WO2001082871A2

WO2001082871A2 - Method of using zinc isotope for therapy and diagnosis of colon cancer

Info

Publication number: WO2001082871A2
Application number: PCT/US2001/014664
Authority: WO
Inventors: David Tsai
Original assignee: Ambryx Biotechnology, Inc.
Priority date: 2000-05-04
Filing date: 2001-05-04
Publication date: 2001-11-08
Also published as: WO2001082871A3

Abstract

This invention relates generally to treatment and diagnosis of cancer, and more specifically to treatment and diagnosis of colon cancer employing a zinc isotope or a combination of a zinc isotope and a phosphate binder.

Description

METHOD OF USING ZINC ISOTOPE FOR THERAPY AND DIAGNOSIS OF COLON CANCER

Inventor: David Tsai

RELATED PATENT APPLICATION This application claims priority to a United States patent application Serial No. 09/374,214, filed August 13, 1999.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to treatment and diagnosis of cancer, and more specifically to treatment and diagnosis of colon cancer.

2. Description of Related Art

Zinc is an important nutrient and is necessary to maintain a multitude of physiologic processes in the body. Mineral supplements are often used by people to ensure that the body has a sufficient level of zinc. Zinc is used in the treatment of two diseases . The only two diseases known to be treated with zinc are Wilson's disease and acrodermatitis enteropathica. The use of zinc therapy for these diseases has demonstrated the exceptional safety and efficacy of zinc when used for a long period of time, namely, forty (40) years. (Anderson, et al . The Annals of Pharmacotherapy, Vol 32, pp. 78-87, January 1998).

Many different types of treatments have been developed to combat cancer, all having different side effects and varying degrees of efficacy. Further, there are many different types of cancer all of which do not respond to the same treatment. Some treatments are effective against breast cancer and not effective against lung cancer.

Before cancer can be treated in a patient, the cancer must first be diagnosed. Early cancer diagnosis is sometimes a critical factor in the treatment of the cancer. If the cancer is detected in its early stages, then there is a greater chance of successful treatment. Thus, the need for new cancer diagnostic techniques is as equally important as the need for new cancer treatment .

Therefore the fight against cancer has many fronts, depending on the type of cancer being treated. Until cancer is no longer a life-threatening disease, the art of cancer treatment and diagnosis will be open to useful refinement.

SUMMARY OF THE INVENTION The present invention introduces such refinement by inducing apoptosis in colon cancer cells by exposing them to zinc. Apoptosis is an active process of gene-directed cellular self- destruction, also called programmed cell death. Programmed cell death is different than cell death caused by cell injury, which is called necrosis. Necrosis is not desirable because cell death through necrosis causes inflammation in the surrounding tissue.

Apoptosis plays an important role in the human body from the early stages of embryonic development to the inevitable decline associated with old age. In normal adult tissue, apoptosis occurs continuously in slowly proliferating cell populations such as hepatic and adrenal cortical epithelium as well as in rapidly proliferating populations such as intestinal crypt epithelium and differentiating spermatogoni .

The idea that cancer may be caused by insufficient apoptosis emerged only recently. (Cope and Willie, "Apoptosis": The Molecular Basis of Cell Death, Cold Spring Harbor Press, p. 61, 1991) . This idea opened the door to a new concept in cancer therapy--cancer cells may be killed by encouraging apoptosis.

The present invention in a first of its preferred embodiments includes a method of inducing apoptosis in colon cancer cells by administering zinc to the colon cancer cells . The low toxicity of zinc and the fact that zinc selectively and rapidly induces apoptosis in colon cancer cells, without having any effect on normal colon cancer cells, indicates that zinc is a therapeutic agent for colon cancer .

It is preferred that the source of the zinc be one or a combination of the following: zinc acetate, zinc chloride, and/or zinc sulfate.

In a second of its preferred embodiments the invention includes a method of inducing apoptosis in colon cancer cells by administering zinc and a phosphate binder to the colon cancer cells. The inclusion of the phosphate binder increases the apoptosis-inducing activity of zinc. It was observed that when zinc acetate was dissolved in phosphate buffer solution, a white precipitate was formed. This suggests that the zinc acetate is instable in phosphate solution. The precipitate that is formed is zinc phosphate, a water-insoluble chemical. Based on this observation, it was reasoned that phosphate interferes with zinc. To test this reasoning, calcium acetate (a phosphate binder) was added to the zinc acetate solution and then administered to the colon cancer cells—the apoptosis-inducing activity of zinc acetate in colon cancer cells increased three to four times.

