WO2001026684A2

WO2001026684A2 - Method for treating hypoxic cells

Info

Publication number: WO2001026684A2
Application number: PCT/EP2000/010422
Authority: WO
Inventors: Constance E. Medlen
Original assignee: Aventis Pharma S.A.
Priority date: 1999-10-14
Filing date: 2000-10-13
Publication date: 2001-04-19
Also published as: AU1386401A; WO2001026684A3

Abstract

The present invention relates to the cytotoxic and radiosensitizing potential of SN-38 (7-ethyl-10-hydroxycamptothecin), a metabolite of CPT-11. SN-38 has been found to be useful for sensitizing hypoxic cells, such as hypoxic cancer cells, to the effects of radiation.

Description

METHOD FOR TREATING HYPOXIC CELLS

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to the use of SN-38 to potentiate the cytotoxic effect of radiation on hypoxic cell populations.

Discussion of the Related Art

The combination of chemotherapy and radiation therapy has become the treatment of choice for a number of advanced human malignancies. Although systemic chemotherapy ideally may control metastatic disease, radiation therapy provides an effective local control at primary tumor sites. A number of chemotherapeutic drugs are known to be able to enhance the cytotoxicity of ionizing radiation. Widely used chemotherapeutic agents, including 5-fluorouracil, etoposide, ad amycin, vinblastine, mytomycin C, cisplatin, bleomycin, and paclitaxel, have all been shown to mediate radiosensitization effects via different mechanisms. However, the potential excessive toxicities imposed by combined modality treatments of normal tissues have dictated the achievable doses and limited their potential efficacies in eradicating cancers. The development of new radiosensitizers that can selectively enhance the cytotoxicity of ionizing radiation in cancer cells remains a challenge in the art of oncology. Camptothecin belongs to a group of anticancer agents with a unique mechanism of action. It inhibits topoisomerase I through the formation of a stable topoisomerase l-DNA cleavable complex. It is a potent cytotoxic agent that is active against a number of experimental tumors. However, some clinical trials with camptothecin have been terminated owing to a severe toxicity profile. Irinotecan, also known as CPT-11 (7-ethyl-10-[4-(1- piperidino)-1 -piperidino]carbonyloxycamptothecin) is a semisynthesized derivative of camptothecin. It has broad antitumor activities and has demonstrated good results in clinical trials against lung, colorectal, cervical, and ovarian carcinomas. Irinotecan is converted by the enzyme carboxylesterase to a more active anti- tumor compound, SN-38 (7-ethyl-10-hydroxycamptothecin), a metabolite of CPT-11 , which has been reported to be 200 to 1000 times more potent than irinotecan (Kawato et al.. Cancer Res. 51 :4187-4191. (1991)). Both irinotecan and SN-38 potentiate the cytotoxic effect of radiation on various carcinoma cell lines under aerobic conditions ((Omura et al., Radiother. Oncol. 43:197- 201 , (1997) ; Sasai et al., Int. J. Radiat. Oncol. Biol. Phvs. 42:785-788, (1998); Chen et al., Cancer Res. 57:1529-1536, (1997)).

Hypoxic cells in solid tumors limit the therapeutic efficacy of radiotherapy of tumors, including carcinomas of the larynx and pharynx

(Overgaard et al., Int. J. Radiat. Oncol. Biol. Phvs. 12:515-521 , (1986)). The identification of drugs that act preferentially on hypoxic cells by sensitizing them to radiation is a challenge to researchers in this field.

SUMMARY OF THE INVENTION Hypoxia refers to a condition whereby body tissues are obtaining insufficient oxygen, for reasons including lack of sufficient blood supply.

