CA2045593C - Use of 1,2-benzopyrone derivatives for the treatment of malignant tumours in humans - Google Patents
Use of 1,2-benzopyrone derivatives for the treatment of malignant tumours in humans Download PDFInfo
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- CA2045593C CA2045593C CA002045593A CA2045593A CA2045593C CA 2045593 C CA2045593 C CA 2045593C CA 002045593 A CA002045593 A CA 002045593A CA 2045593 A CA2045593 A CA 2045593A CA 2045593 C CA2045593 C CA 2045593C
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
- A61K31/37—Coumarins, e.g. psoralen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
The invention relates to the use of 1,2-benzopyrone derivatives for the treatment of malignant tumors in human beings. The derivatives used according to the invention are suitable for both preventive and also therapeutic treatment and they display in particular growth-inhibiting effect vis-à-vis brain-tumor cells and cells from human renal, prostate, skin or lung carcinomas.
Description
~~~~~~~3 ,,_haper & Hruemmex GmbH & Co.
Bahnhofstrasse 35 (P 30579 VOE/wo) 3320 Salzgitter 61 / Ringelheim November 1989 Use of 1.2-benzopyrone derivatives for treatinct malignant tumors in humans The invention relates to the use of 1,2-benzopyrone derivatives for producing medicaments for the preventive and therapeutic treatment of malignant tumors in humans.
The concepts currently available for oncological therapy include therapy with substances or radiation in order to thus achieve an immediate cell death and consequently a reduction in the volumes of primary tumor and/or metastases. With radiation-sensitive tumors this principle can be favourably implemented locally, whilst therapy with cytostatic/cytotoxic substances always acts systemically on the whole organism, if locoregional application is not technically possible. The concept of systemic cytosta-sis/cytotoxicity is limited in its biological efficiency by the range of current side-effects. Thus, attempts have been made, for example by modifying cytotoxic molecules such as cis-platinum to carbo-platinum, to reduce toxic side-effects to a tolerable level. A further modification principle is the addition of sub-stances, such as hyaluronidase, verapramil and the like, which increase the biological effectiveness and others, with the aim of reducing the side-effects, to thus allow for a lower dosage of the cytostatic agent used. This concept has been successfully applied in individual protocols.
It has further been attempted to use substances which act through mechanisms other than direct cytostasis, or have available other mechanisms in addition to cytostasis. Thus it is known of cyclophosphamide that in low concentrations it inhibits the biological activity of T4 lymphocytes. Thus, an immunomodulation can be achieved with a cytostatic.
Bahnhofstrasse 35 (P 30579 VOE/wo) 3320 Salzgitter 61 / Ringelheim November 1989 Use of 1.2-benzopyrone derivatives for treatinct malignant tumors in humans The invention relates to the use of 1,2-benzopyrone derivatives for producing medicaments for the preventive and therapeutic treatment of malignant tumors in humans.
The concepts currently available for oncological therapy include therapy with substances or radiation in order to thus achieve an immediate cell death and consequently a reduction in the volumes of primary tumor and/or metastases. With radiation-sensitive tumors this principle can be favourably implemented locally, whilst therapy with cytostatic/cytotoxic substances always acts systemically on the whole organism, if locoregional application is not technically possible. The concept of systemic cytosta-sis/cytotoxicity is limited in its biological efficiency by the range of current side-effects. Thus, attempts have been made, for example by modifying cytotoxic molecules such as cis-platinum to carbo-platinum, to reduce toxic side-effects to a tolerable level. A further modification principle is the addition of sub-stances, such as hyaluronidase, verapramil and the like, which increase the biological effectiveness and others, with the aim of reducing the side-effects, to thus allow for a lower dosage of the cytostatic agent used. This concept has been successfully applied in individual protocols.
It has further been attempted to use substances which act through mechanisms other than direct cytostasis, or have available other mechanisms in addition to cytostasis. Thus it is known of cyclophosphamide that in low concentrations it inhibits the biological activity of T4 lymphocytes. Thus, an immunomodulation can be achieved with a cytostatic.
2~r ~,~ ;~ 3 New therapy conceptions in oncology have concentrated in partic-ular on natural substances which can be produced as pure sub-stances either by plant extraction or by chemical synthesis . Thus it has been shown that coumarin inhibits malignant cell growth in cell cultures and in animal experiments, empirically verified side-effects in the case of mammals are to be disregarded compared with biological activity (cf. for example K. S.
Zaenker, et al.: "Coumarin in melanoma patients, an experimental and clinical study", Drugs Exptl. Clin. Res. X (II) 767-774 ( 1984 ) ; M. E. Marshall et al . , "Treatment of metastatic renal cell carcinoma with Coumarin ( 1, 2-Benzopyrone ) and Ciinetidine, A pilot study", Journal of Clinical Oncology, 5 , 862-866 (1987); D.
Thornes et al., "Prevention of early recurrence of high risk malignant melanoma by coumarin" European Journal of Surgical Oncology 15, 431-435 (1989)).
It is the problem underlying the present invention to make available substances based on natural substances for the preventive and therapeutic treatment of malignant tumors in humans, said substances being superior with regard to their biological activity to the already tested substances and simultaneously being toxicologically acceptable.
To solve the problem the use of 1,2-benzopyrone derivatives of the general formula R~
HO ~O ~O
is proposed, in which Rl is hydrogen, halogen or a hydroxy, sulphonyl, alkyl, hydroxyalkyl, acyloxy, alkoxy or benzyl group or a glycosidic group.
The alkyl groups in the optionally present alkyl, hydroxyalkyl, acyloxy or alkoxy groups are methyl, ethyl or propyl groups.
Zaenker, et al.: "Coumarin in melanoma patients, an experimental and clinical study", Drugs Exptl. Clin. Res. X (II) 767-774 ( 1984 ) ; M. E. Marshall et al . , "Treatment of metastatic renal cell carcinoma with Coumarin ( 1, 2-Benzopyrone ) and Ciinetidine, A pilot study", Journal of Clinical Oncology, 5 , 862-866 (1987); D.
Thornes et al., "Prevention of early recurrence of high risk malignant melanoma by coumarin" European Journal of Surgical Oncology 15, 431-435 (1989)).
It is the problem underlying the present invention to make available substances based on natural substances for the preventive and therapeutic treatment of malignant tumors in humans, said substances being superior with regard to their biological activity to the already tested substances and simultaneously being toxicologically acceptable.
To solve the problem the use of 1,2-benzopyrone derivatives of the general formula R~
HO ~O ~O
is proposed, in which Rl is hydrogen, halogen or a hydroxy, sulphonyl, alkyl, hydroxyalkyl, acyloxy, alkoxy or benzyl group or a glycosidic group.
The alkyl groups in the optionally present alkyl, hydroxyalkyl, acyloxy or alkoxy groups are methyl, ethyl or propyl groups.
As glycosides, for example alpha and beta D-glucosides, especially beta D-glucopyranoside are considered.
Further, according to the invention, derivatives of 1,2-benzopyrone are especially suitable in which R1 is an acyloxy group of the general formula O H
11 ( ~ RZ
- 0 - C - C,~
wherein R2 is hydrogen, a hydroxy group or an amino group and R3 hydrogen or a methyl, ethyl or propyl group.
According to the invention, especially preferred substances are those in which R1 is hydrogen, a hydroxy group, a methoxy group or a beta D-glucopyranosyl group. Of these the first-named, i.e.
7-hydroxy-1,2-benzopyrone (7-OH-1,2-BP) is the most preferred.
The derivatives of 1,2-benzopyrone used according to the inven-tion can be produced using known methods, c.f. Beilstein, E
III/IV 18, 294 ff.
The compounds especially preferred according to the invention, namely 7-hydroxy-1,2-benzopyrone, 6,7-hydroxy-1,2-benzopyrone, 6-(beta-D-glucopyranosyloxy)-?-hydroxy-1,2-benzopyrone and 6-methoxy-7-hydroxy-1,2-benzopyrone are furthermore commercially available as natural substances.
Further, the synthetic production of the glycosides is possible, for example according to H. Wagner et al., Chem. Ber. 102, 3006 (1969).
Finally, the substances according to the invention can be pro-duced enzymatically in a fermenter using processes well known to the expert.
It was surprisingly found that the derivatives of 1,2-benzopyrone according to the invention develop an unexpectedly high in-vivo I I ~~ iw~
'4 .. CJ <i activity with regard to the inhibition of the growth of tumor cells, the regress of tumors or the inhibition of metastases.
