WO2000018234A1

WO2000018234A1 - Thiazolidenediones alone or in combination with other therapeutic agents for tumor therapy

Info

Publication number: WO2000018234A1
Application number: PCT/US1998/020611
Authority: WO
Inventors: John Alton Copland, Iii; Slavisa Gasic; Randall Urban; Melvyn Soloff
Original assignee: Board Of Regents, The University Of Texas System
Priority date: 1998-09-29
Filing date: 1998-09-29
Publication date: 2000-04-06

Abstract

A purpose of the present invention is to identify and/or develop nontoxic or low toxic chemotherapeutic regiments to treat cancer. Prototype cells tested are derived from an osteosarcoma from an eleven year girl who died after aggressive chemotherapeutic treatment with adriamycin, vincristine, cytoxan, and aramycin-C. The inventive steps described herein include demonstrating that troglitazone inhibits cell proliferation in human osteosarcoma cells, the presence of functional PPAR-η in such cells, and the demonstration of the ability of troglitazone to lower the IC50 of other chemotherapeutic agents such as 5-fluorouracil and doxorubicin when administered in combination therewith as regarding cell proliferation.

Description

DESCRIPTION

THIAZOLIDENEDIONES ALONE OR IN COMBINATION WITH OTHER THERAPEUTIC AGENTS FOR TUMOR THERAPY

BACKGROUND OF THE INVENTION

Troglitazone is a member of the thiazolidinedione class of drugs which act as ligands to activate the transcriptional factor, PPAR-γ. Ligands of PPAR-γ have previously been shown to cause terminal differentiation in human liposarcoma cells in vitro (Tontonoz et al., 1997). Another example of differentiation therapy is the use of all-trans retinoic acid in the treatment of myelocytic lineage derived cancers such as acute promyelocytic leukemia (Warrel et al., 1993). Induction of terminal differentiation of malignant cells is an ideal mechanism of curing cancer as opposed to a cell death mediated mechanisms. PPAR-γ plays a central role in the process of adipocyte differentiation and is expressed in high levels in this tissue. Mechanistically, PPAR-γ heterodimerizes with retinoid X receptor (RXR- α) to form a DNA-binding complex (DR-1) that regulates expression of adipocyte- specific genes (Kliewer et al., 1992; Tontonoz et al, 1994a; Tontonoz et al, 1994b; Tontonoz et al, 1995; Sears et al, 1996). The combination of PPAR-γ ligand (pioglitazone) and RXR ligand (LG268) treatment of liposarcoma cells in vitro result in an additive effect upon terminal differentiation as characterized by accumulation of intracellular lipid, induction of adipocyte-specific genes, and withdrawal from the cell cycle (Tontonoz, 1997). To date, other tissues expressing PPAR-γ include muscle, kidney, liver, and lung, (Tontonoz, 1997).

Besides its ability to bind to PPAR-γ, troglitazone is an antioxidant due to the vitamin E moiety in its structure. Vitamin E has recently been shown to induce apoptosis in human colorectal cancer cell lines (Chinery et al, 1997). However, the IC₅₀ dose of vitamin E for HCT 15 cells was 0.75 mM and for HCT 116 cells was 3 mM. Combinatorial therapy with 5-FU and 3 mM vitamin E reduces the IC₅₀ of 5-FU alone by 2 and 8 fold respectively in HCT 116 and HCT15 cells. The present inventors demonstrate an increase in p21 expression and conclude that vitamin E's inhibitory activity is due to the increase in the cell cycle inhibitor, p21.

Saos-2 cells are an osteogenic sarcoma characterized by a mutant nonfunctional retinoma blastoma (Rb) protein and no p53 protein expression. Both of these proteins play a critical role in regulating cell cycle progression. Loss of function of these two proteins is a partial explanation for the developed resistance to chemotherapeutic agents used to treat patients. The retinoblastoma gene product (pRb) is a critical substrate of the evolutionarily conserved cyclin-dependent kinases (CDK's) which regulate progression through the cell cycle. Progression through the cell cycle is governed by a family of cyclin dependent kinases (CDKs), whose activity is governed by phosphorylation activated by binding of cyclins, and inhibited by the cdk inhibitors (reviewed in Sherr). The CDKs regulate biochemical pathways or checkpoints which integrate mitogenic and growth inhibitory signals, monitor chromosome integrity, and coordinate cell cycle transitions. Passage through Gl and S phase is regulated by the activities of cyclin

