WO1994012186A1 - Prostaglandin derivative having radioprotective effects - Google Patents
Prostaglandin derivative having radioprotective effects Download PDFInfo
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- WO1994012186A1 WO1994012186A1 PCT/EP1993/003359 EP9303359W WO9412186A1 WO 1994012186 A1 WO1994012186 A1 WO 1994012186A1 EP 9303359 W EP9303359 W EP 9303359W WO 9412186 A1 WO9412186 A1 WO 9412186A1
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- nocloprost
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- radiation
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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
- A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
Definitions
- the present invention relates to the new use of nocloprost or a pharmacologically acceptable salt thereof for producing a composition for treating or preventing of irradiation caused damage of human normal cells and to a radioprotective composition which comprises nocloprost or its pharmacologically acceptable salt and a pharmaceutically acceptable carrier.
- 9-chloroprostaglandin derivatives such as nocloprost are already known.
- EP-303377 describes the preparation of nocloprost (9 ⁇ -chloro-16,16- dimethyl-prostaglandin E2) and the pharmacologically acceptable salts thereof and their use in human medicine with substantially improved effectiveness than the corresponding natural prostaglandins.
- Prostaglandins seem to be able to protect gastric and intestinal mucosa from the ulcerogenic effect of ethanol or indomethacin (Robert et al., Gastroenterology, 77, 433- 443, 1979) or from radiation (Prasad, Int. Journal of Radiation Biology, 22, 187-189, 1972) even through this latter effect has not been consistently reported by in vitro studies (Holahan et al., Prostaglandin and Lipid Metabolism in Radiation Injury, 253-262, 1987).
- prostaglandins can also protect cancer cells from radiation injury and thus limit radiotherapy efficacy in cancer patients has not been adequately investigated or excluded.
- Nocloprost itself has already been proved to prevent, in a dose-dependent manner, acute gastric lesions induced by ethanol, aspirin and acidified tarocholate in rats (Kounturek et al., Proceeding of 7th International Conference on Prostaglandins and Related Compounds, 198, 1990) and to promote healing of chronic gastric ulcerations induced by acetic acid and indomethacin.
- nocloprost shows a specific radioprotective effect in normal human cells and no influence on the cytotoxic effect of ionizing radiation on cancer cells.
- Nocloprost or its salts is used according to this invention in amounts which are in the range of the amounts thus far used in human studies.
- nocloprost can be administered to mammals orally or parenterally.
- the dose of nocloprost is from 0.1 to 1500 g/kg/day when administered to human patients.
- the unit dose for the pharmaceutically acceptable carrier is from 0.01 to 100 mg.
- the dosage for i.v. administration as a continuous infusion in customary aqueous solvents is preferably between 0.1 ng/kg/min. and 0.1 ⁇ g/kg/min..
- the active ingredient according to the invention is used in conjunction with the adjuvants known and customary in galenical pharmacy, for example for the preparation of cytoprotective agents.
- suitable compositions are described for example in EP- 30377.
- nocloprost has been demonstrated in pharmacological tests.
- the effect exerted by nocloprost or its salts is investigated on normal human dermal fibroblasts and colon adenocarcinoma cells.
- the compound did not influence proliferation or clonogenic capacity and it did not induce any DNA strand breakage in normal or tumor cells.
- Preincubation with nocloprost induced an enhancement in the fraction of cells able to survive ionizing radiation that was paralleled by a reduction in the amount of radiation induced desoxyribonucleic acid double strand breaks (DNA DSB).
- Normal human dermal fibroblasts are obtained as cryopreserved primary cultures from Promo Cell (Heidelberg, Germany).
- the LoVo cell line is derived from a human colon adenocarcinoma (Drewinko et al., 1977) and mainly consits of epithelial-like cells.
- the LoVo cell line is maintained in a logarithmic phase monolayer culture using Ham's F- 12 medium supplemented with 10% fetal calf serum in a humidified atmosphere containing 5% CO2 and 95% air at 37°C. Under such growth conditions the time for doubling of the cell population is approximately 30 h.
- Nocloprost (9- ⁇ -chloro-16,16-dimethyl PGE2), is dissolved in absolute ethanol at 5.2 mg/ml and stored at -20°C. The compound is diluted with medium immediately prior to addition to cells. In all experiments the final ethanol concentration is less than 0.1%.
- Exponentially growing cells are harvested with trypsin:EDTA (0.005:0.02%) and irradiated with a - ⁇ - ⁇ Cs gamma irradiator (IBL-437, Oris, France) at a dose rate of 10 Gy/min. Irradiations are carried out in ice for filter elution experiments and at room temperature for proliferation and colony formation experiments. For DNA repair studies, cells are added to prewarmed culture medium and incubated at 37°C for the designated time.
