JP2002537273A - New redox clamps and their use - Google Patents

Abstract

  (57) [Summary] A redox clamp that maintains cells in a selected redox state is provided. Also, using redox clamp agents, sensitize cells to chemotherapeutic agents, such as antineoplastic drugs, inhibit cell overgrowth and stabilize the redox state of cells with abnormal fluctuations in these redox states Provide a way to

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Field of the Invention The present invention is a cell in the selected cell redox state "locked (locking)
A new class of chemosensitizers that function by "clamping" or "clamping". The selected cellular redox state in which cells are maintained by these agents is optimal for cellular processes that mediate apoptosis, normal cell growth control and differentiation. Accordingly, these agents are useful for sensitizing cells to the effects of therapeutic agents, including but not limited to antineoplastic agents, such as butyrate and butyrate analogs.

BACKGROUND apoptosis of the invention that has an important role in promoting aspects of cancer development, has become increasingly clear. While suppression of apoptosis has been suggested to be a universal property of cancer promoters, many agents that up-regulate apoptosis in pre-neoplastic and neoplastic cells have therapeutic utility in the treatment of neoplasia. (McCarty, MF, Med. Hypotheses, 1998, 50 (5): 423-33).

[0003] For example, butyrate, which has been shown to induce apoptosis in various types of cancer, is currently considered as a potential cancer chemotherapeutic agent. The mechanism by which butyrate has the function of inducing apoptosis and changing the growth and differentiation of cancer cells is not known. However, treatment with butyrate has been reduced to reduced glutathione (
GSH), has been shown to reduce cellular levels of key determinants of cellular redox status (Benard, O., and Balasubaramanian, KA, Mol. Cell Bioch.
em., 1997, 170: 109-114). This butyrate-induced decrease in cellular GSH levels correlates with the differentiation and apoptosis of cultured cancer cells. Further, the cell GS
Agents known to modulate (ie, decrease) H levels also increase the differentiation effect of butyrate in short-term in vitro assays (Benard, O.,
And Balasubaramanian, KA, Mol. Cell Biochem., 1997, 170: 109-114). Butyrate derivatives have also been shown to exhibit radiation sensitizing properties (Chung et al.
., Radiat. Res., 1998, 149 (2): 187-194). In general, agents that increase the sensitivity of cells to radiation also act by reducing the cell's natural ability to resist radiation-induced oxidative stress. Agents that reduce cellular glutathione or inhibit the action / expression of antioxidant enzymes / proteins sensitize cells to the effects of radiation. Therefore, the radiation sensitizing effect of butyrate is
Reflects the ability of the cell to regulate the redox ability.

However, while butyrate reduces cellular GSH, this short-chain fatty acid also
Induces genes that oppose this change in cellular redox status. In particular, butyrate treatment is associated with elevated expression of a potent antioxidant and metal-chelating protein, metallothionein (MT) (Liu et al., In Vitro Cell Dev. Bi.
ol., 1992, 28A: 320-326). Elevations in MT and other antioxidant generation systems may counter redox conditions that are initially achieved by the effect of butyrate on GSH levels. Disruption of cellular redox state by the effect of butyrate on cellular GSH levels causes cells to readjust redox state by modulating an extra element of redox control. Reconditioning of the redox state in this manner can counteract the therapeutic effects of butyrate. For example, there is evidence that butyrate-induced MT expression partially opposes the redox conditions initially achieved by the effect of butyrate on GSH levels. There is evidence to suggest that butyrate-induced MT expression can, in part, explain the progressive development of butyrate resistance following prolonged treatment. Furthermore, overexpression of the antioxidant MT results in low cellular GS
It has been disclosed to compensate for H levels (Miura et al., Life Sci., 1997 60 (21):
301-309).