E/CDK2, cyclin D/CDK4, and cyclin D/CDK6. Cyclin A/CDK2 promotes passage through the S phase and cyclin B/CDK2 kinase is essential for G2/mitosis transition. Two families of CDK inhibitors (CKIs) mediate cell cycle arrest following growth inhibitory stimuli. The INK4 family members pl5INK4b/MTS2, pl6LNK4/MTSl, pl8, and pl9 bind CDK4 and CDK6 specifically and inhibit cyclin D binding. Members of the KIP family are currently composed of p21KIPl/WAFl/SDII (transcriptional target of p53), p27KIPl, p57KIP2 (reviewed in Harper et al.). These molecules specifically target CDKs that are important to Gl-S transition: CDK2, CDK4, and CDK6. Thus, CKIs bind CDKs inhibiting progression through the cell cycle. Phosphorylation of CKIs lead to degradation of

CKI and to cell cycle progression. Conversely loss of expression of key CKI(s) can lead to continual cell cycle progression. Compounds that can increase CKI levels are potential chemotherapeutic agents against cancer. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A - DNA concentration of Saos-2 cells 3 days after a single treatment of troglitazone. FIG. 1 A shows DNA content per well following a single treatment with troglitazone. * Denotes different from control (P<0.05).

FIG. IB - LDH activity in Saos-2 Cells treated with troglitazone for 3 days.

FIG. IB shows lactate dehydrogenase (LDH) levels as measured in the media after 3 days of treatment.

FIG. 2 - CAT activity in Saos-2 cells transfected with AOX-PPRE-tk-CAT construct. FIG. 2 shows that troglitazone treatment increase CAT activity 4 fold in the absence of transfected PPAR-γ (lane 2 vs lane 1) as well as the presence of transfected PPAR (lanes 3 and 4). This demonstrates the existence of functional

PPAR-γ in Saos-2 cells. * Denotes P<0.05 compared to appropriate control.

FIG. 3 - 3H-Thymidine incorporation into Saos-2 cells. FIG. 3 shows that a dose of 5 μg/ml troglitazone caused a 16% reduction of 3H-uridine uptake while a 10 μg/ml dose caused a 43% reduction. * Denotes statistically different from control (P<0.05).

FIG. 4 - mRNA levels in Saos-2 cells. FIG. 4 shows the effect upon mRNA levels for cell cycle inhibitor genes in response to a single treatment of 1 μM troglitazone for 24 hr. This revealed a 2.6 fold increase in p21 mRNA levels. Levels were normalized to GAPDH. Another control used is L32 which is seen to change very little, pi 8 is shown not change, as well.

FIG. 5 - DNA content of Saos-2 cells 3 days after a single treatment of troglitazone and/or 5-fluorouracil. FIG. 5 shows the effects of toxic chemotherapeutic agent, 5-fluorouracil (5-FU), revealing that combinatorial therapy with 10 μg/ml troglitazone and 1 μM 5-FU was equivalent to 100 μM dose of 5-FU alone. Combinatorial therapy reduces the dose of 5-FU needed by a factor of 100. * Denotes different from control (P<0.05); + Denotes different from corresponding 5-fluorouracil treatment (P<0.05).

FIG. 6 - DNA content of Saos-2 cells. FIG.6 shows that troglitazone demonstrates a similar lowering of the dose of doxorubicin needed to inhibit cell proliferation. * Denotes P<0.05 as compared to control; + denotes P<0.05 as troglitazone/doxorubicin compared to equivalent doxorubicin dose of doxorubicin. DNA was isolated from cells 4 days after a single treatment with the indicated drug.

FIG. 7 - DNA content of Saos-2 cells. FIG. 7 shows that troglitazone may be acting or acting additionally through its vitamin E antioxidant properties in addition to its ability to bind to PPAR-γ.

FIG. 8 - DNA content of Saos-2 cells. FIG.8 shows the results of combinatorial therapy with 5-flourouracil (5-FU) or doxorubicin. P<0.05 as compared to match contol with/without 5-fluorouracil (5-FU).

FIG. 9 DNA content of Saos-2 cells. FIG 9 shows that the thiazolidinediones had no additional inhibitory effect on cell proliferation when administered with methotrexate but did with doxorubicin. * Denotes P<0.05 as compared to its doxorubicin control.

DESCRIPTION OF THE INVENTION

One purpose of the present invention is to identify and/or develop nontoxic or low toxic chemotherapeutic regimens to treat cancer. The cells used are derived from an osteosarcoma from an eleven year girl who died after aggressive chemotherapeutic treatment with adriamycin, vincristine, cytoxan, and aramycin-C.