One aspect of the invention relates to a process for treating cancers wherein at least a portion of the cancer cells are hypoxic, including digestive tract cancers such as, for example, colorectal, gastrointestinal, laryngeal, pharyngeal, and esophageal cancers; and solid tumors such as carcinomas, neuroblastoma, medulloblastoma, rhabdomyosarcoma, and suprotentorial PNEUT, said process comprising administering to a patient in need thereof a radiosensitizing effective amount of SN-38, followed by treatment with radiation. Another aspect of the invention is a process for sensitizing hypoxic cells to radiation, comprising administering to said hypoxic cells a radiosensitizing effective amount of SN-38.

An additional aspect of the invention is a process for potentiating the cytotoxic effect of radiation on a carcinoma cell line under hypoxic conditions, said process comprising administering to said cell line a potentiating effective amount of SN-38. Still another aspect of the invention is a process for inhibiting hypoxic cell growth comprising administering to a patient in need thereof an effective amount of SN-38.

Yet another aspect of the invention is a process for determining the radiosensitizing effect of a compound under hypoxic conditions, said process comprising providing a modular incubator chamber having at least one layer of wax cast in an amount effective to simulate living tissue ; inserting into said modulator incubator chamber at least one container containing : (A) a number of cells upon which the radiosensitizing potential of a compound will be tested ; and (B) the compound for which the radiosensitizing effect is to be determined into the modular incubator chamber ; creating hypoxic conditions in said modular incubator chamber ; sealing said modular incubator chamber ; and subjecting the modular incubator chamber to irradiation.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows survival curves of WH03 cells after a 6 day exposure to irinotecan (Fig. 1A) and SN-38 (Fig. 1 B). WH03 is an esophageal cancer cell line. Each point of Figure 1 represents the mean of three experiments with the standard error.

Figure 2 shows growth fractions of WH03 cells following irradiation under aerobic (■) and hypoxic (▼) conditions.

Figure 3 shows growth fractions of WH03 cells following irradiation under hypoxic conditions in the presence of 3.1 :M irinotecan (T). The control irradiation response under hypoxia (■) is shown for comparison. Each point represents the mean of those three experiments with the standard error.

Figure 4 shows growth fractions of WH03 cells following irradiation under hypoxic conditions in the presence of 0.045 nM SN-38 (T) or 0.92 nM SN-38 (•). The control irradiation response under hypoxia (■) is shown for comparison. Each point represents the mean of these three experiments with the standard error.

Figure 5 shows a modular incubator chamber having a layer of wax cast on the inside and the outside of the chamber. The test tubes are embedded in holes cut into a piece of polystyrene, which is about 5 cm thick and the approximate diameter of the chamber. The polystyrene rests on the top of the inside layer of the wax. This chamber allows for the determination of the radiosensitizing effect of a compound under aerobic or hypoxic conditions.

Figures 6A and 6B show the cytotoxic effects of irinotecan and SN-38 on WH03 cells without irradiation.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention relates to the cytotoxicity and radiosensitizing potential of the topoisomerase I modulator SN-38 on digestive tract cancers such as, for example, colorectal, gastrointestinal, laryngeal, pharyngeal, and esophageal cancers; and solid tumors such as carcinomas, neuroblastoma, medulloblastoma, rhabdomyosarcoma, and suprotentorial PNEUT. It has now been found that SN-38 is 1000 times more active than irinotecan against the human carcinoma cell line WH03. This is in agreement with Kawato et al., Cancer Res. 51 :4187-4191 , (1991 ), who reported SN-38 to be anywhere between 200 and 1000 times more potent than irinotecan. Irinotecan and SN-38, at subtoxic concentrations, did not sensitize proliferating WH03 cells under aerobic conditions. This is consistent with the findings of Falk et al., Int. J. Radiat. Biol. 61 :749-757, (1992), and Omuru et al., Radiother. Oncol. 43:197-201 (1997), who reported that the radiosensitizing effects of camptothecin and SN-38 are restricted to Go phase cells and thus are ineffective against rapidly proliferating tumor populations. However, it has now been found that SN-38, but not irinotecan, sensitizes proliferating WH03 cells to radiation under hypoxic conditions. A concentration of SN-38 as low as 0.046 nM increased the radiation-induced cell lethality significantly. The combined effect of radiation and SN-38 under hypoxic conditions has not previously been described.

