive oil, by oral administration or by injection. Doses of 2-8 mg/day are recommended for humans. The pharmaceutical mixture can also_be used in treatment of animals suffering f om cancerous diseases. The doses given will then be different from the doses recommended for human beings, depending on the weight and species of the animal. For instance for a dog weighing approximately 20 kg the correct dose will be 1-2 mg/day.
In the following the invention will be further explained by the examples and the " attached figures. The examples are just meant to be illustrative for the invention and shall not be considered as limiting.
Fig. 1 shows the percent cell survival of NHIK 3025 (•) , HeLa S 3 (■) , PANC-l (A) , and A549 (♦) cells treated with 9.19-cycloart-23-ene-3β.25-diol (la) as a function of the said cycloartenol derivative concentration. Drug treatment was for 24 hr and each point represents the
mean colony count from five replicate dishes per point. Vertical bars represent standard deviation (SE) when exceeding the size of the symbols.
Fig. 2 shows the percent cell survival of NHIK 3025 cells treated with 9,19-cycloart-23-ene-3β.25-diol (la) as a function of the said cycloartenol derivative concentration. The cells were either established and treated in Eagle's Minimum Essential Medium (MEM) containing 10% fetal calf serum (FCS) (•) or 10% human " serum (HS) (A) . Drug treatment was for 24 hr and each point represents the mean colony count from five replicate dishes per point. Vertical bars represents SE when exceeding the size of the symbol.
Fig. 3 shows the percent cell survival of Ehrlich ascites tumour cells treated with 9,19-cycloart-25-ene- 3β.24-diol (lb) as a function of the said cycloartenol derivative concentration. The cells were cultured in suspension cultures in MEM supplemented with 10% FCS. Drug treatment was for 24 hr with various concentrations of 9,19-cycloart-25-ene-3β.24-diol.
Isolation of active compounds:
Example 1.
9.19-cycloart-23-ene-3β.25-diol (la) and 9.19-cycloart-25-ene- 3β.24-diol (lb) were extracted and isolated from the Poinsettia plant (Euphorbia pulcherrima) . As earlier mentioned these compounds are also present in some other plant materials, and can be extracted from these too.
The compounds (la) and (lb) exist as crystalline structures after the isolation.
The melting point for the compounds was measured to be 198-202°C and 172-175°C for (la) and (lb) , respectively.
The structures were also characterized by NMR (combination of 13 C and l H) , mass spectroscopy (MS) and -X-ray diffraction. The results from mass spectroscopy were: Weak molecular ion observed at M/Z 442 (1,8%) and one peak at M/Z 424. This might be because of splitting off water. High resolution gave the general composition C 30 H 50 O 2 .
The structures of compounds (la) and (lb) were further confirmed by partial synthesis of their acetates from cycloartenol by acetylation followed by photo-oxidation and reduction with lithium aluminium hydride (J.L. Fourrey et al, Tetrahedron, (1970), vol. 26, p. 3839-3847).
Cytotoxic effect:
The cytotoxic effect of the isolated cycloartenol derivatives from the Poinsettia plant (Euphorbia pulcherrima) was investigated using cultured human cells in vitro. Cell inactivation was measured as loss in the ability of single cells to give rise to macroscopic colonies following treatment.
In order to eliminate any variance due to culture conditions, cell lines established in Eagle's Minimum Essential Medium (MEM) or Dulbecco's modification of this medium (DMEM) were used.
Cells of the human line NHIK 3025, established from a cervix carcinoma-.in situ (K. Nordbye and R. Oftebro, "Establishment of four new cell strains from human uterine cervix I and II." , Exp. Cell Res., (1969), vol. 58, p. 458-460), were cultivated as monolayers in MEM supplemented with 10% heat-inactivated fetal calf serum (FCS) .
This same cell line has also been cultured in MEM supplemented with 10% hea -inactivated human serum (HS) .
HeLa S 3 , a human cervix epitheloid carcinoma (ATCC CCL 2.2), was cultivated in MEM supplemented with 10% FCS.
A549, a human lung carcinoma (ATCC CCL 185), was cultivated in DMEM supplemented with 10% FCS.
PANC-1, a human pancreas epitheloid carcinoma (ATCC CRL 1469) , was cultivated in DMEM supplemented with 10% FCS.
Ehrlich ascites tumour cells were cultivated in MEM supplemented with 10% FCS in suspension cultures.
The other cell types were cultivated as monolayers in plastic flasks or Petri dishes. They were recultured every second or third day to ensure continuous, exponential growth.