[0005] A new class of agents having the ability to maintain cells in a selected redox state has now been identified. These agents do not allow cells to successfully compensate for treatment-induced changes in cellular redox conditions. For this reason, these agents are useful for enhancing the therapeutic activity of chemotherapeutic agents (eg butyrate) that depend on the redox state of the cells. These agents are also useful in controlling conditions associated with cell hyperproliferation and abnormal fluctuations in cell redox status.

[0006] While maintaining the summary other redox active system of the present invention in the oxidation state, the reduction of specific cell oxidation system and / or molecular, thus thiol-containing molecule having the ability of a cell is maintained at a selected redox state Where the redox clamp agent (r
A new class of compounds called edox clamping agents has now been identified.

[0007] Accordingly, it is an object of the present invention to provide a method for maintaining cells in a selected redox state, comprising contacting the cells with a redox clamp agent that maintains the cells in a selected redox state. is there.

Another object of the present invention is to provide a chemotherapeutic agent known to elicit a stress response in cells, comprising contacting the selected cells with a chemotherapeutic agent in combination with a redox clamp agent. It is to provide a method for sensitizing selected cells.

Another object of the invention is to treat cancer in a patient, comprising administering to the patient a chemotherapeutic agent known to elicit a stress response in cancer cells in combination with a redox clamp agent. It is to provide a method.

[0010] Another object of the present invention comprises contacting the cell with a redox clamp.
An object of the present invention is to provide a method for inhibiting cell overgrowth.

[0011] Yet another object of the present invention is to provide a method for stabilizing the redox state of a cell having an abnormal change in the redox state, comprising contacting the cell with a redox clamp agent.

[0012] DETAILED DESCRIPTION cell redox state of the invention (i.e., cells were an oxidation / reduction reactions, the ability to maintain an oxidation or reduction state of thiol) is in the cells having a control effect on many cellular processes Environmental perspective (Sun, Y., and Oberly, LW, Free Rad. B
iol. & Med., 1996, 21 (3): 335-348; Monteiro, HP, and Stern, A., Free.
Rad. & Biol. & Med., 1996, 21 (3): 323-333). Redox sensitive pathways range from simple metabolism to critically important cellular signal transduction cascades controlling gene expression, proliferation, differentiation and apoptosis.

Here, a new group of compounds that can lock or clamp cells to a particular redox state has been identified. These compounds, referred to herein as "redox clamps," are thiols that have specific chemical properties, including the ability to reduce certain cellular oxidation systems and / or molecules while maintaining other redox active systems in an oxidized state. (Sulfhydryl) -containing molecules. This is a major distinction between these agents and other agents that affect cellular redox status. These redox clamps are distinct from antioxidants. The reason is that antioxidants allow for major fluctuations in redox status, pro-oxidizing
This is because it can only prevent the development of the condition.

[0014] The function of the redox clamp agent is not merely to reduce cellular levels of antioxidants, thiols or antioxidant proteins / enzymes (ie, metallothionein, glutathione reductase, etc.). Rather, the reason for the application of the redox clamp to the survival system is to (redox defined) adjust and maintain the relative amounts of cellular components so that a particular redox state is maintained. In this regard, a redox clamp agent is different from an agent that merely reduces cellular GSH or decreases the expression or cellular levels of antioxidant proteins or enzymes. Unlike redox clamping agents, such agents do not oppose readjustment of redox conditions following processing.

The ability of these agents to clamp or fix cells to a selected redox state makes them useful in a variety of therapeutic applications. Redox clamp agents can be used in combination with many chemotherapeutic agents for the treatment of cancer and pre-neoplastic diseases and other medical conditions characterized by cell hyperproliferation or abnormal cellular redox conditions.

For example, butyrate shows promise for the clinical control of prostate cancer and benign prostate symptoms. However, the serum levels required to achieve favorable results are high (> 5 mM) and difficult to maintain (Newmark, HL, and Young, CW, J
Cell Biochem. Suppl., 1995, 22: 247-253). In addition, butyrate-resistant cells can develop from tumors following prolonged treatment with this agent (Ho et al., J. Cell Physio.
l., 1994, 160 (2): 213-226). Therefore, there is a need for agents that can sensitize cells to butyrate and prevent the development of butyrate resistance. These agents have the effect of lowering the serum levels of butyrate required for antitumor activity and prolonging the treatment period.