As Tables 1 to 3 show, it was possible to demonstrate the growth-inhibiting activity of the derivatives according to the invention in-vivo on numerous tumor cells of varying origin.
Table 1 Cell lines Conc. ~k Growth inhi-(human) 7-OH-1,2-BP bition compared in growth with ocnt~rol medium after x days Origin Symbol umol/ml prostatic carcinoma LNCap 250 66 x = 10 anaplastic astrocytoma g-CCM 200 44 "
anaplastic astrocytoma g-UVW 200 40 "
breast carcinoma MCF 7 200 64 "
bladder carcinoma EJ 200 26 "
Burkitt-lymphoma (leukemia) Daudi 1.2 94.8 x = 9 glioblastoma U 178 MG 25 74 x = 12 neuroblastoma TP 410 N 25 62 x ~ 12 glioblastoma TP 242 MG 25 79 "
glioblastoma TP 336 MG 10 60 "
epidermoidal carcinoma A 431 10 76 "
It is of particular importance that the 1,2-benzopyrone deriva-tives used according to the invention, i.e. 7-hydroxy-1,2-benzo-pyrone and 6,7-hydroxy-1,2-benzopyrone and its 6-ether and -ester have a strong growth-inhibiting effect on brain-tumor cells (glioblastoma cells) (cf. tables 1 to 3), because previously neither chemotherapy, radiation treatment nor therapy with so-called BRM (Biological Response Modifiers) have had therapeutic success. Apart from the interferons, an in-vivo proliferation-inhibition of brain-tumor cells was shown for the first time with the substances according to the invention.
In connection with the neoplastic transformation and increased proliferation rate of glioblastoma cells, two autocrinic loops, the EGF and the PDGF system, are discussed. It was shown that, through 7-OH-1,2-BP, modifications occur at the level of gene expression of one of these systems. It was demonstrated (Seliger et al., unpublished results) that the transcription of the genes coding for PDFG-A and PDFG-B is inhibited (the corresponding mRNA-levels decrease, cf. Fig. 8), whilst the expression of the PDGF receptors and of the EGF system is not influenced. It is conceivable that the inhibition of the PDGF-mRNA induced by 7-OH-1,2-BP is responsible for the observed growth-inhibition, an autocrinic loop being interrupted.
The 1,2-benzopyrone derivatives according to the invention are further particularly suitable for the treatment of renal, bladder, prostate, skin or lung carcinomas as well as leukemia.
i,: r3 The substances according to the invention can be used alone and also in combination with traditional therapeutics and with other methods for the treatment of malignant tumors.
Thus, a combination of the 1,2-benzopyrone derivatives according to the invention and chemotherapeutics such as cis-platinum and 5-fluorouracil (5-FU) showed unexpected synergistic effects (cf.
Example 6).
The substances according to the invention can further be used in addition to traditional therapy with cytokines~and monokins as well as in conjunction with radiation therapy, in order to in-crease the effectiveness thereof and to diminish the toxicity of the therapy scheme. It was shown on different cell lines from prostate carcinomas that using a combination of 7-OH-1,2-BP and tumor-necrosis-factor (TNF) leads to a super-additive inhibition of cell growth (cf. Example 7).
It was further found that in the case of hormone-dependent tumor cells, such as for example (LNCaP cells), the growth inhibition brought about by 7-OH-1,2-BP can be increased by additional hormone therapy in the traditional sense, for example with tes-tosterone (cf. Example 8).
The use of the substances according to the invention can be considered for the prevention of malignant tumors and also for acute and adjuvant therapy.
In the case of preventive treatment with the substances according to the invention, their capacity for chemoprevention and for the inhibition of the oncogene expression in particular plays an important role. Carriers of oncogenes are subject to an increased risk of cancer, because these genes are activated in numerous ways and the expression products can induce the formation of tumors. The term "chemoprevention" describes the ability of suitable substances to prevent or delay the tumor formation J~ N i9 fJ~'::~:.9L aJ
-induced by oncogene expression. The case of the transgenic mouse offers a particular opportunity to test the chemopreventive effect of test substances. (An oncogene - MTV/ras - is trans-ferred by gene manipulation to the fertile ooeyte of certain breeds of mice and the oocyte is reimplanted and delivered. The fully-grown animal and its offspring carry the oncogene in the somatic cells.) The transgenic mice frequently develop tumors.
According to the invention it was possible to show that with these animals the incidence of tumors was significantly reduced compared with control animals thanks to permanent treatment with 7-OH-1,2-BP (cf. Example 11).
In this connection it is also known that the survival time of patients whose tumors disproportionally express certain oncogenes such as "c-myc" and "H-ras", is lower than the survival time of tumor patients whose tumor does not display any increased oncogene expression. According to the invention it was now shown that the substances according to the invention and in particular 7-OH-1,2-BP inhibit the cell growth of tumor cells with oncogene-overexpression (cf. Example 12).
It was further discovered that low dosages of the 1, 2-benzopyrone derivatives according to the invention have an immunomodulating effect (cf. Example 10 and Figs. 5 to 7). In particular it was shown with human mononuclear cells (MNC) that the stimulation of the cells with combinations comprising 7-OH-BP and endotoxins such as lipopolysaccharides of bacterial origin results in a linked interaction of various cytokines. As Fig. 7 shows, a 10-fold increase in the I1-1 level leads to a 7-fold increase in the TNF-(tumor-necrosis-factor)level. Similar results were obtained for I1-6, although a 10-fold increase in the I1-6 level is accompanied by a 4-fold increase in the TNF-level.
If the substances according to the invention are used in the manner described above for the preventive treatment of malignant tumors, daily dosages of for example 50 to 300 mg per patient are :~ ef usable.
In the case of the therapeutic treatment of malignant tumors, the point of emphasis is the growth-inhibiting properties of the substances according to the invention which come into effect at higher daily dosages of for example 300 to 6000 mg. This applies accordingly to treatment after primary therapy, to prevent a tumor relapse and metastasis formation.
Of particular importance - particularly regarding the hitherto usual therapies of malignant tumors - is the fact that no toxic side-effects of any kind were observed when the substances ac-cording to the invention were used on humans, even with long-term administration of extremely high dosages (7000 mg/daily, cf.
Example 9).
The substances used according to the invention are accordingly, for the purposes of the invention, simultaneously highly effec-tive and non-toxic.
As shown above, the 1,2-benzopyrone derivatives used according to the invention can be applied alone or in combination, preferably as fixed combination, with known cytostatics and/or cytokines. When they are administered alone, the daily dosage amounts vary, depending on the indication and therapeutic goal, between 50 and 300 mg if the immunomodulating effect is to be predominant and between 300 and 6000 mg if emphasis is placed on the cytostatic aspect.
These dosage amounts can be administered in the form of the usual pharmaceutical preparations, such as for example solutions, drag~es, capsules, tablets, injection or infusion solutions, orally or parenterally, such as for example intramuscularly, intra-arterially, intravenously and also topically such as for example in the form of transdermal plasters.
n r~
n ; S '~ n ' :, e.. ~a a~
- g -Sterile aqueous solutions which contain the active agent proposed according to the invention are suitable for application. These solutions can, if necessary, be buffered in a suitable manner;
further, the liquid diluting agent can be isotonically set with sufficient salt solution or glucose.
To manufacture the medicaments on the basis of the 1,2-benzopy-rone derivatives used according to the invention, the usual carriers and additives can be used. Usual carriers are e.g.
water, physiological cooking salt solution, alcohols, polyethyl-ene glycols, glycerin ester, gelatine, carbohydrates such as lactose and starch, calcium carbonate, magnesium stearate, tal-cum. Usual additives are e.g. preservatives, lubricants, wetting agents and emulsifiers, colorants, flavourings and aromatic substances. The choice of the carriers and additives depends on whether the preparations according to the invention are to be applied enterally, parenterally or locally.
The invention will be illustrated in the following with reference to Examples:
- 10 - ~~~~~d~ ~~
Example 1 Growth-inhibiting effect of 7-hydroxy-1,2-benzopyrone on various tumor cell lines - comparison with coumarin The following cell lines were investigated:
Glioblastoma U 178 MG
Neuroblastoma TP 410 N
Glioblastoma TP 242 MG
Glioblastoma TP 336 MG
Epidermoidal carcinoma A 431 The neuroblastoma cell line and the three glioblastoma cell lines were cultivated in Ham's F10 medium enriched with 10 ~ fetal calf serum (FCS) and the usual quantity of penicillin, streptomycin (Pen/Strep) and glutamine. The epidermoidal cell line was culti-vated in modified Eagle's Medium (MEM), likewise enriched with ~k (FCS) and the usual quantity of glutamine and Pen/Strep.
