JP2003516938A - Method for selectively killing cancer cells by reducing cellular coenzyme A level - Google Patents

Abstract

(57) SUMMARY The present invention describes a method of inhibiting or killing the growth of cancer cells by a rapid decrease in free intracellular coenzyme A (CoA). The invention includes: any method of selectively reducing CoA in cancer cells by increasing CoA utilization and / or reducing its synthesis. As shown herein, reduction of free CoA may be achieved by inhibition of fatty acid synthase (FAS). The present invention also includes reducing free intracellular coenzyme A by adding to or alternative to FAS inhibition.

Description

Detailed Description of the Invention

[0001] by many overview of the related field research, very high levels of fatty acid synthase in the following (FAS
, EC2.3.1.85) has been demonstrated: malignant human breast cancer (Alo, PL, Visca)
, P., Marci, A., Mangoni, A., Botti, C., and Di Tondo, U. Expression of fatty acid synthase (FAS) as a predictor of recurrence in patients with stage 1 breast cancer, Cancer
77: 474-482, 1996; Jensen, V., Ladekarl, M., Holm-Nielsen, P., Melsen,
F., and Soerensen, FB node-negative prognostic value of tumor antigen 519 (OA-519) expression and proliferative activity detected by antibody MIB-1 in breast cancer, Jou
rnal of Pathology. 176: 343-352, 1995) and other cancers (Rashid, A., Pizer, E.
.S., Moga, M., Milgraum, LZ, Zahurak, M., Pasternack, GR, Kuhajda
, FP, and Hamilton, SR High fatty acid synthase expression and fatty acid synthesis activity in colorectal tumors, American Journal of Pathology. 150: 201-208, 19
97; Pizer, E., Lax, S., Kuhajda, F., Pastermack, G., and Kurman, R. Fatty acid synthase expression in endometrial cancer: correlation with cell proliferation and hormone receptors, Cancer. 83. : 528-537, 1998). FAS expression has also been identified in breast ductal carcinoma in situ and lobular carcinoma in situ; lesions associated with the development of high-risk invasive breast cancer (Milgraum, LZ, Witters, LA, Pasternack, GR, and Kuhajda,
Enzymes of the FP fatty acid synthesis pathway are highly expressed in carcinoma in situ of the breast, Cli
nical Cancer Research. 3: 2115-2120, 1997). FAS is a major synthase of fatty acid synthesis (FA synthesis), and NADP of malonyl-CoA and acetyl-CoA
It catalyzes H-dependent condensation to produce palmitate, a saturated free fatty acid mainly containing 16 carbons (Wakil, S., Fatty acid synthase, an accomplished multifunctional enzyme, Biochemistry.
28: 4523-4530, 1989). High levels of FA by ex vivo measurement in tumor tissue
Both S and FA synthesis have been revealed, indicating that the overall genetic program consists of approximately 25 enzymes from hexokinase to FAS and is highly active (Rashid et al., 1997).

Treatment of cultured human cancer cells with an inhibitor of FAS containing the fungal product cerulenin and the novel compound C75 resulted in a rapid decrease in FA synthesis followed by DNA.
Synthesis was reduced, the cell cycle was arrested, and finally apoptosis occurred (Pizer, E
S., Jackisch, C., Wood, FD, Pasternack, GR, Davidson, NE, and Kuhajda, F. Inhibition of fatty acid synthesis induces programmed cell death in human breast cancer cells, Cancer Research. 56: 2745- 2747, 1996, Pizer, ES, Chr
est, FJ, DiGiuseppe, JA, and Han, WF Pharmacologic Inhibitors of Mammalian Fatty Acid Synthase Inhibit DNA Replication and Induce Apoptosis in Tumor Cell Lines, Cancer Research. 58: 4611-4615, 1998. ). Pharmacological inhibition of mammalian fatty acid synthase activity induces inhibition of DNA replication within about 90 minutes of drug application. These findings suggest that there is a vital biochemical relationship between FA synthesis and cancer cell proliferation. While of great interest, the question of how inhibition of fatty acid synthase causes this phenomenon remains unknown. Importantly, these effects also occur in the medium from fetal bovine serum in the presence of exogenous fatty acids. In fatty acid-free medium conditions, the cytotoxic effect of cerulenin on certain cells could be restored by the addition of exogenous palmitate, whereas most cancer cells inhibited FA synthesis by the end product of the pathway. Not recovered (data not shown) (Pizer, E
S., Wood, FD, Pastnack, GR, and Kuhajda, FP fatty acid synthase (FAS): a target for cytotoxic antimetabolites in HL60 promyelocytic leukemia cells, Cancer Research. 1996: 745-751, 1996). Therefore, whether the cytotoxic effect of FA synthesis inhibition on most cancer cells is caused by end product starvation or by other biochemical mechanisms remains unsolved. SUMMARY OF THE INVENTION The present invention describes a method of inhibiting the growth or killing of cancer cells by abrupt reduction of free cell coenzyme A (CoA). The invention includes: Any method of selectively reducing CoA by increasing CoA utilization and / or decreasing its synthesis in cancer cells.