It is preferred that the phosphate binder be either calcium acetate and/or calcium carbonate, alone or in combination. Although these are the preferred phosphate binders, any chemical that removes phosphate from the solution and does not interfere with the activity of zinc is acceptable. It is also preferred that the source of the zinc be zinc acetate, zinc chloride, and zinc sulfate, alone or in combination.

In a third of its preferred embodiments the invention includes a method of treating colon cancer by administering isotopic zinc to the colon cancer cells . Isotopic zinc emits radiation energy which can be captured by radiation measuring techniques such as a gamma camera.

To minimize the patient's exposure to the radiation emitted by isotopic zinc, it is preferred that the zinc isotopes have a relatively short half-life such as the zinc isotope, zinc-62. Furthermore, depending on the size and stage of the tumor, it is preferred that the radiation level be about 1000-3000 cGy.

Non-isotopic zinc and isotopic zinc can be combined to treat colon cancer. Further, a phosphate binder can be combined with the treatment using only isotopic zinc or with the combination of isotopic and non-isotopic zinc. The addition of the phosphate binder enhances the apoptotic activity of both the isotopic and non-isotopic zinc.

In a fourth of its preferred embodiments the invention includes a method of diagnosing colon cancer by administering isotopic zinc to the colon cancer cells and using radionuclide imaging techniques to measure the isotopic zinc accumulated in the colon cancer cells. Radionuclide imaging depends on the fact that certain substances selectively accumulate in different parts of the body. (Armstrong, et al. Diagnostic Imaging, 2d ed. , pp. 6- 7) . Zinc selectively accumulates in colon cancer cells and not in normal colon cells. As a result, radionuclide imaging techniques can be used to detect isotopic zinc accumulated in colon cancer cells.

It is preferred that the zinc isotopes have a relatively short half-life to minimize the patient's exposure to the radiation emitted by the zinc isotopes. It is preferred that the source of the zinc isotopes be zinc-62, having a half-life of 9.26 hours. Furthermore, the practitioner will select a radiation level that will allow the selected imaging technique to effectively image the colon cancer and that will minimize the patient's exposure to radiation.

All of the foregoing operational principles and advantages of the present invention will be more fully appreciated upon consideration of the following detailed description, with reference to the appended drawings .

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a photograph taken through a phase-contrast microscope of colon cancer cells (HT-29) in growth medium and control buffer- (saline solution) ;

Fig. 2 is a photograph taken through a phase-contrast microscope of colon cancer cells (HT-29) after zinc acetate (in saline) has been administered and the HT-29 cells have undergone apoptosis;

Fig. 3 is a photograph taken through a fluorescence microscope of colon cancer cells (HT-29) stained with Hoechst dye in saline solution;

Fig. 4 is a photograph taken through a fluorescence microscope of colon cancer cells (HT-29) after zinc acetate (in saline) has been administered and the colon cancer cells have undergone apoptosis, the cells have been stained with Hoechst dye;

Fig. 5 is a photograph taken through a phase-contrast microscope of colon cancer cells (T-84) in growth medium and control buffer (saline solution) ;

Fig. 6 is a photograph taken through a phase-contrast microscope of colon cancer cells (T-84) after having zinc acetate ^, _

(in saline solution) administered to the colon cancer cells;

Fig. 7 is a photograph taken through a phase-contrast microscope of normal colon cells (CCD 18 Co) in growth medium and control buffer (saline solution) ;

Fig. 8 is a photograph taken through a phase-contrast microscope of normal colon cells (CCD 18 Co) after zinc acetate (in saline) was administered to the cells;

DESCRIPTION OF THE PREFERRED EMBODIMENTS

While the invention will be described in connection with preferred embodiments, it will be understood, that it is not intended to limit the invention to those embodiments . On the contrary, it is intended to cover all alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.