The active metabolite of irinotecan, SN-38, induces hypoxia-selective radiosensitization of a rapidly proliferating human esophageal cancer cell line at submicromolar concentrations. Thus, SN-38 may be clinically useful in combination with radiation in hypoxic conditions in the treatment of cancers such as digestive tract cancers, for example, colorectal, gastrointestinal, laryngeal, pharyngeal, and esophageal cancers; and solid tumors such as carcinomas, neuroblastoma, medulloblastoma, rhabdomyosarcoma, and suprotentorial PNEUT.

According to one embodiment of the invention, a cytotoxic effect of SN- 38 may be administered to a patient without radiation treatment. Surprisingly, the present inventor has found that SN-38 significantly controls cell proliferation in hypoxic cell populations even without radiation co-therapy. For example, it has been found that SN-38 exerts greater than 25% control over a hypoxic WH03 cell population at a concentration as low as 0.046 nM (See Fig. 6). SN-38 may be administered to a patient in an amount effective to induce a cytotoxic effect, which amount may be readliy determined through routine experimentation by one of ordinary skill in the art. For example, SN-38 may be administered to a patient in an amount ranging from 100 to 1000 mg/m² in order to induce a cytotoxic effect.

According to one embodiment, a combination therapy comprising SN- 38 and radiation may be useful for treating digestive tract cancers such as, for example, laryngeal cancer, pharyngeal cancer, esophageal cancer, colorectal cancer, and gastrointestinal cancer.

According to another embodiment of the invention, an effective amount of at least one additional chemotherapeutic agent may be administered with a radiosensitizing or cytotoxic effective amount of SN-38. Suitable chemotherapeutic agents include any agent known to be useful for the treatment of solid tumors and cancer generally, and such agents may include, for example, 5-fluorouracil, etoposide, adriamycin, vinblastine, mytomycin C, cisplatin, bieomycin, and paclitaxel. The additional chemotherapeutic agent can be administered simultaneously or sequentially with SN-38 by the same or different routes of administration. The additional chemotherapeutic agent can be administered in an amount effective to treat cancer, including an amount effective to potentiate the cytotoxic or radiosensitizing amount of SN-38.

Over the past 20 years, there has been considerable interest in developing a hypoxic cell radiosensitizer potent enough to radiosensitize hypoxic cells at clinically acceptable doses. For these purposes various procedures have been utilized to achieve an in vitro hypoxic atmosphere (Zeman et al., Radiat. Res. 122:72-76, (1990); Hentosh, Analvt. Biochem. 210:249-252, (1993)). These methods were mostly time consuming and were not suitable for high output.

Surprisingly, the present inventor has found that such regularly encountered problems can be eliminated by the use of a standard modular incubator chamber having a layer of wax equivalent to an amount of soft tissue cast on the inside and/or outside of the modular incubator chamber. Multiple test tubes containing monolayers of proliferating cells may be grouped together for each radiation dosage. In this way, it is possible to perform various drug treatments within any one experiment with an oxygen enhancement ratio (OER) of 2.1.

Cell cultures may be plated in containers such as 5 ml glass tubes at densities of, for example, 600 cells per tube, and incubated for 24 hours at 37°C in a humidified atmosphere of 5% CO2. Solutions of either irinotecan or SN-38 are then added to the tubes before radiation. The tubes are then placed into a modular incubator chamber (Billups-

Rothenburg Inc., Del Mar, California, USA) with a wax build-up equivalent to approximately 2 cm tissue (8 MV d_maχ) cast into the bottom of the chamber. The chamber may then be placed on a shaker incubator for 30 minutes while continuously passing either a mixture of 5% Cθ2/95% air for aerobic conditions, or 5% Cθ2/95% N for hypoxic conditions, through said chamber before sealing. The cells may then incubated for a further period of time, for example 1 hour, at approximately 37°C, in order to deplete residual oxygen by cellular and respiratory metabolism before irradiation (1 -10 Gy).