This therapeutic strategy provides new chemotherapeutic agents for a wide range of human cancers. Furthermore, it is predicted that this therapeutic strategy may increase the efficacy of other commonly used cancer therapeutics, as it is a novel pathway that induces apoptosis that is not shared with other cancer therapeutics. .

In one aspect, the invention provides a method for inhibiting the growth of tumor cells in a living body, which causes the living body to undergo a rapid reduction of intracellular free coenzyme A in cancer cells in the living body. Administering the composition. In particular, upon administration of the composition, intracellular malonyl CoA in the cells of the organism rises rapidly, preferably within 3 hours of administration. Intracellular malonyl CoA is expected to increase prior to cell growth inhibition,
Preferably, an increase in intracellular malonyl CoA correlates with a decrease in malonyl CoA consumption. More preferably, the increase in intracellular malonyl-CoA precedes any increase in consumption rate of malonyl-CoA. In one mode, an increase in intracellular malonyl CoA correlates with a decrease in the intracellular activity of malonyl CoA decarboxylase (MCD) or a decrease in the intracellular activity of fatty acid synthase, and the composition also contains an inhibitor of MCD. Good. In another manner, increased intracellular malonyl CoA correlates with increased malonyl CoA synthesis.

In another aspect, the present invention provides a method for inhibiting the growth of tumor cells in a living body, which causes the living body to undergo a sharp decrease in intracellular free coenzyme A in cancer cells in the living body. Increasing intracellular malonyl CoA correlates with increased intracellular activity of acetyl CoA carboxylase (ACC), including administering the composition. Alternatively, the composition may contain an ACC activator, an activator of citrate synthase, an inhibitor of 5'-AMP dependent protein kinase (AMPK), and / or an inhibitor of acyl CoA synthase. In a preferred manner, the second chemotherapeutic agent is also administered to the organism.

In the method of the invention, intracellular malonyl CoA levels prior to administration of the composition are preferably at least 2-fold higher than normal malonyl CoA levels in non-malignant cells. In general, intracellular levels of malonyl CoA are elevated and intracellular levels of acetyl CoA and free CoA are reduced compared to pre-treatment levels. Preferably, the rate of fatty acid synthesis in living cells is at least 2-fold higher than normal prior to administration of the composition, and administration of the composition is cytotoxic to the cells. A decrease in intracellular free coenzyme A levels can be expected to correlate with apoptosis in cells with reduced coenzyme A.

In a preferred embodiment of the method of the present invention, the composition is an inhibitor of pantothenate kinase,
It contains an inhibitor of phosphopantothenoyl cysteine synthetase, an inhibitor of phosphopantothenoyl cysteine decarboxylase, and / or an inhibitor of phosphopantothein adenylyl transferase. In another preferred mode, the composition is Co
It contains a substrate with the ability to esterify to A. In general, the organism treated according to the present invention has tumor cells with a high rate of fatty acid synthesis, and the cell number of the tumor cells decreases after administration of the composition.

In another aspect, the invention provides a screening method that aids in the detection of a composition having selective cytotoxicity to tumor cells, the method comprising administering a target composition to a target cell. Monitoring the intracellular levels of free and / or derivatized coenzyme A in the cells after the administration, where a sharp decrease in free coenzyme A in the cell indicates selective cytotoxicity.

In yet another aspect, the invention provides a screening method that facilitates the classification of a composition having selective cytotoxicity to tumor cells, the method comprising targeting a target composition to a target cell, Administration in the absence and simultaneous presence of an amount of an ACC inhibitor sufficient to limit the production of malonyl CoA, wherein the difference in cytotoxicity is free and / or derivative coenzyme A. Shows the cytotoxic activity resulting from the action on the intracellular level of. Detailed Description of the Embodiments If fatty acid starvation mediates the cytotoxic effects of cerulenin and C75, any other FA synthesis inhibitor with similar potency should produce a similar effect. To confirm this notion, Applicants compared the effects of inhibition of acetyl-CoA carboxylase (ACC, EC6.4.1.2), an enzyme that limits the rate of fatty acid synthesis, on cancer cells with the effects of FAS inhibitors. The inventors have now demonstrated that inhibition of FAS induces high levels of malonyl CoA, which occurs within 30 minutes of C75 treatment. These physiological excess levels of malonyl CoA (but not low levels of internally synthesized fatty acids) trigger apoptosis of breast cancer cells. Furthermore,
This is a novel pathway that induces selective apoptosis of cancer cells.