In a pre-trial, 5 x 103 cells of each line were in each case initially plated out in 96-well-plates and incubated for 48 hours with differing concentrations, namely 150 ~mol, 75 ~mol, 37.5 umol, 18 ~mol and 9 ~mol for each ml 7-hydroxy-1,2-benzopyrone medium. Untreated cells served as a control. Subsequently, the cells were incubated for 8 hours with 3H-thymidine. The radioac-tivity absorbed by the cells was measured in the usual way. It was shown that the TP 336 and A 431 lines were more sensitive to (7-OH-1,2-BP) than were U 178, TP 242 and TP 410 N. The 37.5 ~M/ml concentration of the active substance was, however, not cytotoxic for any of the cells, but cytostatic.
On the basis of these results a growth curve over a period of 12 days in all was established for the said cell lines, the lines TP 336 MG and A 431 being incubated under the above-described t' ~ '? d e3 ,,,'~~ s3 conditions with 10 ~M/ml 7-OH-1,2-BP in each case, whilst 25 ~M/ml of the active substance was used with the lines TP 242 MG, TP 410 N and U 178 MG. In each case 5 x 104 cells were plated out in T25 bottles, the untreated cells being incubated in analogous conditions as control. The cell count was microscopically determined after 3, 5, 7, 10 and 12 days.
The results are given below in Table 2.
Table 2 Cell Conc. Cell count (x104) % growth in~i-line (~M/ml) after bition compared 7-OH-1,2-BP 3 5 7 10 12 with control days after 12 days U 178 MG 25 15 13 26 2737 74%
Control 23 57 84 91 141 TP 410 N 25 8 20 36 4250 62%
Control 25 45 71 97 132 TP 242 MG 25 4 6 8 10 12 79%
Control 10 15 23 49 57 TP 336 MG 10 7 11 22 18 18 60%
Control 13 25 39 34 45 A 431 10 4 10 3 i2 21 76%
Control 6 24 45 56 87 The table shows that a clear inhibition occurred for all cell lines studied; in the case of TP 242 MG with 25~M/ml, a nearly 80% inhibition of proliferation was observed compared with the ~~ ,!~ ~5 '~~
~, ~.J 'r; j ~.- ~i control. Of particular importance is the growth-inhibition of the brain-tumor cells U 178 MG, TO 410 N, TP 242 MG and TP 336 MG, which - except for interferons - previously could not be achieved with any available substance.
Comparative trial The production of the growth curve was repeated with the exception that, instead of 7-OH-1,2-BP, coumarin was used in the corresponding concentrations.
Not only in the pre-trial, but also in the formulations for the production of a growth curve, no significant difference could be observed between the cell samples treated with coumarin and the control.
Only 7-hydroxy-1,2-benzopyrone, but not the compound unsubstitut-ed in ?-position, developed growth-inhibiting activity on the investigated cell lines.
Example 2 Growth-inhibiting effect of 7-OH-1,2-BP on breast cancer cells The MCF 7 cell line from breast cancer was cultivated in DMEM-S
10, enriched with 10 $ FCS, for 10 days in the usual way. The cells were subsequently incubated with the concentrations of 7-OH-1,2-BP given in Figure 1 and subsequently the survival rate and the cell count were photometrically determined with the ethidium-bromide/acridine orange process in the known manner. The results are given in Figure 1.
They clearly show the cytostatic effect of ?-OH-1,2-BP on breast cancer cells.
~
L
~'.Js~~
Example 3 Antineoplastic effect of 7-OH-1,2-BP on human anaplastic astrocy-toma cells The process as per Example 2 was repeated with the exception that cells of the 6-UVW line from human anaplastic astrocytoma were used and enriched in DMEM:HAM's F 12 medium (1:1) with L-glutamine and 10 % FCS. The results are given in Figure 2.
They show that 7-OH-1,2-BP also has a cytostatic effect on human anaplastic astrocytoma cells.
Example 4 Growth-inhibiting effect of 7-hydroxy-1,2-benzopyrone and deriva-tives of the same on various tumor cell lines The effect of 7-hydroxy-1,2-benzopyrone (7-OH-coumarin), 6,7-hydroxy-1,2-benzopyrone(esculetin),6-beta-D-glucopyranosyloxy-7-hydroxy-1,2-benzopyrone (esculin) and 6-methoxy-7-hydroxy-1,2-benzopyrone (scopoletin) were investigated on the following cell lines:
Glioblastoma Tp242MG
Tp483MG
Neuroblastoma Tp410N
Leukemic K562 Daudi Hypernephroma CAK I-1 Bladder tumor HCV
Breast cancer MCF-7 ~.~ i~~ t~~ r, Prostate carcinoma GV 1 AtT-20 Melanoma A-375 The cell lines were cultivated as indicated in Example 1.
The antiproliferative effect of the 1,2-benzopyrone derivatives according to the invention on the tumor cell lines in each case was shown with the help of the 3H-thymidine insertion and with reference to growth curves.
3H-thymidine assay In a 24-well-plate, in each case 10'~ cells of the line to be investigated were inoculated per well. Approximately 4B hours later the cells were half-confluent; the medium was then changed and replaced by a medium containing the active substance in question (cf Table 3). After a further 48 hours 0.25 a Ci 'H-thymidine (Ammersham) was added to each well and the cells were incubated for a further 24 hours . Subsequently the cultures were washed twice with ice-cold PBS and the high-molecular-weight 3H-radioactivity was precipitated with 5 ~ trichloroacetic acid for one hour at 4 °C. After the washing of the cells in PBS the 3H-radioactivity was solubilized with 0.3M NaOH and measured in the counter. The growth-inhibition was determined by comparing the thymidine insertion of the cells treated with active substance with untreated cells. The results are given in table 3 below.
Table 3 Active substance 3H-thym3.dine insertion (%) of control concentration 7-OH-1,2-BP Esculetin Esculin Scopoletin (~M/ml) A) Glioblastoma Tp242MG/Tp483MG
cells ~
B)'Neuroblastoma cells TP
N
~0~~ ~~~~~
Table 3 (continuation) Active substance 3H-thymidine tracer (%) of control concentration 7-OH-1,2-BP Esculetin Esculin Scopoletin (~M/ml) C) I~eukemic cells Daudi/K-562 ~
8 76/76 75/?7 87/88 79/85 D) CARI cellsCAKI-1/GAKI-2 4 90/88 88/90 92/90 87/$4 - 17 - _h ~r~
~~r::yi:~~'~.
'~' ~~ ~ e9 Table 3 (continuation) Active substance 'H-thymidine tracer (%) of control concentration 7-OH-1,2-BP Esculetin Esculin Scopoletin ( ~rt/ml ) E) HCV cells 8 82 83 89 ' 81 F) Breastcancer cellsMC7/Z-R-75-1 y t ., l' ..:: ~ I,J
Table 3 ~~continuationl Active 3H-thymidine tracer (%) of control substance 7-OH-1,2-BP Esculetin Esculin Scopoletin concentration (NM/ml) G) Prostatecarcinomacells GV-1/AtT 20 25 63/60 75/67 76/69' 78/68 H) Melanomacells 16 72/74 75/72 82/81 "
100 49/53 63/58 64163, 75/76 250 47!42 59/52 60/60 75/71 Producing growth curves The growth curves of the cell lines in each case were determined in the presence of the active substances given below and compared lg -h, ~ r.,.
LY y :I ,:.~ 4' ~ C.~
with controls which had been cultivated without active substance .
The active substance concentration in each case was so chosen that 'H-thymidine insertion was 50 % inhibited. The active sub-stance concentrations used under this aspect far the active sub-stances and cell lines in each case are given in table 4 below in ~M/ml.
Table 4 Cell lines ?-OH-C Ee culetinEsculin Scopoletin Tp 242 MG 25 75 500 500 Tp 483 MG 25 150 100 500 Tp 410 N 25 25 75 500 Daudi 50 250 100 250 CAKI-2 75 500 500 * 500 HCV 500 500 * * 500 MCF-7 100 250 100 * 500 Z-R-75-1 50 175 250 500*
AtT 20 50 250 250 250 A-375 100 500 * * 500 G-361 125 500 500 * 500 * Maximum active-substance dosagewhichcan be used but does not ' lead to a 50 % growth inhibition.
20 - c~~ ~;~.°'F.~e9 ~1 ~ ~- ~ Y j t To produce the growth-curves, in each case 104 cells were inoculated per plate and incubated in the presence of the active substance in question or as a control. After 2, 5, 7 and 10 days the cell count was microscopically determined. Tine results are given in Figures 12 to 24.