HT-29 (ATCC cell line number HTB 38) , is a cell derived from a human colon tumor, and was used to test the biological activity of zinc. In order to test the activity of zinc, two plates of one thousand (1,000) HT-29 cells were seeded in 10 microliters of McCoy 5A medium containing ten percent (10%) fetal bovine serum, penicillin and streptomycin at 37 degrees Celsius in 5% C0₂

microtray plates (25 μl wells, Robbins Scientific Corp.). The plates were incubated for 24 hours. Saline solution (5μl) was then added to the first plate and incubated for eight (8) hours, and a photograph (Fig. 1) of the first plate was taken under phase-contrast microscopy. Fig. 1 shows that the one thousand HT-29 cells in saline solution are healthy and normal.

Zinc acetate dissolved in saline solution (5μl) , at a final concentration of 80μM, were added to the second plate, incubated for eight (8) hours, and a photograph (Fig. 2) of the second plate was taken under phase-contrast microscopy. Fig. 2 clearly demonstrates -the death of the HT-29 colon cancer cells. To confirm that the HT-29 cell death induced by zinc was caused by apoptosis (programmed cell death) rather than necrosis (cell injury), another assay was done.

Two plates were each seeded with one thousand (1,000) HT-29 colon cancer cells in lOμl of McCoy 5A medium containing 10% fetal bovine serum, penicillin and streptomycin at 37 degrees Celsius in 5% CO microtray plates (25 μl wells, Robbins Scientific Corp.).

Both plates were incubated for 24 hours. Saline solution (5μl) , a control, was added to the first tray and incubated for eight (8) hours. After eight hours two μl of Hoechst dye (0.03 ng/ml in PBS) was added to the first tray. The Hoechst dye stains the nucleus of the cells. After two hours the cells were photographed (Fig. 3) under a fluorescence microscope. Fig. 3 shows that the nuclei of the HT-29 cells incubated in the saline solution are normal and healthy. Zinc acetate dissolved in saline solution (5μl, 80μM) was added to the second plate, incubated for eight (8) hours, and then the cells were stained with 2μl of Hoechst dye. Two hours later, a photograph (Fig. 4) of the plate was taken under fluorescence microscope. Fig. 4 confirms that the colon cancer cells have undergone apoptosis because the nuclei of the cells show the characteristics of apoptosis. First, the zinc acetate caused the condensation of the nucleus, this is demonstrated by the more intense fluorescent light displayed by Fig. 4 as compared to Fig. 3. Second, the nuclear condensation is accompanied by the fragmentation of the DNA which is demonstrated by the breakage of the nucleus. Third, some of the nuclear fragments show peripheral crescents of compacted chromatin, a characteristic of a cell that has undergone apoptosis .

T-84 (ATCC cell line number CCL 248) is another type of colon cancer cell that was used to test the biological activity of zinc. In order to test the activity of zinc on the T-84 cells, two plates of T-84 cells were seeded in 10 microliters of 1:1 mixture of Ham's F12 medium and DMEM medium containing ten percent (10%) fetal bovine serum, penicillin and streptomycin at 37 degrees Celsius in 5% CO microtray plates (25 μl wells, Robbins Scientific

Corp.) . The plates were incubated for 24 hours.

Saline solution (5μl) was added to the first plate, incubated for eight (8) hours, and a photograph (Fig. 5) was taken under phase-contrast microscopy. Fig. 5 shows that the one thousand T- 84 cells in saline solution are healthy and normal.

Zinc acetate dissolved in saline solution (5μl), at a final concentration of 80μM, was added to the second plate, incubated for eight (8) hours, and a photograph (Fig. 6) was taken under phase-contrast microscopy. Fig. 6 clearly demonstrates the death of the T-84 colon cancer cells.

In order to determine whether zinc induces apoptosis in normal human colon cells, two plates were seeded with one thousand (1,000) CCD 18 Co (ATCC cell line number CRL 1459), normal human colon cells, in lOμl of 1:1 mixture of Ham's F12 medium and DMEM medium containing 10% fetal bovine serum, penicillin and streptomycin at 37 degree Celsius in 5% CO„ microtray plates (25 μl wells, Robbins Scientific Corp.) . Cells in both plates were grown for 24 hours. Saline solution (5 μl) was added to the first plate and incubated for eight (8) hours. After eight hours the cells were photographed (Fig. 7) under a phase-contrast microscope. Fig. 7 shows that the CCD 18 Co cells are healthy and normal.