FIG. 1A is an overview of a portion of the FA synthesis pathway involving the inhibitor target enzyme used in this study. TOFA (5- (tetradecyloxy) -2-furoic acid) is acetyl Co
Is an allosteric inhibitor of A carboxylase (ACC, EC6.4.1.2),
Inhibits acetyl CoA from being carboxylated to malonyl CoA. Once esterified to coenzyme A, TOFA-CoA allosterically inhibits ACC by a mechanism similar to that of long-chain acyl CoA, an inhibitor that is the physiological end product of ACC (Halvorson, DL and McCune, SA alone). Inhibition of fatty acid synthesis by 5- (tetradecyloxy) -2-furoic acid in isolated adipocytes Lipids. 19: 851
-856, 1984). Cerulenin (Funabashi, H., Kawaguchi, A., Tomoda, H., Omur
a, S., Okuda, S., and Iwasaki, S. Cerulenin binding sites in fatty acid synthetase, J. Biochem. 105: 751-755, 1989) and C75 (Pizer et al., 1998).
Are both FAS inhibitors and inhibit the condensation of malonyl CoA and acetyl CoA into fatty acids. Cerulenin is a suicide inhibitor
And forms a covalent adduct with FAS (Moche,
M., Schneider, G., Edwards, P., Dehesh, K., and Lindqvist, Y. Structure of the complex between the antibiotic cerulenin and its target, beta-ketoacyl carrier protein synthase, J Biol Chem. 274: 6031-6034, 1999), while C75 is thought to be a slow-binding inhibitor (Kuhajda FP, Pizer ES, Mani NS, Pinn ML, Ha).
n WF, Chrest FJ, and CA, T. Synthesis and antitumor activity of novel inhibitors of fatty acid synthase, Proceeding of the American Assicoation for Cancer Research.
40: 121, 1999). Using TOFA, the inventor obtained FA synthesis inhibition comparable to that by cerulenin or C75 in human breast cancer cell lines. However, surprisingly, TOFA was essentially non-toxic to human breast cancer cells. These data indicate that fatty acid starvation is not a major cause of cytotoxicity to cancer cells in serum-supplemented medium. Another effect of FAS inhibition, FAS
The production of high levels of the substrate, malonyl CoA, specifically resulting from the inhibition of C.sup.1 is believed to mediate the cytotoxicity of cerulenin and C75.

Malonyl CoA is acetyl CoA carboxylase (ACC, EC6.4.1.2)
, Which is an enzyme product of, is an important regulatory molecule in cell metabolism. In addition to its role as a substrate in fatty acid synthesis, malonyl CoA regulates fatty acid β-oxidation through its interaction with carnitine palmitoyl transferase-1 (CPT-1) at the mitochondrial outer membrane. CPT-1 is Palmitoyl Co
It regulates β-oxidation of fatty acids in mitochondria by regulating the passage of long-chain acyl-CoA derivatives such as A across the mitochondrial outer membrane. Physiologically, malonyl CoA levels in the cytoplasm are high during fatty acid synthesis. High levels of steady-state malonyl-CoA inhibit long-chain acyl-CoA entry into mitochondria,
This prevents the futile cycle of the oxidation of internally synthesized fatty acids.

Coenzyme A is a biocofactor in cellular processes involved in energy production, lipid biosynthesis, and energy regulation. For example, acetyl CoA and malonyl CoA
Is a substrate for fatty acid and cholesterol synthesis. All fatty acids are taken up by cellular structures or oxidized in mitochondria to energy,
Must be esterified to CoA. Succinyl CoA is an intermediate in the TCA cycle. Therefore, maintaining a sufficient supply of CoA is necessary for cell survival.

Many types of cancer cells synthesize high levels of fatty acids. As expected, cells synthesizing high levels of fatty acids have high steady-state levels of malonyl CoA, at least 6-fold higher than normal cells (see Example 6). In order to reduce the effective amount of free CoA, intracellular levels of malonyl-CoA can be selectively and rapidly raised to physiological excess levels in tumor cells by treating them with an inhibitor of FAS. This treatment is both by inhibiting malonyl-CoA utilization as a substrate in fatty acid synthesis and by stimulating malonyl-CoA synthesis by removing the concomitant fatty acyl-CoA inhibition of ACC.
Increase the level of A (FIG. 1A). Since FAS is preferentially expressed in cancer cells, elevation of malonyl CoA is mainly restricted to tumor cells. by this,
Apoptosis of cancer cells and sparing of normal tissue is induced as occurs in human cancer xenografts treated with FAS inhibitors (see Example 5). Malonyl C
The sharp rise in oA levels does not supply sufficient free CoA to other cellular processes and causes cell death.

Free CoA levels may be manipulated using various methods and targeting enzymes. The examples show a reduction in free CoA associated with elevated malonyl CoA levels due to reduced utilization of malonyl CoA and concomitant increased production. Evidence utilizing metabolic labeling with [U- ^14C ] acetate has demonstrated high levels of fatty acid synthesis in human cancer cells (Kuhajda, FP, Jenner, K., Wood, FD, Henniga).
r, RA, Jacobs, LB, Dick, JD, and Pasternack, GR Fatty acid synthesis: potential alternative targets for anti-cancer therapy, Proceedings of National Acad
emy of Science. 91: 6379-6383, 1994; Rashid, A., Pizer, ES, Moga, M.,
Milgraum, LZ, Zahurak, M., Pasternack, GR, Kuhajda, FP, and H
amilton, SR High fatty acid synthase expression and fatty acid synthesis activity in colorectal tumors, American Journal of Pathology. 150: 201-208, 1997). This high level of fatty acid synthesis in human cancer cells allows the levels of malonyl CoA to be selectively manipulated to induce apoptosis. The sharp rise in malonyl-CoA levels results in the selective destruction of cancer cells via apoptosis, leaving normal cells unaffected. This therapeutic strategy identifies potential new targets and strategies for cancer chemotherapy based on altered malonyl-CoA levels.