The values given there represent the average cell count of three parallel formulations in each case.
Example 5 Tnhibition of the growth of bladder tumor and erythroleukemic cells by 7-OH-1,2-BP .
Cells from 3 different bladder tumors as well as erythroleukemic cells (K 562) were incubated in 3 parallel test-formulations for 20 hours with increasing concentrations of 7-OH-1,2-BP (cf.
Figure 3) in each case in 96-well-plates, washed and mixed with medium. Subsequently the cell count was photometrically determined according to Alley M.C. et al., Cancer Research 48, 589-601 (1988). In the process, a colorant is added to the cells that have been mixed with medium, the colorant being so converted by cells with mitochondrial activity that a photometric determination of the cell count is possible.
The results are given in Figure 3.
They show that, in the case of the cell lines investigated, a dosage-related inhibition of the mitochondrial activity occurs, which can be causally attributed to cell-growth inhibition.
cn ~
~~ ~ ' : a a ~Y ed 3' Example 6.
Synergistic effect of 7-OH-1,2-BP in combination with 5-fluorouracil (5-FU) and with cis-platinum Cells from a colon adenocarcinoma and from a broncho-alveolar carcinoma were used. The cells were cultivated, and the synergis-tic effectiveness during the cell-growth inhibition of 7-OH-1,2-BP, in each case in combination with 5-FU and with cis-platinum, was examined according to the method described by Steel, Int. J.
Radiat. Oncol. Biol./ Phys. 5, 85-91 (1979).
For this purpose, firstly individual dosage-response curves were plotted in each case for 5-FU, cis-platinum and 7-OH-1,2-BP with reference to cell-growth inhibition. Subsequently, fox each of the combinations, the points of equal biological activity were taken from both of the curves and were used as reference points for those dosage amounts which were to be used in a combination test (5-FU + 7-OH-1,2-BP or cis-platinum + 7-OH-1,2-BP). With these concentrations dosage response curves were once again plotted.
The results are given in Figure 4a) and 4b).
They show that both combinations develop a synergistic effect during the inhibition of the cell growth of the cell lines investigated.
Example 7 Synergistic effect of 7-OH-1,2-BP in combination with tumor-necrosis-factor (TNF) Various cell lines (DU-145, PC-3 and LNCaP) from prostate carci-nomas were used.
~d ~ ! ~~ C,D
Cell growth was determined as in Example 1 in a well plate, the cells being cultivated with 7-OH-1,2-BP alone, TNF alone, the combination of 7-OH-1,2-BP and TNF and also without the addition of active substance (control).
The results are given in table 5 below.
Table 5 Cell Control 7-OH-1,2-BP TNF '7-OH-1,2-BP
line (500 ~M/ml) [1 nM/ml] (500 uM/lnMj DU-145 51.86+3.06 38.30+2.92 55.39+0.68 29.97+3.69 PC-3 18.58+1.05 11.51+0.63 18.39+0.74 6.97+0.13 LNCaP 61.40+3.70 11.0+1.10 15.60+2.50 1.60+0.99 Initial cell count: 3 x 104 cells/well; exposure period: DU-145, PC-3: 4 days; LNCaP: 6 days The results show that the combination of 7-OH-1,2-BP with TNF
leads to a super-additive inhibition effect on cell growth.
Example 8 Increasing growth inhibition by combining 7-OH-1,2-BP and testos-terone LNCaP cells were cultivated as described in the previous Examples in a well plate, with the exception that the medium had been previously freed, using charcoal, of any androgynous substances possibly present.
2 3 ' ~~ ~:~ty ,.
Cell growth was determined in the presence of 250 or 500 ~M 7-OH-1,2-BP/ml medium combined with 20 nM testosterone per hole. The results are given in Figure 9. They show that in the presence of 500 ~M 7-OH-1, 2-BP a virtually complete inhibition of cell growth is achieved. Further, the comparison with the compound 3-OH-1,2-BP proves that the latter is essentially ineffective.
Example 9 Toxicity of 7-OH-1,2-BP in human beings 60 patients who were suffering from different, malignant tumors Were treated with 7-OH-1,2-BP in a tolerance study. The initial dosage was 50 mg of the substance/day and the dosage was continu-ously increased up to 2000 mg/day. Over 8 consecutive weeks the patients were examined once a week for signs of systemic toxicity and organic dysfunctions. Because no side-effects at all had oc-curred at the end of the eighth week, the study was continued accompanied by the usual laboratory chemical controls and the daily dosage stepwise increased to 7000 mg/day. Toxic side-effects were not observed even with long-term administration of a daily dosage of 7000 mg/day.
Examx~le 10 Tmmunomodulating effect of 7-OH-1,2-BP
The influence of 7-OH-1,2-BP on the release of lymphokines by human mononuclear cells (MNC) was investigated by incubating the cells for 48 hours with various concentrations (cf . Figures 5 and 6) of 7-OH-1,2-BP dissolved in methanol.for 48 hours. The stock solution contained 10 mg 7-OH-1, 2-BP/ml methanol and was set with RPMI 1640 to the desired concentration in each case. The cells were isolated from the peripheral blood of healthy donors by density-gradient centrifugation according to the method described by Boyum in Scand. J. Clin. Lab. Invest. 21, 77 (1968) and, after - 24 - ~~c~1 fJ'~
z~
washing three times with physiologic salt solution, was suspended in RPMI with a content of 100 M/ml penicillin, 100 ~g/ml strepto-mycin and 2 mM glutamine ( full medium) . The lymphokines interleu-kin-1 (IL 1) and interleukin-6 (IL 6) were measured in the known manner in-vivo following induction with phytohemagglutinin (PHA), concanavalin A (ConA), lipopolysaccharide (LPS) or aKT 3 and adding increasing dosage amounts of 7-OH-1,2-BP. Measurement was carried out in an ELISA using microtiter plates as solid phase (fixed monoclonal antibodies against the lymphokine in question, polyclonal rabbit antibodies against the bound lymphokine, rabbit anti-IgG coupled to alkaline phosphatase as marker and p-nitrophenylphosphate as substrate).
The results are given in Figures 5 and 6.
They show that the release of IL 1 and IL 6, after stimulation with 7-OH-1,2-BP, was increased in the dosage range of 3.6 to 33 ~g/ml. (The inhibition in the higher dosage range is based on the cytotoxic effect of the methanol used as solvent.) At low concentrations, the active substance accordingly stimulates the release of immunomodulators and therefore has a direct immunomodulation effect.
Further, the interaction among various cytokines after stimulation of MNC with 7-OH-1, 2-BP ( 10 ~g/ml ) and LPS ( 10 to 100 pg/ml) was investigated on the MNC of 19 donors. In 6 out of 19 cases (32%) a co-stimulation of TNF-alpha, I1-1 and Il-6 was demonstrated. The calculation of the Spearman correlation coefficient showed a significant correlation between each of the three cytokines (I1-1-beta:Il-6 p = 0.022, I1-1-beta:TNF-alpha p = 0.007, Il-6:TNF-alpha p = 0.007).
The results are shown graphically in Figure 7.
- 2 5 - : , .., .~ ." , C~ ~ , Example 11 Chemopreventive properties of 7-OH-1,2-BP
The chemopreventive properties of 7-OH-1,2-BP were tested on transgenic mice of the Onco Mouses type (mammary glands-neoplasms):
The extent to which 7-OH-1,2-BP is in the position to prevent or delay tumor occurrence and to increase the survival time of the animals was investigated. 7-OH-1,2-BP was fed in drinking water in concentrations of 200 ~rM/ml.
The results are given in table 6 below.
Table 6 Chemoprevention of 7-OH-1,2-BP as demonstrated on transgenic mice No. of mice No. of mice Tumor incidence ~k without 7-OH-coumarin with 7-OH-1.2-BP
Example 12 Growth inhibition of "c-myc"-expressing tumor cells by 7-OH-1,2-BP
a) DUKX cells (ovarian carcinoma cells from Chinese hamsters) which over-express "c-myc" products, were cultivated according to standard conditions, two comparative groups being grown. One group contained the medium 100 ~g/ml 7-OH-1,2-BP, whilst the second group was cultivated as a control group without the active substance.
The development of the cultures was photographically recorded after 24, 48 and 72 hours; the results are given in Figure 10.