[0017] The antitumor effect of butyrate is believed to be sensitive and / or dependent on cellular redox conditions. Here, the redox clamp agent of the present invention may have the effect of increasing the ability of butyrate to inhibit proliferation and induce differentiation and apoptosis of established prostate cancer cell lines (ie, LNCaP and PC-3). Illustrated.

[0018] Thirty redox modulators or cell culture conditions were tested for this ability to affect butyrate-mediated apoptosis. In addition, the effect of some of these agents or conditions on the ability of butyrate to reduce cellular GSH levels and increase cellular MT levels was tested. The purpose of these studies was to examine butyrate GSH
The aim was to identify agents that enable / enhance the reducing effect and counteract the treatment-induced reregulation of the redox state by the cells. Included in this screen were agents that were well established redox regulators. Other agents were selected based on their ability to alter cellular redox status as determined in preliminary studies. A partial listing of the agents tested is provided in the table below.

Table 1

[Table 1]

Although many of the previously listed agents have been found to enhance butyrate-induced apoptosis ( ^* ), enhanced treatment with some of the compounds results in increased levels of GSH and / or MT. Was obtained. In addition, many of these agents have been found to be toxic to cells, while others are LNCa
Inhibited the ability of butyrate to induce apoptosis in P cells. But,
Two compounds have been identified as having the ability to induce apoptosis in LNCaP and PC-3 cells in combination with butyrate.

One compound found to work in combination with butyrate was meso-2,3-dimercaptosuccinic acid (DMSA). This agent is currently used in clinical practice to reduce the burden on the body of toxic, heavy metals following inadvertent human exposure (Miller, AL, Altern.Med. Rev., 1998, 3 ( 3): 199
-207). In this capacity, DMSA functions by chelating heavy metals that accumulate in tissue and promoting its elimination. In these studies, DMS
A alone was found to be a weak inducer of metallothionein (MT), but LNCaP cells treated with DMSA and butyrate showed 75% lower levels of MT than cells treated with butyrate alone. Was. Therefore, DMS
A decreases the ability of butyrate to increase cellular MT levels. The half-life of the MT protein depends on the presence of a transition metal ion, such as zinc. For example, MT
-Metal complexes were shown to exhibit resistance to protease digestion (McKim e
Appl. Pharmacol., 1992, 116 (19): 117-124). Therefore, DMS
A may have the function of reducing butyrate-induced elevation of cellular MT levels by sequestering metal ions required to stabilize the MT protein.

The second compound found to function in combination with butyrate to induce apoptosis was 2-mercaptoethanesulfonic acid (MESNA).
This finding suggests that MESNA has a putative antioxidant property.
otectant) (Holoye et al., Am. J. Clin. Oncol., 19
90, 13 (2): 148-155), which was unexpected. However, while MESNA treatment alone was found to have minimal effect on apoptosis, M
Co-administration of ESNA significantly increased butyrate-induced apoptosis. Interestingly, this agent completely inhibits butyrate-induced MT expression and slightly enhances butyrate-induced reduction of cellular GSH.

[0023] The combination of all three agents was found to be most effective in promoting apoptosis in the LNCaP human prostate cancer cell line.

Based on these experiments, DMSA and MESNA were both identified as members of the group of redox clamps of the present invention. The reason is that both compounds
This is because butyrate-treated cells are believed to maintain a particular redox state that is clearly optimal for promoting differentiation and apoptosis. The results from these in vitro experiments are confirmed in vivo in established mouse models for prostate cancer. When transplanted into nude mice, established human prostate cell lines have been demonstrated to achieve and maintain a redox state that closely resembles that of in situ prostate cancer cells (Canada et al., Biochem. Pharmacol., 1996,
51: 87-90). Therefore, a preclinical evaluation of these agents using a nude mouse model of human prostate cancer will be performed.