Preferably, the manipulation of free coenzyme A levels according to the invention is carried out by administering the composition (or compositions) to the organism in need thereof. Compositions administered to living organisms may contain substances that have a biological effect of reducing the amount of free CoA available. Agents that interfere with the biosynthesis of CoA or that are incorporated into CoA esters that reduce the pool of free CoA may be used alone or with other agents of the invention. Preferred substances have the effect of at least partially increasing intracellular malonyl CoA levels. Generally, the organism is a mammal, such as a mouse, rat, rabbit, guinea pig, cat, dog, horse, cow, sheep, goat, pig, or primate, such as a chimpanzee, baboon, or preferably a human. Usually, a living body has tumor (malignant tumor) cells. The method of the present invention relates to selectively acting on malignant tumor cells, and has less effect (or more preferably no effect) on normal (non-malignant tumor) cells.

The substance in the composition to be administered to an organism preferably increases intracellular malonyl CoA level in at least a part of malignant tumor cells in the organism. Preferably malonyl CoA levels are increased at least 2-fold, more preferably at least 5-fold. Preferably, this substance increases intracellular malonyl CoA concentration in malignant tumor cells to a level higher than that in surrounding normal cells. Suitable substances may elevate malonyl CoA levels by any of a number of methods (see another mechanism below). In some embodiments, more than one substance may be administered and some or all of these substances may affect malonyl CoA levels by different mechanisms. Materials that act by any of the methods in the list below may be used in the compositions of the present invention. Assays for the following activities are available in the literature and confirmation of whether a particular substance exhibits one of these activities is within the skill of the art. Methods of Rapidly Decreasing Free CoA for Cancer Treatment An abrupt (ie, abrupt or abrupt) decrease in free CoA levels causes selective destruction of cancer cells via apoptosis. This therapeutic strategy identifies potential new targets and strategies for cancer chemotherapy based on altered levels of malonyl-CoA that occur selectively in cancer cells, and corresponding altered levels of free CoA. Substances that increase malonyl-CoA production: Effectors of acetyl-CoA carboxylase (ACC) : Substances that increase ACC activity, reduce ACC inhibition, or increase the amount of active ACC enzyme increase the level of malonyl-CoA.

Effector of 5′c-AMP protein kinase: 5′c-AMP protein kinase inhibits ACC by phosphorylation, causing a sharp drop in malonyl CoA. Inhibitors of this kinase cause a rapid rise in the level of malonyl CoA by removing the inhibition of ACC.

Citrate synthase effector: Increased mitochondrial citrate provides a substrate for fatty acid synthesis, and citrate acts as a “feed-forward” activator of ACC to increase malonyl CoA synthesis.

Effector of acyl CoA synthase: Inhibition of acyl CoA synthase lowers the concentration of fatty acid acyl CoA in cells and releases inhibition of ACC. This increases ACC activity and malonyl CoA levels. Substances that reduce malonyl-CoA utilization: Malonyl-CoA decarboxylase (MCD) effector: This enzyme is malonyl-C
It catalyzes the ATP-dependent decarboxylation reaction from oA to acetyl CoA. Malonyl CoA levels are rapidly elevated by inhibition of MCD. Concomitant reduced malonyl CoA utilization and increased production: Inhibition of the effector FAS of fatty acid synthase (FAS) results in reduced utilization of malonyl CoA by inhibiting its uptake into fatty acids. Inhibition of FAS also reduces fatty acid acyl CoA levels, which activates ACC. Representative FAS inhibitors may be obtained as described in US Pat. Nos. 5,759,837 and 5,981,575, which patents are hereby incorporated by reference.

These strategies for rapidly increasing malonyl CoA levels may be used with or in cooperation with other agents to promote apoptosis of cancer cells. Preferably, at least one substance in the composition of the invention elevates the level of malonyl CoA by a mechanism other than inhibiting FAS. Decreased CoA synthesis: Pantothenate kinase (PanK) effector: This enzyme catalyzes the ATP-dependent phosphorylation of pantothenate (the first step of coenzyme A synthesis), and its reduced activity reduces total CoA. However, it is considered that the available free CoA decreases accordingly.

Effector of phosphopantothenoylcysteine synthetase: This enzyme adds an cysteine to 4-phosphopantothenic acid to produce 4-phosphopantothenoyl-L-cysteine (the first of the coenzyme A synthesis It is considered that the total amount of CoA is reduced by catalyzing the two stages), and the available free CoA is reduced accordingly.

Effector of phosphopantothenoylcysteine decarboxylase: This enzyme removes the alpha-carboxyl group of cysteine from 4-phosphopantothenoyl-L-cysteine to produce 4-phosphopantothein (coenzyme It is believed that the 3rd stage of A synthesis) is catalyzed, and that its activity lowers the total amount of CoA, which in turn reduces the available free CoA.