They show the dramatic inhibition of cell growth in the presence of 7-OH-1,2-BP (Figure 10, bl to b3).
b) The investigations as per a) were repeated, with the differ-ence that the cells cultivated in the presence of 7-OH-1,2-BP
were freed of the active substance after 24 hours, treated with trypsin and freshly inoculated, and the cell growth was photographically recorded after a further 48 hours in 7-OH-1,2-BP-free medium.
For comparison, corresponding cultures were cultivated for 24 hours with 7-OH-1,2-BP, treated with trypsin and freshly inoculated in a 7-OH-1,2-BP-containing medium.
Cell growth was likewise photographically recorded after 48 hours.
The results are given in Figure 11.
They show a considerable cell growth of those cultures from which the 7-OH-1,2-BP was removed after 24 hours and not replaced during the consequent cultivation period (cf. Figure llbl). By comparison, cell growth was virtually completely inhibited in those cultures for which the second cultivation period also took place in the presence of 7-OH-1,2-BP (Figure 11b2).
The results suggest that the permanent availability of the active substance 7-OH-1,2-BP is necessary for growth-inhibition of tumor cells with oncogenic overexpression.
Further, according to the invention, derivatives of 1,2-benzopyrone are especially suitable in which R1 is an acyloxy group of the general formula O H
11 ( ~ RZ
- 0 - C - C,~
wherein R2 is hydrogen, a hydroxy group or an amino group and R3 hydrogen or a methyl, ethyl or propyl group.
According to the invention, especially preferred substances are those in which R1 is hydrogen, a hydroxy group, a methoxy group or a beta D-glucopyranosyl group. Of these the first-named, i.e.
7-hydroxy-1,2-benzopyrone (7-OH-1,2-BP) is the most preferred.
The derivatives of 1,2-benzopyrone used according to the inven-tion can be produced using known methods, c.f. Beilstein, E
III/IV 18, 294 ff.
The compounds especially preferred according to the invention, namely 7-hydroxy-1,2-benzopyrone, 6,7-hydroxy-1,2-benzopyrone, 6-(beta-D-glucopyranosyloxy)-?-hydroxy-1,2-benzopyrone and 6-methoxy-7-hydroxy-1,2-benzopyrone are furthermore commercially available as natural substances.
Further, the synthetic production of the glycosides is possible, for example according to H. Wagner et al., Chem. Ber. 102, 3006 (1969).
Finally, the substances according to the invention can be pro-duced enzymatically in a fermenter using processes well known to the expert.
It was surprisingly found that the derivatives of 1,2-benzopyrone according to the invention develop an unexpectedly high in-vivo I I ~~ iw~
'4 .. CJ <i activity with regard to the inhibition of the growth of tumor cells, the regress of tumors or the inhibition of metastases.
As Tables 1 to 3 show, it was possible to demonstrate the growth-inhibiting activity of the derivatives according to the invention in-vivo on numerous tumor cells of varying origin.
Table 1 Cell lines Conc. ~k Growth inhi-(human) 7-OH-1,2-BP bition compared in growth with ocnt~rol medium after x days Origin Symbol umol/ml prostatic carcinoma LNCap 250 66 x = 10 anaplastic astrocytoma g-CCM 200 44 "
anaplastic astrocytoma g-UVW 200 40 "
breast carcinoma MCF 7 200 64 "
bladder carcinoma EJ 200 26 "
Burkitt-lymphoma (leukemia) Daudi 1.2 94.8 x = 9 glioblastoma U 178 MG 25 74 x = 12 neuroblastoma TP 410 N 25 62 x ~ 12 glioblastoma TP 242 MG 25 79 "
glioblastoma TP 336 MG 10 60 "
epidermoidal carcinoma A 431 10 76 "
It is of particular importance that the 1,2-benzopyrone deriva-tives used according to the invention, i.e. 7-hydroxy-1,2-benzo-pyrone and 6,7-hydroxy-1,2-benzopyrone and its 6-ether and -ester have a strong growth-inhibiting effect on brain-tumor cells (glioblastoma cells) (cf. tables 1 to 3), because previously neither chemotherapy, radiation treatment nor therapy with so-called BRM (Biological Response Modifiers) have had therapeutic success. Apart from the interferons, an in-vivo proliferation-inhibition of brain-tumor cells was shown for the first time with the substances according to the invention.
In connection with the neoplastic transformation and increased proliferation rate of glioblastoma cells, two autocrinic loops, the EGF and the PDGF system, are discussed. It was shown that, through 7-OH-1,2-BP, modifications occur at the level of gene expression of one of these systems. It was demonstrated (Seliger et al., unpublished results) that the transcription of the genes coding for PDFG-A and PDFG-B is inhibited (the corresponding mRNA-levels decrease, cf. Fig. 8), whilst the expression of the PDGF receptors and of the EGF system is not influenced. It is conceivable that the inhibition of the PDGF-mRNA induced by 7-OH-1,2-BP is responsible for the observed growth-inhibition, an autocrinic loop being interrupted.
The 1,2-benzopyrone derivatives according to the invention are further particularly suitable for the treatment of renal, bladder, prostate, skin or lung carcinomas as well as leukemia.
i,: r3 The substances according to the invention can be used alone and also in combination with traditional therapeutics and with other methods for the treatment of malignant tumors.
Thus, a combination of the 1,2-benzopyrone derivatives according to the invention and chemotherapeutics such as cis-platinum and 5-fluorouracil (5-FU) showed unexpected synergistic effects (cf.
Example 6).
The substances according to the invention can further be used in addition to traditional therapy with cytokines~and monokins as well as in conjunction with radiation therapy, in order to in-crease the effectiveness thereof and to diminish the toxicity of the therapy scheme. It was shown on different cell lines from prostate carcinomas that using a combination of 7-OH-1,2-BP and tumor-necrosis-factor (TNF) leads to a super-additive inhibition of cell growth (cf. Example 7).
It was further found that in the case of hormone-dependent tumor cells, such as for example (LNCaP cells), the growth inhibition brought about by 7-OH-1,2-BP can be increased by additional hormone therapy in the traditional sense, for example with tes-tosterone (cf. Example 8).
The use of the substances according to the invention can be considered for the prevention of malignant tumors and also for acute and adjuvant therapy.
In the case of preventive treatment with the substances according to the invention, their capacity for chemoprevention and for the inhibition of the oncogene expression in particular plays an important role. Carriers of oncogenes are subject to an increased risk of cancer, because these genes are activated in numerous ways and the expression products can induce the formation of tumors. The term "chemoprevention" describes the ability of suitable substances to prevent or delay the tumor formation J~ N i9 fJ~'::~:.9L aJ
-induced by oncogene expression. The case of the transgenic mouse offers a particular opportunity to test the chemopreventive effect of test substances. (An oncogene - MTV/ras - is trans-ferred by gene manipulation to the fertile ooeyte of certain breeds of mice and the oocyte is reimplanted and delivered. The fully-grown animal and its offspring carry the oncogene in the somatic cells.) The transgenic mice frequently develop tumors.
According to the invention it was possible to show that with these animals the incidence of tumors was significantly reduced compared with control animals thanks to permanent treatment with 7-OH-1,2-BP (cf. Example 11).
In this connection it is also known that the survival time of patients whose tumors disproportionally express certain oncogenes such as "c-myc" and "H-ras", is lower than the survival time of tumor patients whose tumor does not display any increased oncogene expression. According to the invention it was now shown that the substances according to the invention and in particular 7-OH-1,2-BP inhibit the cell growth of tumor cells with oncogene-overexpression (cf. Example 12).
It was further discovered that low dosages of the 1, 2-benzopyrone derivatives according to the invention have an immunomodulating effect (cf. Example 10 and Figs. 5 to 7). In particular it was shown with human mononuclear cells (MNC) that the stimulation of the cells with combinations comprising 7-OH-BP and endotoxins such as lipopolysaccharides of bacterial origin results in a linked interaction of various cytokines. As Fig. 7 shows, a 10-fold increase in the I1-1 level leads to a 7-fold increase in the TNF-(tumor-necrosis-factor)level. Similar results were obtained for I1-6, although a 10-fold increase in the I1-6 level is accompanied by a 4-fold increase in the TNF-level.
If the substances according to the invention are used in the manner described above for the preventive treatment of malignant tumors, daily dosages of for example 50 to 300 mg per patient are :~ ef usable.
In the case of the therapeutic treatment of malignant tumors, the point of emphasis is the growth-inhibiting properties of the substances according to the invention which come into effect at higher daily dosages of for example 300 to 6000 mg. This applies accordingly to treatment after primary therapy, to prevent a tumor relapse and metastasis formation.