Zinc acetate dissolved in saline solution (5μl), at a final concentration of 80μM, was added to the second plate and incubated for eight (8) hours. A photograph of the second plate (Fig. 8) was taken under a phase-contrast microscope. Fig. 8 shows that the normal colon cells have not been affected by the zinc, they are normal and healthy.

To determine the types of cells zinc acetate would induce apoptosis in, a variety of different cell lines were tested. As shown in the chart below, zinc acetate induced apoptosis selectively in human colon cancer cells .

Cell lines: I .Choriocarcinoma (JEG-3). 2. Prostate cancer (LNCaP). 3.Hepatocarcinoma (Hep G2).

4. Prostate-cancer (DU-l 45).

5. Colon carcinoma (T-84). δ.Colon adenocarcinoma (HT-29).

7. Colon adenocarcinoma (Colo 205).

8. norma! colon fibroblast (CCD 18Co).

9. Osteosarcoma (MG-62).

10. Sarcoma (XC).

I I .Human lung fibroblast (CCD 39 Lu). 12. Human lung (Wi 38).

CHART I % of apoptotic cells = Number of cells with DNA. condensation and fragmentation

Total number of cells

It is important to note that the zinc acetate did not induce apoptosis in normal cells such as the human lung fibroblast (CCD 39 Lu) , the human lung cells (Wi 38) or the normal colon fibroblast (CCD 18Co) .

It was also determined that the apoptosis-inducing activity of zinc was increased three to four fold when a phosphate binder was added to the assay. The phosphate binder, calcium acetate, is preferred, although others such as calcium carbonate can be used. Chart II demonstrates that zinc induces apoptosis in a dose- dependent manner and that addition of a phosphate binder increases the apoptosis-inducing activity of zinc. Two different assays were done, and the results are illustrated in Chart II. In the first assay the HT-29 cells were incubated with various concentrations of zinc acetate for six (6) hours. In the second assay the HT-29 cells were incubated with various concentrations of zinc acetate and calcium acetate for six (6) hours.

CHART II

The percentage of apoptotic cells depending on the concentration in uM was then determined. As Chart II indicates, as the concentration of zinc is increased the percentage of apoptotic cells also increased. Chart II also indicates that the addition of calcium acetate to the zinc acetate solution significantly increases the apoptosis-inducing activity of the zinc. Specifically, zinc acetate alone has an LD_g(} for induction

of cell death in HT-29 cells of 68μM. In contrast, when calcium acetate and zinc acetate are used together the LD is 24μM.

CHART III

Chart III is a similar assay using T-84 colon cancer cells. Chart III shows the dose-dependence of inducing apoptosis in T-84 cells by zinc acetate. It also shows that adding a phosphate binder to the assay greatly increases the apoptosis-inducing activity of the zinc. In the absence of calcium acetate (a phosphate binder), the LD for the induction of apoptosis in the T-84 cells is a dose of zinc of 50uM. In the presence of calcium acetate the LD is 25μM.

Therefore, it is apparent that zinc induces apoptosis in colon cancer cells in a dose dependent manner and that a phosphate binder enhances the apoptosis-inducing activity of zinc by two to three fold.

CHART IV

Chart IV demonstrates that zinc acetate rapidly induces apoptosis in colon cancer cells. The concentration of the zinc acetate used in this assay was 68μM. In the absence of calcium acetate (a phosphate binder) the zinc acetate induced apoptosis in fifty percent (50%) of the colon cancer cells (HT-29) in six (6) hours. In the presence of calcium acetate, the zinc acetate induced apoptosis in ninety-five percent (95%) of the colon cancer ce]_,ls in six (6) hours.

The concentration of zinc that appears to be useful in inducing apoptosis in colon cancer cells, in the absence of a phosphate binder, is from about 60μM to about 80μM. The concentration of zinc that appears to be useful in inducing apoptosis in colon cancer cells, in the presence of a phosphate binder, is from about 20μM to about 50μM.