The butyrate / DMSA / MESNA combination, compared to the other test groups,
It is predicted to inhibit human prostate tumor growth in a nude mouse model. Other combinations (ie butyrate / DMSA, butyrate / MESNA) are also expected to be more effective therapies than butyrate alone. Examination of tumors from butyrate-treated animals showed an increase in PSA mRNA, Bcl-2 / B
It is expected to show a reduced ax ratio and reduced cell proliferation compared to tumors from untreated animals. These indicators, reflecting increased differentiation and decreased tumor growth, improve in tumors from animals treated with various combination therapies. In addition, these indicators must correlate with reduced cellular levels of GSH and MT. For example, highest PSA mRNA levels, lowest Bcl-2 / Ba
Tumors with x-ratio and lowest growth index must be within a certain range and contain levels of cells GSH, MT and other redox-defining molecules that define a particular redox state. Histological examination should reveal characteristics of relatively non-mitotic morphology, normal chromatin condensation and tumor differentiation isolated from animals treated with the combination as compared to control and animals treated with butyrate alone Must. Thus, these studies not only provide insight into the ways in which DMSA and MESNA have the ability to or interact with butyrate's antitumor activity, but they also provide insights in a similar manner. Provides a means to identify and develop additional redox clamping agents that work. Further, along with the data from the in vitro experiments described here, the results from the in vivo studies
It provides insights into the effective concentrations, toxicity and therapeutic efficacy of butylene chemosensitizers for use in designing human clinical trials.

Thus, as exemplified herein, in one embodiment, the redox clamp agent of the present invention is used in combination with a chemotherapeutic agent known to elicit a stress response in cancer cells to treat neoplasia. It is useful in. An example of such a chemotherapeutic agent is butyrate for the treatment of prostate cancer. Cancer cells respond to chemotherapeutic agent-induced stress by upregulating various cellular processes that oppose stress. The drug-induced stress response makes cancer cells surviving from initial stress more robust, which makes them grow in a less controlled manner and resists subsequent treatment with the same or different chemotherapeutic agents Make it possible. However, in the present invention, a redox clamp agent is co-administered with a chemotherapeutic agent known to elicit a stress response in cancer cells. The redox clamp agent is administered in combination either before and / or during and / or after administration of the chemotherapeutic agent. Redox clamp agents adjust the redox state of cancer cells to areas where the cell death machinery remains active, thereby making the cancer cells more sensitive to being killed by the chemotherapeutic agent, It has the ability to counteract the stress response associated with the treatment of cancer cells by increasing efficiency and inhibiting the development of resistance.

In another embodiment, a redox clamp is used as an antiproliferative to be administered prior to an angioplasty procedure. Angioplasty is a medical procedure performed to open blocked blood vessels. This allows for increased blood flow to the deoxygenated tissue. However, physical trauma is applied to the inner wall of the blood vessel during angioplasty, and a sudden change in oxygen concentration in the blood vessel following this procedure triggers a transitional proliferation of the cells making up the blood vessel. This proliferative response can be significant, resulting in post-procedural thickening of blood vessels and reduced blood flow. This proliferative response makes the angiogenesis procedure more inefficient. The redox clamp can be administered before and / or during and / or after the angioplasty procedure. These agents induce a redox state of vascular cells that is inconsistent with proliferation, thus minimizing cell post-procedural proliferation and making the angiogenic procedure more effective.

Redox clamps can also be used to treat hyperproliferative conditions of the skin. Increased proliferation and / or altered differentiation of epidermal cells in various skin conditions (ie, psoriasis) is associated with major changes in the redox state of the cells. Similarly, elevated cellular levels of the antioxidant glutathione are observed in skin cells following exposure to sunlight (UV light). This change in redox status is a major promoter of skin cell proliferation. Redox clamps, taken orally or contained in a topical ointment, reduce cell proliferation and normalize the differentiation process.