Troglitazone (prototypical thiazolidinedione) is used clinically to treat Type II diabetics and has been shown to have little toxic activity except in rare instances of idiopathic hepatic toxicity. This invention demonstrates that troglitazone alone is not toxic and inhibits cell proliferation in Saos-2cells by inhibiting cell cycle progression. Another important discovery described herein is that combinatorial therapy using troglitazone requires 100 fold less toxic chemotherapeutic agent 5- fluorouracil (5-FU) for equivalent inhibition of cell proliferation (as compared to 5- FU treatment alone). 5-FU is toxic to cells because it is a DNA base analog which inhibits DNA synthesis. Thus, its mechanism of action differs from that of troglitazone. This latter discovery could prove to be a powerful technique allowing the use of lower concentrations of toxic chemotherapeutic agents while providing successful therapeutic results in causing tumor regression and curing cancer.

It is novel to use troglitazone in inhibiting tumor cell growth in osteosarcoma cells. It is also novel to use the nontoxic troglitazone in combination with powerful and potentially toxic chemotherapeutic agents for the purpose of using lower doses of the toxic agent to effectively cause tumor regression. Other thiazolidinediones usable in the practice of the present invention are seen in U.S. Patent No. 5,478,852, issued December 26, 1995 and assigned to Sankyo

Company, Limited, Tokyo, Japan.

It was not apparent that troglitazone alone would inhibit cell proliferation of human osteosarcoma cells. One known mechanism of action of troglitazone is its action through binding to the peroxisome proliferator activated receptor γ (PPAR-γ). PPAR-γ is a transcriptional factor that can cause terminal differentiation of adipocytes. PPAR expression has not been previously demonstrated in osteosarcomas. It is also not obvious that troglitazone would lower the IC₅₀ of toxic chemotherapeutic compounds when administered in combination with these toxic agents.

The inventive steps described herein include demonstrating that troglitazone inhibits cell proliferation in human osteosarcoma cells, the presence of functional PPAR-γ in such cells, and the demonstration of the ability of troglitazone to lower the IC₅₀ of other chemotherapeutic agents when administered in combination therewith as regarding cell proliferation

FIG. 1A shows DNA content per well following a single treatment with troglitazone. Cells were exposed for 3 days and cell number determined by measuring DNA content. Cells were exposed for 3 days and cell number determined by measuring DNA content. The IC₅₀ for troglitazone is between about

1 μg/ml and about 5 μg/ml.

FIG. IB shows lactate dehydrogenase (LDH) levels measured in the media after 3 days of treatment. LDH levels are indicative of cell death either through apoptosis or necrosis. LDH levels are not altered at any concentration of troglitazone used, as compared to controls, indicating that troglitazone, while not being toxic to cells, inhibits cell proliferation.

Examination of the mechanism of action of troglitazone was performed by using the reporter construct AOX-PPRE-tk-CAT. This expression vector contains a DNA sequence which specifically binds PPAR-γ thereby activating chloramphenicol acetyltransferase (CAT) expression. FIG.2 shows troglitazone treatment to increase CAT expression 4-fold (lane 2 vs lane 1) in the absence of transfected PPAR-γ as well as the presence of 0.67ug transfected PPAR (lanes 3 and 4) thus first demonstrating the existence of functional PPAR in Saos-2 cells.

Northern analysis of PPARγ revealed high expression in rat adipocytes, moderate levels in fibroblasts and osteoblasts and low levels in other tissues. (Figure 2B)

3-H uridine incorporation was used to determine whether troglitazone inhibited cell cycle progression. Cells (80% confluent) were treated with troglitazone for 18 hours and examined for 3H-uridine uptake into cells lysates. As shown in figure 3, a dose of 5 μg/ml troglitazone caused a 16% inhibition of 3H- uridine uptake while a 10 μg/ml dose caused a 43% inhibition.

FIG. 4 shows the effect upon mRNA levels for cell cycle inhibitor genes in response to a single treatment of 1 μM troglitazone for 24 hr which revealed a 2.6 fold increase in p21 mRNA levels. Levels were normalized to GAPDH. Another control used is L32 which is seen to change very little, pi 8 is shown not change. Other cell cycle inhibitor genes demonstrating little change in response to troglitazone treatment include pi 30, retinoma blastoma, pi 07, p27, pi 6, and -15.