Zinc induces apoptosis in colon cancer cell lines in vitro. The concentration of zinc for the induction of 50% cell death in colon cancer cells is estimated to be about 68μM without a phosphate binder, and about 20μM with a phosphate binder. Published studies reveal that these concentrations can be easily reached in vivo by oral administration of zinc acetate. For example, plasma zinc concentration was found to reach 169 μM by oral administration of 200 mg of zinc per day in a dog. (Brewer et al., Use of Zinc Acetate to Treat Copper Toxicosis in Dogs, JAVMA 1992, pp. 564-567, Vol. 201 no. 4). Further, there were no adverse effects observed in the dogs even after a 2-year period of administration of zinc at such a high dose. The concentration of serum zinc, in humans, was found to reach 30μM to ^'40μM with a single dose of 50mg of zinc. (Hwang et al., Comparisons of the Effects of Calcium Carbonate and Calcium Acetate on Zinc Tolerance Test in Hemodialysis Patients, American J. Kidney Disease, 1992, pp. 57-60, Vol. XIX, No. 1).

Phosphate interferes with the apoptosis-inducing activity of zinc. Hence, phosphate may diminish the effect of zinc on colon cancer cells. In our assay system, the phosphate concentration was about 3 to 5 mM, whereas the serum phosphate concentration is 2mM. (Ramirez et al., The absorption of dietary phosphorous and^' calcium in hemodialysis patents, Kidney International, 1986, pp. 753-756, Vol. 30). Hence, zinc will probably perform an even _{, „}

stronger anticancer effect in vivo than in vitro, especially when a phosphate binder is combined with zinc.

In order to determine whether zinc selectively accumulates in normal colon cells-, normal colon cells (CCD 18 Co) were grown in two plates. In both plates, the normal colon cells were grown in Eagle's MEM with non-essential amino acids and Earle's BSS containing 10% fetal bovine serum. In the second plate, the cells were incubated with zinc acetate at a final concentration of 25 μM for 15 hours. After incubation, the cells of both plates were washed with PBS three times. The cells were then grown in the same growth media and stained with BTC-5 dye at a final concentration of 1 μM. BTC-5 is a cell-permeant dye that emits fluorescent light when it is exposed to heavy metals such as zinc and mercury. BTC-5 dye can be used to determine intracellular zinc.

The first tray containing the normal colon cells not incubated with zinc and stained with BTC-5 was photographed under a fluorescent microscope. The second tray containing the normal colon cells incubated with zinc and stained with BTC-5 was photographed under a fluorescent microscope. Comparing the photograph of the second tray to the photograph of the first tray shows no significant increase in fluorescent light. Thus, it can be concluded that no significant amount of zinc was taken up by the normal colon cells (CCD 18 Co) after 15 hours of incubation with 25 uM zinc acetate.

In order to determine whether zinc selectively accumulates in _

colon cancer cells, colon cancer cells (HT-29) were grown in McCoy's 5a medium containing 10% fetal bovine serum in a 96 wells plate. In one of two plates containing colon cancer cells, the colon cancer cells were incubated with zinc acetate at a final concentration of 25 uM for 15 hours. After the incubation, the colon cancer cells were washed with PBS three times. The cells were then grown in the same medium and stained with the BTC-5 dye.

The first tray containing the colon cancer cells not incubated with zinc and stained with BTC-5 was photographed under a fluorescent'"microscope. The second tray containing the colon cancer cells incubated with zinc acetate and stained with BTC-5 was photographed under a fluorescent microscope . The photograph of the second tray, as compared to the photograph of the first tray, shows a significant increase in fluorescent light. It can be concluded that a significant amount of zinc was taken up by the colon cancer cells after 15 hours of incubation with 25 μM of zinc acetate. The results suggest that zinc selectively accumulates in colon cancer cells, and not normal colon cells.

The observation that zinc selectively accumulates in colon cancer cells, and not other human cells, indicates that zinc may be used in the diagnosis of colon cancer. Various cancer imaging techniques, such as radionuclide imaging technique, depend on the fact that certain substances accumulate in certain parts of the body.

Radionuclide imaging technique is a medical diagnostic imaging tool that works with radioactive isotopes. Radioactive isotopes emit gamma rays that can be detected by a gamma camera. When the gamma rays strike the gamma camera, an image is eventually produced. Imaging techniques that use gamma rays to image can be used to image colon cancer cells .