In yet other embodiments, these redox clamps are of type II (adult onset)
Used for the treatment of diabetes. In type II diabetes, the cellular processes of basal and insulin-induced uptake of glucose from serum are altered. Glucose uptake by cells is mediated by glucose transport agents. In diabetic individuals, the expression / function of certain types of glucose transport agents is reduced. This contributes to the inability of individuals suffering from diabetes to remove serum glucose levels after and between meals. It is known that the expression of these transport agents is uniquely affected by the redox state of the cell. Dramatic fluctuations in the redox state of diabetic tissue (due to reduced glucose uptake and utilization) tend to down-regulate glucose transporter expression and function. Such a deficiency exacerbates the diabetic condition and promotes a rise in serum glucose. Treatment of type II diabetes with the redox clamp agent of the present invention stabilizes the redox state of cells and normalizes glucose transporter expression / function. With this treatment, individuals with diabetes can more easily control the disease by normalizing serum glucose levels, thereby slowing the progression of the disease.

The following non-limiting examples are provided to further illustrate the present invention. EXAMPLES Example 1 Effect of Agent / Combination on LNCaP Cell Apoptosis LNCaP cells were treated with the agent (alone or in combination) for 48 hours. In these studies, the concentrations used were as follows: MESNA 50 μM; D
MSA 50 μM; butyrate 5 mM. Next, cells are harvested and sub-diplo
id) The percentage of apoptotic cells appearing at the peak was determined by flow cytometry.

Example 2 Effect of Agent on Cellular Reduced Glutathione (GSH) Levels A species-specific limitation of the method of GSH determination is that the method of GSH detection requires that the dye be GSH
The use of human LNCaP cells for these studies is hampered by the dependence on the isoform of glutathione-S-transferase, which is uncommon in human cells to bind to and form fluorescent products. Therefore, the effects of butyrate, MESNA and DMSA on cellular GSH levels were examined using the H4 rat liver cancer cell line. GSH-specific fluorescence of butyrate-treated cells was found to be lower than the control. In addition, DMSA and MESNA treatment reduced basal GSH levels, while other redox modulators (ie, lipoic acid) increased cellular GSH levels. This is interesting because DMSA and MESNA enhanced butyrate-induced apoptosis, while lipoic acid was found to be a potent inhibitor. In additional studies, different cytometric based methods are used to determine reduced glutathione cellular levels in human prostate tumor cells. Also, both reduced and oxidized glutathione are quantified using an HPLC-based method.

Example 3 Effect of MESNA on Cell MT Levels LNCaP cells were treated as described in Example 1. However, cells were harvested over a 24 hour period. The cells were then fixed (paraformaldehyde), stained with a metallothionein-specific (fluorescent binding) antibody and analyzed by flow cytometry. These data indicate that both DMSA and MESNA can inhibit butyrate-induced elevation in cellular MT levels. This observation is consistent with the characterization of DMSA and MESNA as redox clamps: they inhibit butyrate-treated cells from expressing antioxidant proteins, such as MT, while reducing the low GSH levels induced by butyrate. It is believed that maintaining / enhancing will counter trials that result in pro-reducing.

Example 4 In Vivo Model for Prostate Cancer An animal model used to test the effects of butyrate combination therapy on human prostate cancer is described by Raffo et al., Cancer Res., 1995, 55: 4438-4445. As described above, male athymic (nude) mice were treated with human prostate cancer cells (LNCaP).
)) (Sc / flank). This method has been shown to obtain 100% prostate cancer establishment at the site of injection for about 3 weeks. Following a short (4 days) post-injection period, mice are randomly assigned to treatment groups (see Table 2), where they receive treatment with one or more of the described agents. Approximately 40 days after injection, tumor growth was measured (13) and tumors were excised and processed to characterize the effect of the treatment combination on various aspects of tumor growth and differentiation (see Table 2).