Effector of phosphopantothein adenylyltransferase (also called dephospho-CoA pyrophosphorylase): This enzyme adds adenine to 4-phosphopantothein and consumes ATP to produce dephospho-CoA and pyrophosphate. It catalyzes the reaction (4th step of coenzyme A synthesis). The final step of CoA synthesis, ATP-dependent phosphorylation of dephospho-CoA to CoA
Done by A-kinase, perhaps this is the additional catalytic activity of the phosphopantotheine adenylyl transferase enzyme. Inhibition of PanK, phosphopantothenoylcysteine synthetase, phosphopantothenoylcysteine decarboxylase, or phosphopantothein adenylyltransferase sharply inhibits CoA synthesis or reduces free CoA. Intracellular CoA sequestration in the form of stable CoA esters: Certain synthetic substances are taken up by cells and esterified with CoA by various cellular enzymes to produce stable CoA esters. The direct effect of those substances is
The reduction of free CoA by the amount of CoA incorporated into the stable CoA ester. These CoA esters may or may not have additional bioactivity within the cell. Two examples of those synthetics are TOFA and etomoxir. By administering a sufficient amount of these substances to tumor cells, sufficient CoA is sequestered into its stable CoA ester form, resulting in a functionally significant reduction in free CoA. Administration of Components The therapeutic agents of the present invention are preferably formulated into pharmaceutical compositions containing the substance and a pharmaceutically acceptable carrier. The pharmaceutical composition may contain other ingredients as long as the other ingredients do not diminish the effectiveness of the agents of the invention such that they render treatment ineffective. Pharmaceutically acceptable carriers are well known and one of skill in the art can readily select a suitable carrier for a particular route of administration (Remington's Pharmacy, Mack Publishin
Company g, Easton, PA 1985).

A pharmaceutical composition containing any of the agents of the invention may be administered parenterally (subcutaneously, intramuscularly, intravenously, intraperitoneally, intrapleurally, intravesically, as required by the agent of choice). Or intrathecal), topical, oral, rectal, or nasal routes.
The concentration of active agent in the pharmaceutically acceptable carrier may be in the range of 0.01 mM to 1 M or more as long as the concentration does not exceed the acceptable level of toxicity at the time of administration.

The dose and duration of treatment depend on various factors, including the therapeutic index of the drug, the type of disease, the age of the patient, the weight of the patient, and the tolerable dose of toxicity. The dose is generally selected so that the serum concentration is about 0.1 μg / ml to about 100 μg / ml. Preferably, the initial dose level is based on its ability to reach ambient concentrations in vitro models, as described herein, and in in vivo models and clinical trials that have been found to be effective, and the maximum permissible level. To choose between. In standard clinical practice, chemotherapy is preferably tailored to the individual patient and systemic concentrations of chemotherapeutic agents are regularly monitored. The dosage and duration of treatment of a particular drug for a particular patient can be determined by the clinician using standard pharmacological methods, taking into account the above factors. Analysis of blood or fluid levels of the agents of the invention, measurement of the activity or level of the agent in tissues of interest, or monitoring of a medical condition in a patient may be performed to monitor response to treatment. The clinician will adjust the dose and duration of treatment based on the response to treatment as revealed by these measurements. EXAMPLES In order to facilitate a more complete understanding of the invention, a number of examples are set forth below. However, the scope of the invention is not limited to the particular embodiments disclosed in these examples, which are merely for purposes of illustration. Example 1. Inhibition of FAS in cells in vitro TOFA, cerulenin, and C75 all inhibited fatty acid synthesis in human breast cancer cells. The human breast cancer cell lines SKBR3 and MCF7 were maintained in RPMI containing 10% fetal bovine serum. Cells were regularly screened for mycoplasma contamination (Gen-probe). All inhibitors were added as 5 mg / ml stock solutions in DMSO. 5x1 in 24-well plates for measuring fatty acid synthesis activity
0 ⁴ cells / well were pulsed with [U- ¹⁴ C] acetate after exposure to the drug, to extract the fat was quantified as described previously (Pizer, et al., 1988). For MCF7 cells, pathway activity was measured 2 hours after inhibitor exposure. SKBR3 cells are F
There was a slow response to the AS inhibitor, probably due to the very high FAS content, so the activity of the pathway was measured 6 hours after inhibitor exposure.

In a standard pulse labeling experiment, the breast cancer cell lines SKBR3 and MCF7 were labeled for 2 hours after exposure to the FA synthesis inhibitors TOFA, C75, and cerulenin, but all [U ¹⁴ C] acetate to lipids. Uptake was inhibited to the same extent (FIGS. 1B and D). In many similar experiments (not shown), TOFA maximally inhibited FA synthesis in a dose range of 1-5 μg / ml in all cell lines tested, while cerulenin and C75 produced FA synthesis in the 10 μg / ml range. To the maximum. Example 2. Effect of the same inhibitors on cell proliferation TOFA, cerulenin, and C75 all inhibited fatty acid synthesis in human breast cancer cells, but showed specific cytotoxicity. Cells and inhibitors are as described in Example 1. In a clonogenic assay, 4x10 ⁵ cells were seeded in a 25 cm ² flask and inhibitors were added at the concentrations listed for 6 hours. 60m of equal number of treated cells and controls
m seeded in a petri dish. After 7 to 10 days, clones were stained and counted.