- normal human fibroblasts are plated at appropriate concentrations in 25-mm ⁇ plastic flasks in supplemented medium. After 7 days, cells are harvested, assayed for cell viability (trypan blue dye-exclusion test) and counted (Coulter Counter, Kontron, Everett, Mass., USA).
- LoVo cells are plated at appropriate concentrations in plastic dishes and incubated at 37°C in an atmosphere of 5% CO2- 95% air for 12-14 days. Colonies, consisting of 50 or more cells, are stained with crystal violet in 70% v/v ethanol/water and counted under a microscope.
- Cells are labeled with 0.1 ⁇ d/ml of [ ⁇ C] TdR for 24 h and then washed and cultured in non-radioactive medium for 12 h before treatment with nocloprost and/or radiation.
- the frequency of desoxyribonucleic acid double strand breaks is measured by the neutral elution method as described by Bradley and Kohn (Nucleic Acids Research, 7, 793-804, 1979).
- Cells are transferred to 25 mm-diameter (2.0 ⁇ m pore size) polycarbonate filters and lysed with a solution containing 0.05 M Tris, 0.05 M glycine, 0.025 M disodium EDTA 2% SDS and 0.5 mg/ml proteinase K (pH 9.6).
- DNA is then slowly eluted from the filters using the same solution, minus proteinase K, using a Miniplus 2 peristaltic pump (Gilson, Middletown, WI, USA). In both cases, elution is continued for 15 h, and the eluted DNA is collected in five fractions.
- the [l ⁇ C] activity is analyzed by liquid scintillation counting after adding Ready Gel (Beckman Instruments, Inc. Fullerton, C USA). DNA remaining on the filter at the end of the elution is removed by hydrolysis in 0.4 ml of 1 M HC1 at 80°C for 1 h, followed by treatment with 0.6 ml of 1 M NaOH at room temperature for 30 min and mechanical agitation.
- SSF strand scission factor
- the amount af DSB remaining after irradiation is calculated as the ratio of SSF values obtained in cells allowed no time for rejoining to those of cells allowed different intervals of time to repair after irradiation.
- DNA DSB is determined after an exposure to each nocloprost concentration for 120 min.
- the elution kinetics of DNA for treated normal and tumor cells is superimposable on that observed for controls.
- Pretreatment with nocloprost causes an enhancement in the fraction of normal fibroblasts surviving 19 Gy irradiation compared to samples treated with radiation alone.
- the protective effect increases as a function of nocloprost concentration (Table 3)
- compositions containing nocloprost as active ingredient should be useful in preventing damage of human normal cells by irradiation.
- Fig. 1 Radiation survival curves for LoVo cells.
- Solid line Solid line, radiation alone; broken line, 10.0 ⁇ g/ml of nocloprost for 120 min.
- the points represent mean values based on 3 independent experiments. The SD is always within 5%.
- Fig. 2. and Fig. 3. Representative neutral elution profiles depicting the elution kinetics of DNA from human normal dermal fibroblasts and LoVo cells exposed to 50 Gy with or without a 120 min preincubation with 10 ⁇ g/ml of nocloprost.
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Abstract
The present invention relates to a composition having radioprotective action in human cells which contains nocloprost as active ingredient.
Description
PROSTAGLANDIN DERIVATIVE HAVING RADIOPROTECTIVE EFFECTS
The present invention relates to the new use of nocloprost or a pharmacologically acceptable salt thereof for producing a composition for treating or preventing of irradiation caused damage of human normal cells and to a radioprotective composition which comprises nocloprost or its pharmacologically acceptable salt and a pharmaceutically acceptable carrier.
Therapeutic uses of 9-chloroprostaglandin derivatives such as nocloprost are already known. For example EP-303377 describes the preparation of nocloprost (9β-chloro-16,16- dimethyl-prostaglandin E2) and the pharmacologically acceptable salts thereof and their use in human medicine with substantially improved effectiveness than the corresponding natural prostaglandins.
Prostaglandins seem to be able to protect gastric and intestinal mucosa from the ulcerogenic effect of ethanol or indomethacin (Robert et al., Gastroenterology, 77, 433- 443, 1979) or from radiation (Prasad, Int. Journal of Radiation Biology, 22, 187-189, 1972) even through this latter effect has not been consistently reported by in vitro studies (Holahan et al., Prostaglandin and Lipid Metabolism in Radiation Injury, 253-262, 1987).