Immediately after irradiation, performed at room temperature using an 8 MV photon beam, cell cultures are placed in an incubator at 37°C and 5% CO2 for a further period of time, for example 4 hours, after which the medium containing the drugs is replaced with complete medium and the tubes incubated for a further period of time, for example, 6 days. Cell survival may then be assessed using the MTT assay.

The compounds and/or compositions administered according to the invention can be administered orally and/or parenterally, such as intravenously or intraperitoneally. According to a certain embodiment, the compounds and/or compositions are administered via hepatic arterial infusion.

The compositions for oral administration comprise tablets, pills, powders, or granules. In these compositions, the active product according to the invention is mixed with one or more inert diluents, such as sucrose, lactose, or starch. These compositions can comprise substances other than diluents, for example, a lubricant such as magnesium stearate.

Pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or elixirs containing inert diluents, such as water or liquid paraffin, can be used as liquid compositions for oral administration. These compositions can also comprise substances other than diluents, for example, wetting, sweetening or flavoring products.

The compositions for parenteral administration according to the invention can be aqueous or nonaqueous sterile solutions, suspensions, or emulsions. Propylene glycol, a polyethylene glycol, plant oils, such as olive oil, or injectable organic esters, for example, ethyl oleate, can be used as a solvent or vehicle. These compositions can also contain adjuvants, such as wetting agents, emulsifiers, and dispersing agents. Sterilization can be carried out in many ways, for example, using a bacteriological filter, by incorporating sterilizing agents into the composition, or by heating. These compositions for parenteral use can also be prepared in the form of sterile solid compositions which can be dissolved at the time of use in sterile water or any other injectable sterile medium.

The doses used to carry out the methods according to the invention are those which allow a maximum therapeutic response in terms of radiosensitization and cytotoxicity. The doses vary according to the form of administration, the particular product selected, and the personal characteristics of the individual to be treated. In general, the doses are those which are therapeutically effective for the treatment of disorders due to abnormal cell proliferation, and in particular a cytostatic treatment. The products according to the invention can be administered as often and for as long as necessary to obtain the desired therapeutic effect.

Generally, in humans, the doses range from 100 to 1000 mg/m². In a certain embodiment, the therapeutic dose is approximately 80 mg/m²/day for 5 consecutive days, or 30 mg/m /day for 14 consecutive days. It is understood that, in order to select the most appropriate dosage, the route of administration, the patient's weight, general state of health, age, and all the factors which may influence the effectiveness of the treatment will have to be taken into account. In general, the doctor will make this determination based on such factors relevant to the individual to be treated. The example which follows is given by way of non-limiting illustration of the present invention.

EXAMPLE

The following example demonstrates the potential of irinotecan and SN-38 in enhancing the cytotoxic effects of radiation on a human esophageal carcinoma cell line under both aerobic and hypoxic conditions.

Both irinotecan and SN-38, supplied by Aventis Pharma S.A., were solubilized in dimethylsulfoxide at a concentration of 2 mg/ml and diluted in medium to the required concentration immediately before use.

WH03 (human esophageal carcinoma cell line) was maintained in a mixture of MEM (Minimal Essential Medium) and HamsF12 supplemented with 10% heat inactivated fetal calf serum (FCS) and 0.1 mg/ml of penicillin and streptomycin.

This was performed using a metabolic assay based on the reactivity of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) (Sigma Diagnostics Inc.) with viable cells (Twentyman et al., Radiother. Oncol. 43:197-201 (1992)). Cells were seeded at 3 x 10³/well in 96-well microtiter plates in a final volume of 200:1 complete medium in the presence or absence of different concentrations of either irinotecan or SN-38.