Of particular importance - particularly regarding the hitherto usual therapies of malignant tumors - is the fact that no toxic side-effects of any kind were observed when the substances ac-cording to the invention were used on humans, even with long-term administration of extremely high dosages (7000 mg/daily, cf.
Example 9).
The substances used according to the invention are accordingly, for the purposes of the invention, simultaneously highly effec-tive and non-toxic.
As shown above, the 1,2-benzopyrone derivatives used according to the invention can be applied alone or in combination, preferably as fixed combination, with known cytostatics and/or cytokines. When they are administered alone, the daily dosage amounts vary, depending on the indication and therapeutic goal, between 50 and 300 mg if the immunomodulating effect is to be predominant and between 300 and 6000 mg if emphasis is placed on the cytostatic aspect.
These dosage amounts can be administered in the form of the usual pharmaceutical preparations, such as for example solutions, drag~es, capsules, tablets, injection or infusion solutions, orally or parenterally, such as for example intramuscularly, intra-arterially, intravenously and also topically such as for example in the form of transdermal plasters.
n r~
n ; S '~ n ' :, e.. ~a a~
- g -Sterile aqueous solutions which contain the active agent proposed according to the invention are suitable for application. These solutions can, if necessary, be buffered in a suitable manner;
further, the liquid diluting agent can be isotonically set with sufficient salt solution or glucose.
To manufacture the medicaments on the basis of the 1,2-benzopy-rone derivatives used according to the invention, the usual carriers and additives can be used. Usual carriers are e.g.
water, physiological cooking salt solution, alcohols, polyethyl-ene glycols, glycerin ester, gelatine, carbohydrates such as lactose and starch, calcium carbonate, magnesium stearate, tal-cum. Usual additives are e.g. preservatives, lubricants, wetting agents and emulsifiers, colorants, flavourings and aromatic substances. The choice of the carriers and additives depends on whether the preparations according to the invention are to be applied enterally, parenterally or locally.
The invention will be illustrated in the following with reference to Examples:
- 10 - ~~~~~d~ ~~
Example 1 Growth-inhibiting effect of 7-hydroxy-1,2-benzopyrone on various tumor cell lines - comparison with coumarin The following cell lines were investigated:
Glioblastoma U 178 MG
Neuroblastoma TP 410 N
Glioblastoma TP 242 MG
Glioblastoma TP 336 MG
Epidermoidal carcinoma A 431 The neuroblastoma cell line and the three glioblastoma cell lines were cultivated in Ham's F10 medium enriched with 10 ~ fetal calf serum (FCS) and the usual quantity of penicillin, streptomycin (Pen/Strep) and glutamine. The epidermoidal cell line was culti-vated in modified Eagle's Medium (MEM), likewise enriched with ~k (FCS) and the usual quantity of glutamine and Pen/Strep.
In a pre-trial, 5 x 103 cells of each line were in each case initially plated out in 96-well-plates and incubated for 48 hours with differing concentrations, namely 150 ~mol, 75 ~mol, 37.5 umol, 18 ~mol and 9 ~mol for each ml 7-hydroxy-1,2-benzopyrone medium. Untreated cells served as a control. Subsequently, the cells were incubated for 8 hours with 3H-thymidine. The radioac-tivity absorbed by the cells was measured in the usual way. It was shown that the TP 336 and A 431 lines were more sensitive to (7-OH-1,2-BP) than were U 178, TP 242 and TP 410 N. The 37.5 ~M/ml concentration of the active substance was, however, not cytotoxic for any of the cells, but cytostatic.
On the basis of these results a growth curve over a period of 12 days in all was established for the said cell lines, the lines TP 336 MG and A 431 being incubated under the above-described t' ~ '? d e3 ,,,'~~ s3 conditions with 10 ~M/ml 7-OH-1,2-BP in each case, whilst 25 ~M/ml of the active substance was used with the lines TP 242 MG, TP 410 N and U 178 MG. In each case 5 x 104 cells were plated out in T25 bottles, the untreated cells being incubated in analogous conditions as control. The cell count was microscopically determined after 3, 5, 7, 10 and 12 days.
The results are given below in Table 2.
Table 2 Cell Conc. Cell count (x104) % growth in~i-line (~M/ml) after bition compared 7-OH-1,2-BP 3 5 7 10 12 with control days after 12 days U 178 MG 25 15 13 26 2737 74%
Control 23 57 84 91 141 TP 410 N 25 8 20 36 4250 62%
Control 25 45 71 97 132 TP 242 MG 25 4 6 8 10 12 79%
Control 10 15 23 49 57 TP 336 MG 10 7 11 22 18 18 60%
Control 13 25 39 34 45 A 431 10 4 10 3 i2 21 76%
Control 6 24 45 56 87 The table shows that a clear inhibition occurred for all cell lines studied; in the case of TP 242 MG with 25~M/ml, a nearly 80% inhibition of proliferation was observed compared with the ~~ ,!~ ~5 '~~
~, ~.J 'r; j ~.- ~i control. Of particular importance is the growth-inhibition of the brain-tumor cells U 178 MG, TO 410 N, TP 242 MG and TP 336 MG, which - except for interferons - previously could not be achieved with any available substance.
Comparative trial The production of the growth curve was repeated with the exception that, instead of 7-OH-1,2-BP, coumarin was used in the corresponding concentrations.
Not only in the pre-trial, but also in the formulations for the production of a growth curve, no significant difference could be observed between the cell samples treated with coumarin and the control.
Only 7-hydroxy-1,2-benzopyrone, but not the compound unsubstitut-ed in ?-position, developed growth-inhibiting activity on the investigated cell lines.
Example 2 Growth-inhibiting effect of 7-OH-1,2-BP on breast cancer cells The MCF 7 cell line from breast cancer was cultivated in DMEM-S
10, enriched with 10 $ FCS, for 10 days in the usual way. The cells were subsequently incubated with the concentrations of 7-OH-1,2-BP given in Figure 1 and subsequently the survival rate and the cell count were photometrically determined with the ethidium-bromide/acridine orange process in the known manner. The results are given in Figure 1.
They clearly show the cytostatic effect of ?-OH-1,2-BP on breast cancer cells.
~
L
~'.Js~~
Example 3 Antineoplastic effect of 7-OH-1,2-BP on human anaplastic astrocy-toma cells The process as per Example 2 was repeated with the exception that cells of the 6-UVW line from human anaplastic astrocytoma were used and enriched in DMEM:HAM's F 12 medium (1:1) with L-glutamine and 10 % FCS. The results are given in Figure 2.
They show that 7-OH-1,2-BP also has a cytostatic effect on human anaplastic astrocytoma cells.
Example 4 Growth-inhibiting effect of 7-hydroxy-1,2-benzopyrone and deriva-tives of the same on various tumor cell lines The effect of 7-hydroxy-1,2-benzopyrone (7-OH-coumarin), 6,7-hydroxy-1,2-benzopyrone(esculetin),6-beta-D-glucopyranosyloxy-7-hydroxy-1,2-benzopyrone (esculin) and 6-methoxy-7-hydroxy-1,2-benzopyrone (scopoletin) were investigated on the following cell lines:
Glioblastoma Tp242MG
Tp483MG
Neuroblastoma Tp410N
Leukemic K562 Daudi Hypernephroma CAK I-1 Bladder tumor HCV
Breast cancer MCF-7 ~.~ i~~ t~~ r, Prostate carcinoma GV 1 AtT-20 Melanoma A-375 The cell lines were cultivated as indicated in Example 1.
The antiproliferative effect of the 1,2-benzopyrone derivatives according to the invention on the tumor cell lines in each case was shown with the help of the 3H-thymidine insertion and with reference to growth curves.
3H-thymidine assay In a 24-well-plate, in each case 10'~ cells of the line to be investigated were inoculated per well. Approximately 4B hours later the cells were half-confluent; the medium was then changed and replaced by a medium containing the active substance in question (cf Table 3). After a further 48 hours 0.25 a Ci 'H-thymidine (Ammersham) was added to each well and the cells were incubated for a further 24 hours . Subsequently the cultures were washed twice with ice-cold PBS and the high-molecular-weight 3H-radioactivity was precipitated with 5 ~ trichloroacetic acid for one hour at 4 °C. After the washing of the cells in PBS the 3H-radioactivity was solubilized with 0.3M NaOH and measured in the counter. The growth-inhibition was determined by comparing the thymidine insertion of the cells treated with active substance with untreated cells. The results are given in table 3 below.