The possibility that prostaglandins can also protect cancer cells from radiation injury and thus limit radiotherapy efficacy in cancer patients has not been adequately investigated or excluded.
Nocloprost itself has already been proved to prevent, in a dose-dependent manner, acute gastric lesions induced by ethanol, aspirin and acidified tarocholate in rats (Kounturek et al., Proceeding of 7th International Conference on Prostaglandins and Related Compounds, 198, 1990) and to promote healing of chronic gastric ulcerations induced by acetic acid and indomethacin.
It has now surprisingly been found that nocloprost shows a specific radioprotective effect in normal human cells and no influence on the cytotoxic effect of ionizing radiation on cancer cells.
Nocloprost or its salts is used according to this invention in amounts which are in the range of the amounts thus far used in human studies. In medical practice nocloprost can be
administered to mammals orally or parenterally. The dose of nocloprost is from 0.1 to 1500 g/kg/day when administered to human patients. The unit dose for the pharmaceutically acceptable carrier is from 0.01 to 100 mg.
The dosage for i.v. administration as a continuous infusion in customary aqueous solvents, for example 0.9% sodium chloride solution, is preferably between 0.1 ng/kg/min. and 0.1 μg/kg/min..
To form the compound of the present invention into pharmaceutical preparations, conventional methods in the field of making pharmaceutical preparations are employed e.g. the mentioned compound and customary adjuvants and carriers are mixed and formed into pharmaceutical preparations.
The active ingredient according to the invention is used in conjunction with the adjuvants known and customary in galenical pharmacy, for example for the preparation of cytoprotective agents. Examples of suitable compositions are described for example in EP- 30377.
It is thus an object of the present invention to provide an agent for the treatment of irradiation caused damage of normal cells, which does not influence tumor cells. The activity of nocloprost has been demonstrated in pharmacological tests. The effect exerted by nocloprost or its salts is investigated on normal human dermal fibroblasts and colon adenocarcinoma cells. When given alone, the compound did not influence proliferation or clonogenic capacity and it did not induce any DNA strand breakage in normal or tumor cells. Preincubation with nocloprost induced an enhancement in the fraction of cells able to survive ionizing radiation that was paralleled by a reduction in the amount of radiation induced desoxyribonucleic acid double strand breaks (DNA DSB).
In contrast, preincubation of LoVo cells with nocloprost did not influence the radiation effect on clonogenic cell survival. Moreover, in adenocarcinoma cells, nocloprost did not interfere with the formation and repair of radiation-induced DNA DSB.
The results therefore show a differential effect of the PGE2 analog on radiosensitivity of normal and cancer cells. Certain tests are described below:
Cell systems
Normal human dermal fibroblasts are obtained as cryopreserved primary cultures from Promo Cell (Heidelberg, Germany).
The LoVo cell line is derived from a human colon adenocarcinoma (Drewinko et al., 1977) and mainly consits of epithelial-like cells.
Example
After thawing, normal human fibroblasts are cultured in a monolayer in 25-mm^ plastic flasks using Fibroblast Growth Medium (Promo Cell) supplemented with 1,5% dialyzed fetal bovine serum, 1 ng/ml recombinant human basic fibroblast growth factor, 5 μg/ml insulin, and antibiotics. In order to maintain original plating efficiency, growth rate and biological responsiveness, all the experiments are performed during the first two in vitro subcultures.
The LoVo cell line is maintained in a logarithmic phase monolayer culture using Ham's F- 12 medium supplemented with 10% fetal calf serum in a humidified atmosphere containing 5% CO2 and 95% air at 37°C. Under such growth conditions the time for doubling of the cell population is approximately 30 h.
Nocloprost (9-β-chloro-16,16-dimethyl PGE2), is dissolved in absolute ethanol at 5.2 mg/ml and stored at -20°C. The compound is diluted with medium immediately prior to addition to cells. In all experiments the final ethanol concentration is less than 0.1%.
Irradiation
Exponentially growing cells are harvested with trypsin:EDTA (0.005:0.02%) and irradiated with a -^-^Cs gamma irradiator (IBL-437, Oris, France) at a dose rate of 10 Gy/min. Irradiations are carried out in ice for filter elution experiments and at room temperature for proliferation and colony formation experiments. For DNA repair studies, cells are added to prewarmed culture medium and incubated at 37°C for the designated time.
Cell proliferation assay
The effect of nocloprost on in vitro proliferation of normal human dermal fibroblasts and colon adenocarcinoma cells is investigated by exposing cells to 0.1-10 g/ml of nocloprost for intervals of up to 120 min..