Cell cultures were plated in 5 ml glass tubes at densities of 600 cells per tube and incubated for 24 hours at 37°C in a humidified atmosphere of 5% C0₂. Solutions of either irinotecan (3.1 :M) or SN-38 (0.046 nM and 0.092 nM) were added to the tubes before radiation. The concentrations of the experimental compounds used caused 25% or less inhibition of cell growth under the experimental conditions used without the effect of radiation. The tubes were placed into a modular incubator chamber (Billups-Rothenburg Inc., Del Mar, California, USA) with a wax build-up equivalent to 2 cm tissue (8 MV d_ma_x) cast into the bottom of the chamber. The chamber was placed on a shaker incubator for 30 minutes, continuously passing a mixture of 5% Cθ₂/95% N₂ through said chamber before sealing. The cells were then incubated for a further 1 h at 37°C to deplete residual oxygen by cellular and respiratory metabolism before irradiation (1-10 Gy). Immediately after irradiation, performed at room temperature using an 8 MV photon beam, cell cultures were placed in an incubator at 37°C and 5% CO2 for a further 4 hours after which the medium containing the drugs was replaced with complete medium and the tubes incubated for a further 6 days. Cell survival was assessed using the MTT assay.

Results

The cell growth fraction (S), relative to that of control samples, for cells treated with a dose (D) of radiation, was fitted to a linear-quadratic equation as log_e= -AxD - BxD² - C. In cases where one of the inactivation parameters

(A or B) was found to be negative, it was set to zero and the other parameter re-estimated. Overall cellular responses were expressed in terms of the mean inactivation dose calculated from the respective inactivation parameters (Kellerer et al., Encvclop. Med. Radiol. Ill 1 :42, (1972)). This parameter quantifies radiosensitivity in a one-dimensional manner and is proportional to the area under the dose-response curve. As the mean inactivation dose is determined predominantly by the response of cells within the first order of magnitude, it is particularly suitable for use with the current set of data where cell growth in most measurements was not less than 10% of control samples. Furthermore, the ratio of mean inactivation dose values obtained with the MTT assay yield dose modifying factors similar to those obtained using conventional colony forming methods (Stabbert et al., Strahlenther. Onkol. 10:567-572, (1996)).

As displayed in Figure 1 , which shows the cell survival curves of WH03 monolayer cultures exposed to either irinotecan or SN-38 for 6 days as a function of the untreated control cells, WH03 cells were only marginally sensitive to irinotecan (activity between 1.5 and 2.5 :M) (Figure 1A), whereas SN-38 possessed activity against WH03 cells at concentration of between 0.003 and 0.012 :M (Figure 1 B).

Exposure of WH03 cells to hypoxic conditions greatly reduced the sensitivity of WH03 cells for radiation (Figure 2). The mean oxygen enhancement ratio for these experiments was 2.1 (Table 1 ).

Irinotecan at 3.1 :M and SN-38 at 0.046 and 0.092 nM had no effect on the sensitivity of WH03 cells for radiation under aerobic conditions (results not shown). However, under hypoxic conditions, SN-38, but not irinotecan, was able to sensitize WH03 cells to radiation at 0.092 nM (Figures 3 and 4). An additive effect was observed between radiation and either SN-38 at 0.046 nM or irinotecan at 3.1 :M (Figures 3 and 4).

The dose modifying factor for irinotecan at 3.1 :M was 1.5 and for SN-38 at 0.046 and 0.092 nM were 1.6 and 2.1 respectively (Table 1 ). Table 1

Mean inactivation doses are calculated from the response of WH03 cells following treatment with radiation and/or different drugs under aerobic or hypoxic conditions. The dose modifying factor is stated as the ratio of mean inactivation doses.

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects as illustrative only and not restrictive. The scope of the invention is, therefore, indicated by the appended claims, rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

1. A process for treating solid tumors wherein at least a portion of the tumor cells are hypoxic, said process comprising administering to a patient in need thereof a radiosensitizing effective amount of SN-38, followed by treatment with radiation.