Although all inhibitors reduced FA synthesis to the same extent, TOFA was either non-toxic or stimulatory to the growth of cancer cells in the dose range of ACC inhibition as measured by the clonogenic assay. On the other hand, cerulenin and C75 showed significant cytotoxicity in the dose range of FAS inhibition (FIGS. 1C and E). The large difference between the cytotoxic effects of ACC and FAS inhibition indicates that the sharp decline in fatty acid production itself is not a significant cause of cell damage after FAS inhibition. Example 3. Measurement of Malonyl CoA The most obvious difference in the predicted results of inhibition of these two enzymes is the malonyl CoA
A level decreased after ACC inhibition but increased after FAS inhibition. Although not previously investigated in eukaryotes, recent data in E. coli demonstrate elevated malonyl CoA levels caused by exposure to cerulenin (Chohnan et al., 1997, “Aerobic”). Changes in size and composition of intracellular pools of non-esterified coenzyme A and coenzyme A thioesters in fungi and facultative anaerobes "
, Applied and Enviromental Microbiology, 63: 555-560). Malonyl CoA levels were measured in cells subjected to FAS inhibition and TOFA inhibition under the conditions described in Example 2.

Corkey et al. HPLC method (“Analysis of Acyl Coenzyme A Esters in Biological Samples” Met
hods in Enzymology, 166: 55-70) using malonyl C in MCF-7 cells.
The oA level was measured. Briefly, 2.5x10 ⁵ cells / 24-well plate
The wells were treated with various agents and then 1.2 ml of 10% TCA was applied at 4 ° C. The mass of the pellet was recorded and the supernatant was washed 6 times with 1.2 ml of ether and vacuum dried at 25 ° C. using vacuum centrifugation. Coenzyme A ester was separated and quantified using reverse phase HPLC (5 μ Supelco C18 column, Waters HPLC system, operated with Millenium ³² software, coenzyme A absorbance maximum monitored at 254 nm). The following gradients and buffers were used: Buffer A: 0.1M potassium phosphate (pH 5
.0), buffer B: 0.1 M potassium phosphate (pH 5.0) (containing 40% acetonitrile). After isocratically eluting with 92% A, 8% B for 20 min at 0.4 ml / min, the flow rate was increased to 0.8 ml / min over 1 min, immediately followed by a linear gradient to 10% B for 24 min. And then held at 10% B for up to 50 minutes, followed by a linear gradient to 100% B in 55 minutes and completed in 60 minutes. The following coenzyme A ester (Sig
ma) was eluted as a standard: malonyl CoA, acetyl CoA, glutathione CoA, succinyl CoA, HMG-CoA, and free CoA. Samples and standards were dissolved in 50 μl buffer A. The coenzyme A ester was eluted sequentially as follows: malonyl CoA, glutathione CoA, free CoA, succinyl CoA, HMG-CoA, and acetyl CoA. Quantification of coenzyme A ester is Mil
Performed with lenium ³² software.

Acid eluate from drug treated cells was analyzed by reverse phase HPLC to directly measure the coenzyme A derivative in MCF-7, although both cerulenin and C75 induced a rapid rise in malonyl CoA levels. , TOFA was confirmed to reduce malonyl CoA levels. FIG. 2A is a representative chromatograph showing the separation and identification of coenzyme A derivatives important in cell metabolism. Malonyl CoA elutes first of these with a column retention time of 19-22 minutes. The overlaid chromatograph in FIG. 2B shows that cerulenin treatment significantly increases malonyl CoA over control but TOFA significantly decreases it. The chemical identity of malonyl CoA was confirmed individually by spiked samples with standards (not shown).

Malonyl CoA levels were markedly elevated by FAS inhibition and reduced by TOFA. Analysis of multiple experiments shown in FIG. 2C shows that malonyl-CoA levels are 930% and 370% higher than controls, respectively, after exposure to cerulenin or C75 at 10 μg / ml for 1 hour, whereas TOFA treatment (20 μg / ml) caused malonyl-coA levels to rise. CoA level is 60
It became clear that it fell%. In Figures 1B and D, the concentration of TOFA required to maximally reduce malonyl-CoA levels was 4-fold higher than the dose to inhibit the pathway. However, the optimal medium for the extraction of CoA derivatives has a cell density higher than that used in other biochemical and viability assays for substrates.
It was twice as expensive.

It can be considered that the remarkable increase of malonyl CoA after FAS inhibition is partly due to the cancellation of the long-chain fatty acid acyl CoA inhibition of ACC and the increase of ACC activity (FIG. 1A). In addition, an increase in malonyl-CoA levels induced by cerulenin occurred within 30 minutes after treatment (930 +/- 15% increase over controls, data not shown),
This is within the time frame for FA synthesis inhibition, well before the onset of DNA synthesis inhibition or early apoptotic events (Pizer et al., 1998). Thus, high levels of malonyl CoA are a characteristic effect of FAS inhibitors, temporally preceding other cellular responses, including apoptosis.