The cells were trypsinized and a known number was seeded into 60 * 15 mm style plastic Petri dishes (Nunclon, Denmark) . The dishes were kept in a C0 2 incubator at 37°C and an atmosphere of 5% C0 2 . The current cycloartenol derivative was added to exponentially growing, asynchronous cells 2 hours after the cells were seeded by replacing the medium in the dishes with medium containing the desired concentrations of the derivative. Following a 24 hr treatment period, the dishes were rinsed with warm (37°C) Hanks' balanced salt solution before fresh medium was added. After 10 to 14 days, colonies of cells were fixed in ethanol and stained with methylene blue. Only colonies containing more than 40 cells were counted as survivors, and data are expressed as the percent cell survival, i.e. the percentage of cells surviving treatment and giving rise to macroscopic colonies relative to control cells.
The cycloartenol derivatives, compound (la) and (lb) , were in an ethanolic solution containing 20 mM of the active cycloartenol
derivative. The compounds were added to cell culture medium without additional sterile filtration, but .using sterile automatic pipette tips.
In example 2 and 3 the compound 9.19-cycloart-23-ene-3β.25-diol (la), isolated in example 1, was used for practical reasons.
Example 2.
The cytotoxic, cell inactivating effect induced by the cycloartenol derivative 9,19-cycloart-23-ene-3β.25-diol, on four established cell lines is given in Table 1. All these experiments were carried out using medium supplemented with fetal calf serum.
Table 1.
The table shows the percent cell survival of each cell line treated for 24 hr with 9.19-cycloart-23-ene-3β.25-diol (la) versus the concentration of the said cycloartenol derivative.
Each value represents the mean ± SE of five replicate dishes per derivative concentration, and survival is relative to control cells.
These data are graphically presented as dose-response curves in Fig. 1.
The greatest cytotoxic effect was induced in cells originating
from human cervix carcinoma. Both NHIK 3025 and HeLa S 3 displayed similar dose-response curves following 24 hr treatment. The IC 90 was found to be approximately 10 μM for these two cell lines, while 20 μ was found to approximate the IC 99 .
Carcinoma of pancreas, represented by the PANC-1 cell line, was less sensitive to treatment than the cervix carcinoma cell line. For PANC-1 the ICg o was found to be approximately 20 μM.
The cell line displaying least sensitivity to treatment was the lung carcinoma cell line A549. .A 24 hr treatment with 20 μM derivative resulted in 50% cell survival.
Example 3.
In order to determine if the nature of the serum supplement would affect the cytotoxic effect induced by the cycloartenol derivative 9,19-cycloart-23-ene-3β.25-diol, an additional set of experiments were performed. The cervix carcinoma cell line NHIK 3025 had previously been established in MEM medium supplemented with both 10% FCS and 10% human serum. Treatment of NHIK 3025 cells under each of these two culture conditions-for 24 hr with 9,19-cycloart-23-ene-3β.25-diol resulted in the cell survival described in Table 2.
Table 2 .
The table shows the percent cell survival of NHIK 3025 cells cultured in MEM medium supplemented with either FCS or human serum and treated for 24 hr with 9,19-cycloart-23-ene-3β.25-diol (la) versus the concentration of the said cycloartenol derivative. Each value represents the mean ± SE of five replicate dishes per derivative concentration, and survival is relative to control cells.
These data are graphically presented as dose-response curves in Fig. 2.
As can be seen, cells established in medium supplemented with human serum were less sensitive than cells cultured in medium "supplemented with FCS. While the IC 90 for NHIK 3025 FCS cells was approximately lOμM, the IC 90 increased to approximately 20 μM for NHIK 3025 HS cells.
Example 4.
The cytotoxic, cell inactivating effect induced by the cycloartenol derivative 9,19-cycloart-25-ene-3β.24-diol, was examined on Ehrlich ascites tumour cells. 9,19-cycloart-25-ene- 3β.24-diol is compound (lb) isolated in example 1.
The results from these experiments are shown in Fig. 3.
The IC 90 was found to be approximately 10 μM.
Reference example.
Cycloartenol (l) which is a triterpene present in most plants, does not have any effect on tumour cells. Experiments have been performed on the cell lines HeLa S 3 and Ehrlich ascites but no cytotoxic, cell inactivating effect was induced by cycloartenol. The cells were treated with cycloartenol in concentrations up to 20 μM without reducing the viability of the tumour cells.
Example 5.
In in vivo experiments with dogs over several months with doses of 1-2 mg/day, p.o. in cod liver oil or olive oil, no side effects were evident.
There is disclosed a pharmaceutical mixture containing compounds defined as formula (I) having cytotoxic, cell inactivating effect on human tumour cells.- These compounds are cycloartenol derivatives. Especially allyl alcohols of these cycloartenol derivatives seem to be effective in treatment of tumour cells. In vivo experiments with dogs show no side effects.