2. The process according to claim 1 , wherein the tumor cells are chosen from digestive tract cancer cells.

3. The process according to claim 2, wherein the digestive tract cancer is chosen from laryngeal cancer, pharyngeal cancer, esophageal cancer, colorectal cancer, and gastrointestinal cancer.

4. The process according to claim 1 , wherein the tumor cells are chosen from carcinomas, neuroblastoma, medulloblastoma, rhabdomyosarcoma, and suprotentorial PNEUT.

5. The process according to claim 1 , wherein the tumor cells are esophageal cancer cells.

6. The process according to claim 1 , wherein the SN-38 is administered in an amount ranging from 100 to 1000 mg/m².

7. The process according to claim 1 , wherein the SN-38 is administered in an amount of 80 mg/m²/day for 5 consecutive days.

8. The process according to claim 1 , wherein the SN-38 is administered in an amount of 30 mg/m²/day for 14 consecutive days.

9. A process for sensitizing hypoxic cells to radiation, comprising administering to said hypoxic cells a radiosensitizing effective amount of SN-38.

10. The process according to claim 9, wherein said hypoxic cells are cancer cells.

11. The process according to claim 9, wherein the cancer cells are chosen from digestive tract cancer cells.

12. The process according to claim 11 , wherein the digestive tract cancer is chosen from laryngeal cancer, pharyngeal cancer, esophageal cancer, colorectal cancer, and gastrointestinal cancer.

13. The process according to claim 9, wherein the hypoxic cells are cells chosen from carcinomas, neuroblastoma, medulloblastoma, rhabdomyosarcoma, and suprotentorial PNEUT.

14. The process according to claim 9, wherein said hypoxic cells are esophageal cancer cells.

15. A process for potentiating the cytotoxic effect of radiation on a carcinoma cell line under hypoxic conditions, said process comprising administering to said cell line a potentiating effective amount of SN-38.

16. The process according to claim 15, wherein the SN-38 is administered at a concentration ranging from 0.04 to 1.00 nM.

17. The process for inhibiting hypoxic cell growth comprising administering to a patient in need thereof an effective amount of SN-38.

18. The process according to claim 17, wherein the hypoxic cell growth is a cancerous cell growth.

19. The process according to claim 17, wherein the hypoxic cell growth is chosen from digestive tract cancer cells.

20. The process according to claim 19, wherein the digestive tract cancer is chosen from laryngeal cancer, pharyngeal cancer, esophageal cancer, colorectal cancer, and gastrointestinal cancer.

21. The process according to claim 17, wherein the hypoxic cell growth is chosen from carcinomas, neuroblastoma, medulloblastoma, rhabdomyosarcoma, and suprotentorial PNEUT.

22. The process according to claim 17, wherein the SN-38 is administered in an amount ranging from 100 to 1000 mg/m².

23. A process for determining the radiosensitizing effect of a compound, said process comprising providing a modular incubator chamber containing at least one layer of wax in an amount effective to simulate living tissue ; inserting into said modulator incubator chamber at least one container containing :

(A) a number of cells upon which the radiosensitizing potential of a compound will be tested; and

(B) the compound for which the radiosensitizing effect is to be determined into the modular incubator chamber ; sealing said modular incubator chambe r; and subjecting the modular incubator chamber to irradiation.

24. The process according to claim 23, wherein prior to sealing the modular incubator chamber hypoxic conditions are created by passing through said chamber a flow of gas comprising at least one gas chosen from air, carbon dioxide, nitrogen, and a mixture thereof.

25. The process according to claim 23, wherein prior to sealing the modular incubator chamber aerobic conditions are created by passing through said chamber a flow of gas comprising at least one gas chosen from air, carbon dioxide, nitrogen, and a mixture thereof.

26. The process according to claim 24, wherein the flow of gas through said chamber is maintained continuously for a period of time prior to said chamber being sealed.

27. The process according to claim 25, wherein the flow of gas through said chamber is maintained continuously for a period of time prior to said chamber being sealed.

28. The process according to claim 23, wherein the container is a test tube.