Table 3 Active substance 3H-thym3.dine insertion (%) of control concentration 7-OH-1,2-BP Esculetin Esculin Scopoletin (~M/ml) A) Glioblastoma Tp242MG/Tp483MG
cells ~
B)'Neuroblastoma cells TP
N
~0~~ ~~~~~
Table 3 (continuation) Active substance 3H-thymidine tracer (%) of control concentration 7-OH-1,2-BP Esculetin Esculin Scopoletin (~M/ml) C) I~eukemic cells Daudi/K-562 ~
8 76/76 75/?7 87/88 79/85 D) CARI cellsCAKI-1/GAKI-2 4 90/88 88/90 92/90 87/$4 - 17 - _h ~r~
~~r::yi:~~'~.
'~' ~~ ~ e9 Table 3 (continuation) Active substance 'H-thymidine tracer (%) of control concentration 7-OH-1,2-BP Esculetin Esculin Scopoletin ( ~rt/ml ) E) HCV cells 8 82 83 89 ' 81 F) Breastcancer cellsMC7/Z-R-75-1 y t ., l' ..:: ~ I,J
Table 3 ~~continuationl Active 3H-thymidine tracer (%) of control substance 7-OH-1,2-BP Esculetin Esculin Scopoletin concentration (NM/ml) G) Prostatecarcinomacells GV-1/AtT 20 25 63/60 75/67 76/69' 78/68 H) Melanomacells 16 72/74 75/72 82/81 "
100 49/53 63/58 64163, 75/76 250 47!42 59/52 60/60 75/71 Producing growth curves The growth curves of the cell lines in each case were determined in the presence of the active substances given below and compared lg -h, ~ r.,.
LY y :I ,:.~ 4' ~ C.~
with controls which had been cultivated without active substance .
The active substance concentration in each case was so chosen that 'H-thymidine insertion was 50 % inhibited. The active sub-stance concentrations used under this aspect far the active sub-stances and cell lines in each case are given in table 4 below in ~M/ml.
Table 4 Cell lines ?-OH-C Ee culetinEsculin Scopoletin Tp 242 MG 25 75 500 500 Tp 483 MG 25 150 100 500 Tp 410 N 25 25 75 500 Daudi 50 250 100 250 CAKI-2 75 500 500 * 500 HCV 500 500 * * 500 MCF-7 100 250 100 * 500 Z-R-75-1 50 175 250 500*
AtT 20 50 250 250 250 A-375 100 500 * * 500 G-361 125 500 500 * 500 * Maximum active-substance dosagewhichcan be used but does not ' lead to a 50 % growth inhibition.
20 - c~~ ~;~.°'F.~e9 ~1 ~ ~- ~ Y j t To produce the growth-curves, in each case 104 cells were inoculated per plate and incubated in the presence of the active substance in question or as a control. After 2, 5, 7 and 10 days the cell count was microscopically determined. Tine results are given in Figures 12 to 24.
The values given there represent the average cell count of three parallel formulations in each case.
Example 5 Tnhibition of the growth of bladder tumor and erythroleukemic cells by 7-OH-1,2-BP .
Cells from 3 different bladder tumors as well as erythroleukemic cells (K 562) were incubated in 3 parallel test-formulations for 20 hours with increasing concentrations of 7-OH-1,2-BP (cf.
Figure 3) in each case in 96-well-plates, washed and mixed with medium. Subsequently the cell count was photometrically determined according to Alley M.C. et al., Cancer Research 48, 589-601 (1988). In the process, a colorant is added to the cells that have been mixed with medium, the colorant being so converted by cells with mitochondrial activity that a photometric determination of the cell count is possible.
The results are given in Figure 3.
They show that, in the case of the cell lines investigated, a dosage-related inhibition of the mitochondrial activity occurs, which can be causally attributed to cell-growth inhibition.
cn ~
~~ ~ ' : a a ~Y ed 3' Example 6.
Synergistic effect of 7-OH-1,2-BP in combination with 5-fluorouracil (5-FU) and with cis-platinum Cells from a colon adenocarcinoma and from a broncho-alveolar carcinoma were used. The cells were cultivated, and the synergis-tic effectiveness during the cell-growth inhibition of 7-OH-1,2-BP, in each case in combination with 5-FU and with cis-platinum, was examined according to the method described by Steel, Int. J.
Radiat. Oncol. Biol./ Phys. 5, 85-91 (1979).
For this purpose, firstly individual dosage-response curves were plotted in each case for 5-FU, cis-platinum and 7-OH-1,2-BP with reference to cell-growth inhibition. Subsequently, fox each of the combinations, the points of equal biological activity were taken from both of the curves and were used as reference points for those dosage amounts which were to be used in a combination test (5-FU + 7-OH-1,2-BP or cis-platinum + 7-OH-1,2-BP). With these concentrations dosage response curves were once again plotted.
The results are given in Figure 4a) and 4b).
They show that both combinations develop a synergistic effect during the inhibition of the cell growth of the cell lines investigated.
Example 7 Synergistic effect of 7-OH-1,2-BP in combination with tumor-necrosis-factor (TNF) Various cell lines (DU-145, PC-3 and LNCaP) from prostate carci-nomas were used.
~d ~ ! ~~ C,D
Cell growth was determined as in Example 1 in a well plate, the cells being cultivated with 7-OH-1,2-BP alone, TNF alone, the combination of 7-OH-1,2-BP and TNF and also without the addition of active substance (control).
The results are given in table 5 below.
Table 5 Cell Control 7-OH-1,2-BP TNF '7-OH-1,2-BP
line (500 ~M/ml) [1 nM/ml] (500 uM/lnMj DU-145 51.86+3.06 38.30+2.92 55.39+0.68 29.97+3.69 PC-3 18.58+1.05 11.51+0.63 18.39+0.74 6.97+0.13 LNCaP 61.40+3.70 11.0+1.10 15.60+2.50 1.60+0.99 Initial cell count: 3 x 104 cells/well; exposure period: DU-145, PC-3: 4 days; LNCaP: 6 days The results show that the combination of 7-OH-1,2-BP with TNF
leads to a super-additive inhibition effect on cell growth.
Example 8 Increasing growth inhibition by combining 7-OH-1,2-BP and testos-terone LNCaP cells were cultivated as described in the previous Examples in a well plate, with the exception that the medium had been previously freed, using charcoal, of any androgynous substances possibly present.
2 3 ' ~~ ~:~ty ,.
Cell growth was determined in the presence of 250 or 500 ~M 7-OH-1,2-BP/ml medium combined with 20 nM testosterone per hole. The results are given in Figure 9. They show that in the presence of 500 ~M 7-OH-1, 2-BP a virtually complete inhibition of cell growth is achieved. Further, the comparison with the compound 3-OH-1,2-BP proves that the latter is essentially ineffective.
Example 9 Toxicity of 7-OH-1,2-BP in human beings 60 patients who were suffering from different, malignant tumors Were treated with 7-OH-1,2-BP in a tolerance study. The initial dosage was 50 mg of the substance/day and the dosage was continu-ously increased up to 2000 mg/day. Over 8 consecutive weeks the patients were examined once a week for signs of systemic toxicity and organic dysfunctions. Because no side-effects at all had oc-curred at the end of the eighth week, the study was continued accompanied by the usual laboratory chemical controls and the daily dosage stepwise increased to 7000 mg/day. Toxic side-effects were not observed even with long-term administration of a daily dosage of 7000 mg/day.
Examx~le 10 Tmmunomodulating effect of 7-OH-1,2-BP
The influence of 7-OH-1,2-BP on the release of lymphokines by human mononuclear cells (MNC) was investigated by incubating the cells for 48 hours with various concentrations (cf . Figures 5 and 6) of 7-OH-1,2-BP dissolved in methanol.for 48 hours. The stock solution contained 10 mg 7-OH-1, 2-BP/ml methanol and was set with RPMI 1640 to the desired concentration in each case. The cells were isolated from the peripheral blood of healthy donors by density-gradient centrifugation according to the method described by Boyum in Scand. J. Clin. Lab. Invest. 21, 77 (1968) and, after - 24 - ~~c~1 fJ'~
z~
washing three times with physiologic salt solution, was suspended in RPMI with a content of 100 M/ml penicillin, 100 ~g/ml strepto-mycin and 2 mM glutamine ( full medium) . The lymphokines interleu-kin-1 (IL 1) and interleukin-6 (IL 6) were measured in the known manner in-vivo following induction with phytohemagglutinin (PHA), concanavalin A (ConA), lipopolysaccharide (LPS) or aKT 3 and adding increasing dosage amounts of 7-OH-1,2-BP. Measurement was carried out in an ELISA using microtiter plates as solid phase (fixed monoclonal antibodies against the lymphokine in question, polyclonal rabbit antibodies against the bound lymphokine, rabbit anti-IgG coupled to alkaline phosphatase as marker and p-nitrophenylphosphate as substrate).