When isotopic zinc is injected into the body, the isotopic zinc selectively accumulates within colon cancer cells. The isotopic zinc accumulated within colon cancer cells emit gamma rays. The gamma rays can then be captured by a gamma camera, or other gamma ray capturing devices . The resultant images from the gamma camera can be used to diagnose the colon dancer cells.

Further, the observation that zinc selectively accumulates in colon cancer cells, and not other human cells, indicates that isotopic zinc may also be used in treating colon cancer. Isotopic zinc emits radioactive energy. Administered to cells, the radiation from isotopic zinc kills the cells.

When isotopic zinc is injected into the body, the isotopic zinc selectively accumulates in colon cancer cells. Once accumulated in the colon cancer cells, the radiation from the isotopic zinc kills the colon cancer cells.

When administering isotopic zinc to a patient for diagnosis and treatment of colon cancer, it is important to keep the radiation dosage at a minimum. For treatment purposes, the radiation level that appears to be safe is from about 1000-3000 cGy, depending on the size and stage of the tumor. Although a^' radiation level above 3000 cGy may be used, such level may have deleterious effects on healthy human cells. For diagnosis purposes, the radiation level that a practitioner selects will vary depending upon the imaging technique used. As the practitioner strives to minimize the patient's exposure to radiation, the practitioner will select a radiation level that will yield effective imaging of the colon cancer. As cancer imaging techniques advance and improve, lower radiation levels will be needed for effective imaging.

It is also preferred that the isotopic zinc have a half-life of a few hours or a few days, such as the zinc isotope zinc-62. Zinc-62 has a-half-life of 9.26 hours. Zinc-62 emits gamma radiation that can be detected by a gamma camera and thus imaged.

Claims

I Claim:

1. A method of diagnosing colon cancer comprising the steps of: administering zinc isotope to colon cancer cells; and detecting the zinc isotope present in the colon cancer cells.

2. The method of claim 1, wherein the zinc isotope is selected from the group consisting of zinc isotopes having a short half-life.

3. The method of claim 1, wherein the zinc isotope has a half-life of less than about 2 days.

4. The method of claim 1, wherein the zinc isotope is zinc-62.

5. The method of claim 1, wherein the step of detecting the zinc isotope comprises using a gamma camera to measure the zinc isotope present in the colon cancer cells.

6. The method of claim 1, wherein the step of detecting the zinc isotope comprises using radionuclide imaging technique to measure the zinc isotope accumulated in the colon cancer cells^'.

7. A method of inducing apoptosis in colon cancer cells by administering zinc isotope to the colon cancer cells.

8. The method of claim 7, wherein the zinc isotope is selected from the group consisting of zinc isotopes having a short half-life.

9. The method of claim 7, wherein the zinc isotope has a half-life of less than about 2 days.

10. The method of claim 7, wherein the zinc isotope is zinc-62.

11. A method of treating colon cancer by administering zinc isotope to colon cancer cells.

12. The method of claim 11, wherein the zinc isotope is selected from the group consisting of zinc isotopes having a short half-life.

13. The method of claim 11, wherein the zinc isotope has a half-life of less than about 2 days.

14. The method of claim 11, wherein the zinc isotope is zinc-62.

15. A method of treating colon cancer by administering a combination comprising zinc and zinc isotope to colon cancer cells.

16. The method of claim 15, wherein the zinc is selected from the group consisting of zinc acetate, zinc chloride, and zinc sulfate.

17. The method of claim 15, wherein the zinc isotope is selected from-the group consisting of zinc isotopes having a short half-life.

18. The method of claim 15, wherein the zinc isotope has a half-life of less than about 2 days.^'

19. The method of claim 15, wherein the zinc isotope is zinc-62.

20. A method of treating colon cancer by administering a combination comprising zinc, zinc isotope, and a phosphate binder to colon cancer cells.

21. The method of claim 20, wherein the zinc is selected from the group consisting of zinc acetate, zinc chloride, and zinc sulfate.

22. The method of claim 21, wherein the zinc isotope is selected from the group consisting of zinc isotopes having a short half-life.

23. The method of claim 21, wherein the zinc isotope has a half-life of less than about 2 days.

24. The method of claim 21, wherein the zinc isotope is zinc-62.

25. The method of claim 21, wherein the phosphate binder is selected from the group consisting of calcium acetate and calcium carbonate .