FIG. 5 shows the effects of toxic chemotherapeutic agent, 5-fluorouracil (5- FU), revealing that combinatorial therapy with 10 μg/ml troglitazone and 1 μM 5-

FU was equivalent to 100 μM dose of 5-FU alone. Combinatorial therapy reduces the dose of 5-FU needed by a factor of 100.

In FIG. 6, troglitazone demonstrates a similar lowering of the dose of doxorubicin needed to inhibit cell proliferation. Note that troglitazone is being administered in μM quantities as opposed to μg/ml (10 μg/ml = 22.7 μM).

Examining compounds similar to troglitazone for inhibitory activity upon cell proliferation should further delineate the mechanism of action of troglitazone. The thiazolidinediones, pioglitazone and BRL49653 have a 10-100 fold greater affinity for PPAR-γ but do not contain the vitamin E moiety in their structures. Comparing these compounds as well as vitamin E succinate revealed that troglitazone was much more potent in inhibiting cell proliferation than the other thiozolidinediones after a single treatment (FIG. 7). Vitamin E succinate demonstrates a dose responsive inhibition of cell proliferation somewhat less effective than troglitazone.. At 100 μM vitamin E, cells are being killed as evidenced by microscopic examination and LDH levels in the media. This effect appears specific since 100 μM succinic acid had no effect upon cell proliferation (last column of FIG. 7). The data from FIG. 7 are indicative that troglitazone may be acting or acting additionally more through its vitamin E antioxidant properties as opposed to its ability to bind to PPAR-γ. Further comparisons with the thiazolidinediones in combinatorial therapy reveal that troglitazone is superior to pioglitazone and BRL49653 in its ability to inhibit cell proliferation. The results of combinatorial therapy with 5-FU are depicted in Figure 8 and those of doxorubicin are depicted in FIG. 9. As also seen in FIG. 9, none of the thiazolidinediones had any additional inhibitory effect on cell proliferation when administered with methotrexate.

Taken together, this data demonstrate that functional PPAR-γ exist in human osteosarcoma, Saos-2, cells. This is also the first demonstration of PPAR-γ mRNA expression is normal osteoblasts. However, thiozolidinediones with a higher affinity for PPAR-γ were less effective than troglitazone in inhibiting cell proliferation suggesting that troglitazone may work as an antioxidant. However, higher doses of vitamin E were needed to cause similar growth inhibitory repsonses when compared to troglitazone. Thus, it is plausible that troglitazone utilizes both functions in inhibiting cell proliferation. Troglitazone because of its unique properties of causing terminal differentiation and low toxicity may be an excellent candidate for prophylactic therapy for individuals at high risk for cancer.

References

Chinery, Brockman, Peeler, Shyr, Beauchamp, Coffey, Nature Med., 3:1233-1241,

1997. Harper and Elledge, Curr. Opin. Genet. Dev., 6:54-56, 1996. Kliewer, Umesona, Noonan, Heyman, Evans, Nature, 358:771-774, 1992.

Sears, MacGinnitie, Kovacs, Graves, Mol Cell Biol, 16:3410-3418, 1996. Sherr, Science, 274:1672-1677, 1996.

Tontonoz, Graves, Budavari, Erdjument-Bromage, Lui, Hu, Tempst, Spiegelman, Nucleic Acids Res., 22:5628-5634, 1994B. Tontonoz, Graves, Hu, Budavari, Spiegelman, Genes Dev., 8:1224-1234, 1994 A.

Tontonoz, Hu, Devine, Beale, Spiegelman, Mol Cell Biol, 15:351-357, 1995. Tontonoz, Singer, Forman, Sarraf et al, Proc. Natl. Acad. Sci. USA, 94:237-241,

1997. Warrel, de The, Wang, Degos, N. Engl J. Med, 329:177-189, 1993.

Claims

Claims:

1. Use of troglitazone alone as a chemotherapeutic agent for the treatment of bone cancer.

2. Use of troglitazone alone as a chemotherapeutic agent for the treatment of tumor expressing PPAR-γ.

3. Use of troglitazone for the treatment of tumors derived from precursor cells to osteosarcomas.

4. Use troglitazone in combination with at least one other chemotherapeutic agent for the treatment of bone cancer, other cancer types expressing PPAR-γ, or cancer derived from the precursor cell type to osteosarcoma.

5. The use of claim 1 or 4 where the bone cancer is osteosarcoma.

6. The use of claim 4 where the other chemotherapeutic agent is 5-fluorouracil or doxorubicin.