The results are given in Figures 5 and 6.
They show that the release of IL 1 and IL 6, after stimulation with 7-OH-1,2-BP, was increased in the dosage range of 3.6 to 33 ~g/ml. (The inhibition in the higher dosage range is based on the cytotoxic effect of the methanol used as solvent.) At low concentrations, the active substance accordingly stimulates the release of immunomodulators and therefore has a direct immunomodulation effect.
Further, the interaction among various cytokines after stimulation of MNC with 7-OH-1, 2-BP ( 10 ~g/ml ) and LPS ( 10 to 100 pg/ml) was investigated on the MNC of 19 donors. In 6 out of 19 cases (32%) a co-stimulation of TNF-alpha, I1-1 and Il-6 was demonstrated. The calculation of the Spearman correlation coefficient showed a significant correlation between each of the three cytokines (I1-1-beta:Il-6 p = 0.022, I1-1-beta:TNF-alpha p = 0.007, Il-6:TNF-alpha p = 0.007).
The results are shown graphically in Figure 7.
- 2 5 - : , .., .~ ." , C~ ~ , Example 11 Chemopreventive properties of 7-OH-1,2-BP
The chemopreventive properties of 7-OH-1,2-BP were tested on transgenic mice of the Onco Mouses type (mammary glands-neoplasms):
The extent to which 7-OH-1,2-BP is in the position to prevent or delay tumor occurrence and to increase the survival time of the animals was investigated. 7-OH-1,2-BP was fed in drinking water in concentrations of 200 ~rM/ml.
The results are given in table 6 below.
Table 6 Chemoprevention of 7-OH-1,2-BP as demonstrated on transgenic mice No. of mice No. of mice Tumor incidence ~k without 7-OH-coumarin with 7-OH-1.2-BP
Example 12 Growth inhibition of "c-myc"-expressing tumor cells by 7-OH-1,2-BP
a) DUKX cells (ovarian carcinoma cells from Chinese hamsters) which over-express "c-myc" products, were cultivated according to standard conditions, two comparative groups being grown. One group contained the medium 100 ~g/ml 7-OH-1,2-BP, whilst the second group was cultivated as a control group without the active substance.
The development of the cultures was photographically recorded after 24, 48 and 72 hours; the results are given in Figure 10.
They show the dramatic inhibition of cell growth in the presence of 7-OH-1,2-BP (Figure 10, bl to b3).
b) The investigations as per a) were repeated, with the differ-ence that the cells cultivated in the presence of 7-OH-1,2-BP
were freed of the active substance after 24 hours, treated with trypsin and freshly inoculated, and the cell growth was photographically recorded after a further 48 hours in 7-OH-1,2-BP-free medium.
For comparison, corresponding cultures were cultivated for 24 hours with 7-OH-1,2-BP, treated with trypsin and freshly inoculated in a 7-OH-1,2-BP-containing medium.
Cell growth was likewise photographically recorded after 48 hours.
The results are given in Figure 11.
They show a considerable cell growth of those cultures from which the 7-OH-1,2-BP was removed after 24 hours and not replaced during the consequent cultivation period (cf. Figure llbl). By comparison, cell growth was virtually completely inhibited in those cultures for which the second cultivation period also took place in the presence of 7-OH-1,2-BP (Figure 11b2).
The results suggest that the permanent availability of the active substance 7-OH-1,2-BP is necessary for growth-inhibition of tumor cells with oncogenic overexpression.
Claims (9)
1. Use of 7-OH-1,2-benzopyrone for producing medicaments for the preventive and/or therapeutic treatment of malignant tumors in humans.
2. Use according to claim 1 in combination with usual cytostatics.
3. Use according to claim 1 in combination with cytokines.
4. Use according to claims 1 to 3 in a daily dosage of 50 to 300 mg for the preventive treatment of malignant tumors in humans.
5. Use according to claims 1 to 3 in a daily dosage of 300 to 6000 mg for the therapeutic treatment of malignant tumors in human beings.
6. Use according to claims 1 to 5 for producing medicaments for the treatment of brain-tumors.
7. Use according to claims 1 to 5 for producing medicaments for the treatment of human renal, prostate, skin or lungcarcinomas or leukemia.
8. Use according to claims 1 to 5 for producing medicaments for the treatment of tumors induced by oncogene expression.
9. Use according to claim 1 in combination with hormones for producing medicaments for the treatment of hormone-dependent tumors.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3938902.2 | 1989-11-24 | ||
| DE3938902A DE3938902C1 (en) | 1989-11-24 | 1989-11-24 | |
| PCT/EP1990/002003 WO1991007959A1 (en) | 1989-11-24 | 1990-11-23 | Use of 1,2-benzopyrone derivatives for the treatment of malignant tumours in humans |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2045593A1 CA2045593A1 (en) | 1991-05-25 |
| CA2045593C true CA2045593C (en) | 2000-09-05 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002045593A Expired - Lifetime CA2045593C (en) | 1989-11-24 | 1990-11-23 | Use of 1,2-benzopyrone derivatives for the treatment of malignant tumours in humans |
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| Country | Link |
|---|---|
| EP (1) | EP0455769B1 (en) |
| JP (1) | JP3053645B2 (en) |
| KR (1) | KR0177821B1 (en) |
| AT (1) | ATE128354T1 (en) |
| AU (1) | AU6739690A (en) |
| BR (1) | BR9007080A (en) |
| CA (1) | CA2045593C (en) |
| DE (2) | DE3938902C1 (en) |
| DK (1) | DK0455769T3 (en) |
| FI (1) | FI913531A0 (en) |
| WO (1) | WO1991007959A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| FR2822379B1 (en) * | 2001-03-20 | 2007-06-15 | Sederma Sa | COSMETIC AND DERMOPHARMACEUTICAL COMPOSITIONS CONTAINING UMBELLIFERONE |
| US7148252B2 (en) * | 2001-10-03 | 2006-12-12 | Signal Pharmaceuticals, Llc | Use of benzopyranones for treating or preventing a primary brain cancer or a brain metastasis |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1985003865A1 (en) * | 1984-03-01 | 1985-09-12 | Casley Smith John Royle | Treatment of high protein oedemas in animals |
-
1989
- 1989-11-24 DE DE3938902A patent/DE3938902C1/de not_active Expired - Lifetime
-
1990
- 1990-11-23 WO PCT/EP1990/002003 patent/WO1991007959A1/en active IP Right Grant
- 1990-11-23 AU AU67396/90A patent/AU6739690A/en not_active Abandoned
- 1990-11-23 DK DK90917032.6T patent/DK0455769T3/en active
- 1990-11-23 BR BR909007080A patent/BR9007080A/en not_active Application Discontinuation
- 1990-11-23 EP EP90917032A patent/EP0455769B1/en not_active Expired - Lifetime
- 1990-11-23 AT AT90917032T patent/ATE128354T1/en not_active IP Right Cessation
- 1990-11-23 FI FI913531A patent/FI913531A0/en not_active Application Discontinuation
- 1990-11-23 DE DE59009724T patent/DE59009724D1/en not_active Expired - Fee Related
- 1990-11-23 CA CA002045593A patent/CA2045593C/en not_active Expired - Lifetime
- 1990-11-23 JP JP2515771A patent/JP3053645B2/en not_active Expired - Fee Related
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1991
- 1991-07-22 KR KR1019910700768A patent/KR0177821B1/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| KR920700636A (en) | 1992-08-10 |
| JPH04505020A (en) | 1992-09-03 |
| BR9007080A (en) | 1991-12-24 |
| EP0455769A1 (en) | 1991-11-13 |
| DE59009724D1 (en) | 1995-11-02 |
| KR0177821B1 (en) | 1999-03-20 |
| EP0455769B1 (en) | 1995-09-27 |
| FI913531A7 (en) | 1991-07-23 |
| WO1991007959A1 (en) | 1991-06-13 |
| CA2045593A1 (en) | 1991-05-25 |
| AU6739690A (en) | 1991-06-26 |
| ATE128354T1 (en) | 1995-10-15 |
| DE3938902C1 (en) | 1991-05-08 |
| JP3053645B2 (en) | 2000-06-19 |
| DK0455769T3 (en) | 1996-02-05 |
| FI913531A0 (en) | 1991-07-23 |
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