[0034] Steel and Torri (1980, Principals and Procedures of Statistics: A Bio
In a study following the principles set forth by the metrical Approach. New York: McGraw-Hill Book Company, Inc., it was evaluated to use at least 15 mice / group. Such an assessment predicts a pooled standard deviation of the tumor volume of 0.8 and a minimum predicted difference between the control and test groups of 40%.

Example 5: Administration of a therapeutic agent to mice DMSA and MESNA are administered orally to mice in drinking water. Oral D
The pharmacokinetics of MSA and MESNA have been extensively characterized in humans (Apo
shian, HV, and Aposhian, NM, Annu. Rev. Pharmacol. Toxicol., 1990,
30: 279-306; and James et al., Br. J. Clin. Pharmacol., 1987, 23 (5): 56.
1-568). Stability studies show that DMSA and MESNA are both stable in simple liquid and solid preparations. Two dose levels are used for DMSA and MESNA (ie, low and high). "Low dose" is determined by extrapolation using an effective dose in humans. "High dose" is three times higher than "low dose". Butyrate is administered to certain dosage groups in the form of the orally active prodrug, tributyrin. Pharmacokinetic data ranged from 5 to 5
Show that an oral dose of 10 g of tributyrin results in a peak plasma butyrate level of 1-2 mM (Yuan et al., Proc. Annu. Meet. Am. Assoc. Cancer Re.
s., 1994, 35: A2556). This serum concentration range was shown to significantly reduce LNCaP tumor growth in nude mice treated with another butyrate prodrug, isobutylamide (Gleave et al., J. Cell. Biochem., 1998, 69 (3): 2
71-281). The mice are monitored for food / water intake. If the intake changes significantly in all treatment groups relative to the control or other test groups, paired feeding can be performed. The 14 proposed treatment groups are listed in the table below.

Table 2

[Table 2]

Example 6: Tumor processing Tumor volumes were calculated and tumors were excised using standard methods. Tumor pieces are extracted for Western or Northern blot analysis. For flow cytometric analysis, using protease XIV according to established protocols,
A single cell suspension is generated from a piece of tumor. Various aspects of these cells (ie, GSH, MT, proliferation index, etc.) are determined as described in the examples below. In addition, sections of the tumor are stored and processed for histological examination. Kidneys and liver from each animal are saved to determine treatment-related organ pathology. If specific indicators of toxicity are observed or suspected, other organs are preserved and tested.

Example 7: Human prostate cancer cell line The human prostate cancer cell line, LNCaP, is used in these animal studies.
This cell line is an androgen-sensitive prostate cancer cell line that is widely used in animal models of human prostate cancer. However, after the optimal dosage and combination has been determined for LNCaP tumors, additional studies in other tumor model systems can be performed. With the LNCaP cell line widely characterized, standard biomarkers reflecting aspects of proliferation, apoptosis and differentiation were determined. These biomarkers were used in this study to determine the anti-tumor effect of the therapeutic combination, to gain insight into the mechanisms by which DMSA and MESNA function to enhance the effect of butyrate, and to add additional redox clamp agents. Used to identify.

Example 8: Effect of therapeutic combination on apoptosis of LNCaP tumors Apoptosis of LNCaP cells as well as apoptosis of other cell types
It was associated with changes in the ratio of cl-2, Bcl-X and Bax. This mechanism of apoptosis is in contrast to the apoptotic process with altered (decreased) expression / function of p53. Using standard western blotting techniques,
By measuring the relative amount of the Bcl-2 family of proteins in control and treated tumor cells, the ability of therapeutic combinations to affect the mechanism of apoptosis can be characterized. Also, other related cellular proteins that mediate / reflect or inhibit the apoptotic process and cell cycle arrest (ie, p53, p2
1 / waf1, p27Kip1) can be characterized by either Western or Northern blot analysis to address unexpected treatment-related phenomena. Detection / quantification of these proteins or mRNA is determined using standardized techniques. The percentage of apoptotic cells in tumors from the various treatment groups is determined flow cytometrically using a standard TUNEL assay. For some tumors, a TUNEL assay is performed using tissue sections.