After treatment with nocloprost, radiation, or both, normal human fibroblasts are plated at appropriate concentrations in 25-mm^ plastic flasks in supplemented medium. After 7 days, cells are harvested, assayed for cell viability (trypan blue dye-exclusion test) and counted (Coulter Counter, Kontron, Everett, Mass., USA).
Colony formation assay
After treatment with nocloprost and/or radiation, LoVo cells are plated at appropriate concentrations in plastic dishes and incubated at 37°C in an atmosphere of 5% CO2- 95% air for 12-14 days. Colonies, consisting of 50 or more cells, are stained with crystal violet in 70% v/v ethanol/water and counted under a microscope.
Neutral elution
Cells are labeled with 0.1 μd/ml of [^C] TdR for 24 h and then washed and cultured in non-radioactive medium for 12 h before treatment with nocloprost and/or radiation. The frequency of desoxyribonucleic acid double strand breaks (DNA DSB) is measured by the neutral elution method as described by Bradley and Kohn (Nucleic Acids Research, 7, 793-804, 1979).
Cells are transferred to 25 mm-diameter (2.0 μm pore size) polycarbonate filters and lysed with a solution containing 0.05 M Tris, 0.05 M glycine, 0.025 M disodium EDTA 2% SDS and 0.5 mg/ml proteinase K (pH 9.6). DNA is then slowly eluted from the filters using the same solution, minus proteinase K, using a Miniplus 2 peristaltic pump (Gilson, Middletown, WI, USA). In both cases, elution is continued for 15 h, and the eluted DNA is collected in five fractions.
The [l^C] activity is analyzed by liquid scintillation counting after adding Ready Gel (Beckman Instruments, Inc. Fullerton, C USA). DNA remaining on the filter at the end of the elution is removed by hydrolysis in 0.4 ml of 1 M HC1 at 80°C for 1 h, followed by treatment with 0.6 ml of 1 M NaOH at room temperature for 30 min and mechanical agitation.
Data are plotted as the relative amount of DNA retained on the filter versus the elution time. The strand scission factor (SSF) is determined from the relationship SSF = -
loglQ[(fx)/(ϊo)], where fo and fx are the fractions of DNA retained on the filter at the 12th hour of elution for non-irradiated controls and the corresponding treated samples, respectively (Meyn and Jenkins, Cancer Research, 4^ 5668-5672, 1983).
The amount af DSB remaining after irradiation is calculated as the ratio of SSF values obtained in cells allowed no time for rejoining to those of cells allowed different intervals of time to repair after irradiation.
Table 1. Effect of nocloprost on cell proliferation of human normal fibroblasts
Cone, No. of viable cells/flask (αg/ml) (mean ± SD)
Controls 0.96 ± 0.13xl06 Nocloprost* 0.1 0.92 ± 0.16xl06 1.0 0.89 ± 0.09xl06
10.0 0.88 ± 0.18xl06
* Exponentially growing cells are exposed to nocloprost for 120 min, plated in 25-mm3 plastic flasks and counted seven days after seeding.
Table 2.
Effect of nocloprost on clonogenic capacity of human colon adenocarcinoma cells
Cone, Cloning efficiency tøg/ml) (mean ± SD)
Controls 50.0 ± 6.7 Nocloprost* 0.1 48.2 ± 10.3 1.0 51.3 ± 7.1
10.0 46.3 ± 5.3
* Exponentially growing cells are exposed for 120 min to nocloprost and plated in plastic dishes. Colonies are counted after 12-14 days.
As shown in the previous tables nocloprost does not influence either the rate of fibroblast proliferation (Table 1) or the capacity of LoVo cells to form colonies (Table 2) at all the concentrations and even for the longest treatment time.
DNA DSB is determined after an exposure to each nocloprost concentration for 120 min. The elution kinetics of DNA for treated normal and tumor cells is superimposable on that observed for controls. Pretreatment with nocloprost causes an enhancement in the fraction of normal fibroblasts surviving 19 Gy irradiation compared to samples treated with radiation alone. The protective effect increases as a function of nocloprost concentration (Table 3)
Table 3.
Effect of nocloprost on the surviving fraction of human normal dermal fibroblasts after exposure to ionizing radiation
10 Gy
Nocloprost + 10 Gy:
* No. of treated cells per flask/no. of control cells per flask.
This protective effect is not observed in adenocarcinoma cells in the same treatment conditions (see Fig. 1).
The formation of DNA DSB induced by 50 Gy in both normal and tumor cells is investi¬ gated as a function of nocloprost pretreatment. As shown in Fig. 2 and 3, the amount of radiation-induced DNA DSB is reduced by nocloprost in normal but not in tumor cells. The possible influence of nocloprost on the rejoining of DNA DSB is evaluated in cells allowed to recover in the presence of lO g/ml of the compound after irradiation. A reduction in the fraction of DNA damage repaired is observed in normal fibroblasts but not in colon tumor cells (Table 4).