The levels of cerulenin or C75 that induce high levels of malonyl CoA were measured in human breast cancer cells as determined by apoptotic assay with clonogenic assay and flow cytometric analysis (using merocyanin 450 staining). In contrast, it showed cytotoxicity (Pizer et al., 1998). FAS inhibition increased malonyl-CoA levels by inhibiting its consumption by FAS inhibition, and concomitantly stimulating synthesis by removing the inhibitory effect of long-chain acyl CoA on ACC activity (Fig. 2). Example 4. TOFA Relief of FAS Inhibition: Relief of FAS Inhibition by TOFA results in high levels of malonyl CoA
Is the cause of the cytotoxicity of cancer cells. Given that high levels of malonyl CoA due to FAS inhibition are responsible for cytotoxicity, it should be possible to rescue cells from FAS inhibition by reducing malonyl CoA accumulation in TOFA. Co-administration of TOFA and cerulenin to SKBR3 cells (Fig. 3
A) eliminated the cytotoxic effect of cerulenin alone in the clonogenic assay performed as described in Example 2. In MCF7 cells (Fig. 3C), TO
FA rescued both cerulenin and C75 under similar experimental conditions.

Representative flow cytometric analysis of SKBR3 cells (FIG. 3B) and MCF7 (FIG. 3D) demonstrated these findings, and TOFA rescued cells from cerulenin-induced apoptosis. Apoptosis was measured by multiparameter flow cytometry using a FACStar ^plus flow cytometer with argon and krypton laser (Becton Dickinson)
. Apoptosis was quantified using merocyanine 540 staining (Sigma), which detects changes in phospholipid packing of the cytoplasmic membrane that occur early in apoptosis and is added directly to cells from culture medium (Pizer Et al., 1998; Mower et al., 1994 “
Reduced membrane phospholipid packing and reduced cell size precede DNA cleavage in B cell apoptosis in adult mice ”, J. Immunol., 152: 4832-4842). In some experiments, changes in apoptotic chromatin structure were observed. Simultaneously measured as a decrease in staining with LDS-751 (Exciton) (Frey et al., 1995, “Nucleic acid stain for detection of apoptosis in living cells” Cytometry, 21: 265-274) .Merocyanine 540 [10 μg / ml
] Was added as a 1 mg / ml stock solution in water. Cells were stained with LDS-751 from a 1 mM stock DMSO solution at a final concentration of 100 nM. Merocyanine 540 positive cells were labeled by an increase in red fluorescence and collected at 575 +/- 20 nm, 0.5 to 2 logs relative to merocyanine 540 negative cells. Similarly, LDS-751 dim cells were harvested at 660 nm with a DF20 bandpass filter and showed a 0.5 to 1.5 log reduction in fluorescence relative to normal cells. Collect data and use CellQuest software (Becton Dickinson
) Was used for analysis.

In these experiments, all LDS-751 turbid cells were merocyanine 540 positive, but it was observed that some merocyanine 540 positive cell populations were not LDS-751 turbid. All merocyanine 540 positive cells were classified as apoptotic. These experiments also confirmed different cytotoxicity between TOFA (<5% increased apoptosis; no clonogenic decline) and cerulenin (> 85% apoptosis; clonogenic 70% reduction). Taken together, these studies indicate that high levels of malonyl CoA play a role in the cytotoxic effect of FAS inhibition on cancer cells. Example 5. Effect of FAS Inhibitors on Tumor Cell Proliferation In Vivo To determine whether the effects of FAS inhibition seen in vitro can transfer to an in vivo environment that requires systemic activity, athymic nude mice At C7
Five MCF-7 subcutaneous xenografts were tested to quantify their effects on FA synthesis and the growth of established solid tumors. Previous studies have demonstrated local efficacy of cerulenin on human cancer xenografts (Pizer et al., 1996, "Inhibition of fatty acid synthesis delays disease progression in xenograft models of ovarian cancer", Cancer Res
., 56: 1189-1193), which was limited by the inability of cerulenin to act systemically. in vi
Similar responses of breast cancer cells to cerulenin and C75 in vitro have been demonstrated in vivo.
Suggest that C75 may be effective against xenografted breast cancer cells.

The antitumor effect of C75 in vivo was studied using a subcutaneous flank xenograft of a human breast cancer cell line, MCF-7, in female nu / nu mice (Harlan). All animal experiments followed the Institute's Animal Care Guidelines. All mice were anteriorly flanked with 90 days of sustained release subcutaneous estrogen pellets (Innovative Research) and tumor inoculated 7 days later. 10 ⁷ MCF-7 cells were supplemented with DME supplemented with 10% FBS and insulin 10 μg / ml.
Xenotransplanted from M medium.