Example 9: Effect of therapeutic combination on LNCaP tumor differentiation The differentiation marker known as PSA (prostate specific antigen)
The cell line was characterized. Increased expression of this protein has been linked to an increase in the differentiation status of this cell line as well as other human prostate cell lines and actual prostate tumors (Gleave et al., J. Cell. Biochem., 1998, 69 (3). : 271-281). LNC
PSA mRNA expression in aP tumors is determined in this study by conventional Northern blot analysis. This information is used to determine the effect of the treatment on the differentiation status of the human tumor.

Example 10: Effect of therapeutic combination on LNCaP tumor growth Several hours before sacrifice, several mice in each treatment group were injected with bromodeoxyuridine (BrdU). The introduction of this thymidine analog into the DNA of growing cells allows the determination of the growth index of the tumor to be determined flow cytometrically.
Human tumor cells can be split cytometrically from contaminated mouse cells. This is because the DNA-specific dye used in the assay (ie, propidium iodide) stains cells based on total DNA content (human cells have significantly more genomic DNA than mouse cells). ). The reduction in the percentage of cells that transduce BrdU in tumors from mice receiving a particular combination of agents is
Interpreted as a treatment-related inhibition of tumor growth. These findings suggest other characteristics of the tumor (
That is, size / volume, expression of differentiation markers, etc.).

Example 11 Effect of Therapeutic Combination on Redox Status of LNCaP Tumors Determinants of differentiation, apoptosis and proliferation correlate with treatment-related changes in redox status of tumor cells. Currently, redox status is defined in relative terms based on reduced glutathione and MT cellular levels. M decreased
T expression and lower cytoreduced glutathione levels indicate that combination therapy (ie butyrate and ME) is compared to untreated or treated with butyrate alone.
(SNA and / or DMSA).
To observe elevated GSH and MT levels from butyrate-induced antioxidant repulsion based on our predictions in tumors from animals receiving butyrate alone versus tumors from untreated animals Is possible. For this analysis, the levels of GSH and MT in the tumor cells are determined flow cytometrically and by Western blot analysis. In addition, an HPLC-based method of quantifying cellular levels of oxidized and reduced glutathione is used.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 31/194 C12N 5/00 E (81) Designated country EP (AT, BE, CH, CY, DE, DK) , ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR) , NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Marmel Stein, Fred H. United States New Jersey 08876, Hillsborough, Woods Road 123 F-term (reference) 4B065 AA93X AC20 BD25 CA44 4C084 AA17 AA20 NA05 ZB261 ZB262 4C206 AA02 JA52 NA05 ZB26

Claims (5)

[Claims] 1. A method for maintaining a cell in a selected redox state, comprising:
Contacting a cell with a redox clamp agent that maintains the cell in a selected redox state. 2. A method of sensitizing a selected cell to a chemotherapeutic agent known to elicit a stress response in the cell, comprising combining the selected cell with a redox clamp agent. Such a method, comprising contacting with a chemotherapeutic agent known to elicit a stress response. 3. A method of treating cancer in a patient, the method comprising administering to the patient a chemotherapeutic agent known to elicit a stress response in cancer cells in combination with a redox clamp agent. . 4. A method of inhibiting cell overgrowth, comprising contacting a cell with a redox clamp agent to maintain the cell in a redox state that is inconsistent with cell growth. 5. A method for stabilizing the redox state of a cell having abnormal fluctuations in the redox state, comprising contacting the cell with a redox clamp agent that maintains the cell in a selected redox state. Method.