Table 4.
Effect of nocloprost on the rate of DNA double-strand break rejoining
Strand scission factor
Normal fibroblasts Adenocarcinoma cells
o 4120 to tl20
50 Gy 0.310 0.146 (53)! 0.349 0.036 (90)
50 Gy + nocloprost2 0.310 0.067 (78)! 0.349 0.038 (89)!
! In parantheses, percentage of damage repaired.
2 Immediately after irradiation, cells are incubated with lOμg/ml nocloprost for 120 min.
These results show a differential effect of nocloprost on radiosensitivity of normal and cancer cells.
Thus compositions containing nocloprost as active ingredient should be useful in preventing damage of human normal cells by irradiation.
Fig. 1. Radiation survival curves for LoVo cells.
Solid line, radiation alone; broken line, 10.0 μg/ml of nocloprost for 120 min. The points represent mean values based on 3 independent experiments. The SD is always within 5%.
Fig. 2. and Fig. 3. Representative neutral elution profiles depicting the elution kinetics of DNA from human normal dermal fibroblasts and LoVo cells exposed to 50 Gy with or without a 120 min preincubation with 10 μg/ml of nocloprost.
Claims
C l a i m s
l.Use of nocloprost or its pharmaceutically acceptable salts for the preparation of a composition for the treatment or prevention of irradiation caused damage of human normal cells.
2.Radioprotective composition comprising nocloprost or a pharmaceutically acceptable salt thereof and a pharmaceutically accetable excipient thereof.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ITMI922749A IT1257479B (en) | 1992-12-01 | 1992-12-01 | PROSTAGLANDINIC DERIVATIVES WITH RADIOPROTECTIVE EFFECTS |
| ITMI92A002749 | 1992-12-01 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1994012186A1 true WO1994012186A1 (en) | 1994-06-09 |
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ID=11364389
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/EP1993/003359 WO1994012186A1 (en) | 1992-12-01 | 1993-11-30 | Prostaglandin derivative having radioprotective effects |
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| Country | Link |
|---|---|
| IT (1) | IT1257479B (en) |
| WO (1) | WO1994012186A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996003992A1 (en) * | 1994-08-04 | 1996-02-15 | Loyola University Of Chicago | Protective prostaglandins for use in conjunction with chemotherapeutic agents |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0030377A1 (en) * | 1979-12-10 | 1981-06-17 | Schering Aktiengesellschaft | 9-Chloro-prostaglandin derivatives, process for their preparation and use as medicines |
-
1992
- 1992-12-01 IT ITMI922749A patent/IT1257479B/en active IP Right Grant
-
1993
- 1993-11-30 WO PCT/EP1993/003359 patent/WO1994012186A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0030377A1 (en) * | 1979-12-10 | 1981-06-17 | Schering Aktiengesellschaft | 9-Chloro-prostaglandin derivatives, process for their preparation and use as medicines |
Non-Patent Citations (3)
| Title |
|---|
| PRASAD K.D.: "Radioprotective effect of prostaglandin and an inhibitor of cyclic nucleotide phosphodiesterase on mammalian cells in culture", INT. J. RADIAT. BIOL., vol. 22, no. 2, 1972, pages 187 - 189 * |
| VIGNEULLE M., ET AL.: "Intestinal radioprotection using a combined treatment with the PGE2 analog nocloprost and the thiol derivative WR2721 in irradiated dogs", FASEB J., vol. 4, no. 4, 1990, pages A984 * |
| ZAFFARONI N., ET AL.: "Differential effect of 9beta-chloro-16,16 dimethyl prostaglandin E2 (Nocloprost) on the radiation response of human normal fibroblasts and colon adenocarcinaoma cells", RADIATION RESEARCH, vol. 135, July 1993 (1993-07-01), pages 88 - 92 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5578643A (en) * | 1992-05-20 | 1996-11-26 | Loyola University Of Chicago | Protective prostaglandins for use in conjunction with chemotherapeutic agents |
| WO1996003992A1 (en) * | 1994-08-04 | 1996-02-15 | Loyola University Of Chicago | Protective prostaglandins for use in conjunction with chemotherapeutic agents |
Also Published As
| Publication number | Publication date |
|---|---|
| ITMI922749A1 (en) | 1994-06-01 |
| IT1257479B (en) | 1996-01-25 |
| ITMI922749A0 (en) | 1992-12-01 |
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