Treatment was started about 10 days after inoculation, when measurable tumors developed. Eleven mice (divided into two different experiments, 5 and 6 samples, respectively) were treated with 0.1 C75 weekly.
It was administered intraperitoneally at a dose of 30 mg / kg in ml RPMI. The dose is in BALB / c mice
Based on single dose LD ₁₀ assay of 40 mg / kg; 30 mg / kg is well tolerated in outbred nude mice. The RPMI alone was administered to 11 control mice (divided in the same manner as the treatment group). Tumor volume was measured in three dimensions with calipers. The experiment was terminated when the control reached the surrogate endpoint.

In a parallel experiment to determine fatty acid synthesis activity in treated and control tumors, groups of MCF-7 xenograft mice were treated with the above doses of C75 or vehicle for 3 hours. Later killed. Tumor and liver tissues were labeled with [U ¹⁴ C] ex vivo and lipids extracted and counted as reported (Rashid et al., 1997).

In a further experiment performed in parallel to perform histological examination of treated and control tumors, 6 C75 treated mice and 6 vehicle control mice were killed 6 hours after treatment. Tumors and normal tissues were fixed in neutral buffered formalin, processed for routine histology, and FAS immunohistochemistry was performed. The immunohistochemistry of FAS
MCF-7 xenografts were performed with mouse monoclonal anti-FAS antibody (Alo et al., 1996).
Used at 1: 2000 and used LSAB2 detection kit on Dako Immunostainer.

The activity of the fatty acid synthesis pathway in xenografted mouse tissues was measured by ex vivo pulse labeling with [U- ^14C ] acetate. Tumor xenografts had 10-fold higher FA synthesis activity than liver, highlighting the difference in pathway activity between benign and malignant tissues (Fig. 4A). High levels of FAS expression in MCF-7 xenografts
It was similar to A synthetic activity (Fig. 4B). Intraperitoneal injection of 30 mg / kg C75 reduced fatty acid synthesis within 3 hours by 76% in ex vivo labeled liver and 70% in MCF-7 xenografts (FIG. 4A). These changes in FA synthesis preceded cytotoxic histological evidence in xenografts and became apparent 6 hours after treatment (FIGS. 4C and 4D). C75-treated xenografts showed a large number of apoptotic bodies throughout the tumor tissue, which were absent in vehicle-treated tumors. Histological analysis of liver and other host tissues following C75 treatment showed no evidence of short-term or long-term toxicity (not shown).

C40 treatment of xenografts elicited cytotoxicity and reduced tumor growth without damaging normal tissues. Six hours after administration of 30 mg / kg C75, tumor histology revealed significant cytotoxicity compared to control tumors (FIGS. 4C and 4D, accompanying preprints). Note that the liver and other organs show no evidence of tissue damage, whereas C75-treated xenografts show evidence of apoptotic bodies (data not shown). Weekly intraperitoneal C75 treatment blocked the growth of established subcutaneous MCF-7 tumors compared to vehicle controls, indicating a systemic antitumor effect (FIG. 4E). After 32 days of weekly treatment, tumor growth in treated groups was more than 8-fold different compared to vehicle controls. Similar to cerulenin, it was the only toxicity with only transient and reversible weight loss (Pizer et al., 1996).
.

A first analysis of the results of systemic FAS inhibitor treatment by the systemic pharmacological activity of C75 was performed. The significant antitumor effect of C75 on human breast cancer xenografts in the environment of physiological levels of ambient fatty acids was similar to the in vitro results in serum-supplemented medium, consistent with a mechanism of cytotoxicity independent of fatty acid starvation. . Example 6. Human cancer cells have high levels of steady-state malonyl C in vivo.
Having oA The results of Example 5 indicate that malonyl CoA accumulation may not be a significant issue in normal tissues, presumably because the activity of the FA synthetic pathway is usually low even in adipogenic organs such as the liver. Suggests that there is. More interestingly, in these experiments malonyl CoA is the major low molecular weight CoA conjugate detected in breast cancer cells, while other studies report predominant succinyl CoA and acetyl CoA in cultured hepatocytes. (Corkey,
1988). High levels of malonyl CoA in tumor tissue reflect high levels of fatty acid synthesis in tumor cells compared to liver (Rashid et al., 1997).

Using the MCF-7 human breast cancer xenograft model of Example 5, tumor xenografts from the same animal and malonyl Co in liver using high performance liquid chromatography.
The A level was measured. Figure 3 below shows elevated levels of malonyl CoA in tumor tissue compared to liver. Furthermore, the distribution of other CoA derivatives is clearly changing. For example, the liver had about 10-fold lower malonyl CoA, about 10-fold higher acetyl-CoA levels, and higher levels of other CoA derivatives, especially succinyl-CoA, compared to xenografts. The difference in the profile of the CoA derivative may indicate a large difference in energy metabolism between cancer cells and hepatocytes.

For purposes of better understanding, the above invention has been described in some detail by way of illustration and example with reference to specific embodiments, but other aspects, advantages, and modifications are within the skill of the art to which this invention pertains. Obvious to anyone. The above description and examples are intended to be illustrative and not limiting the scope of the invention. Modifications of the above modes for carrying out the invention that are obvious to those skilled in the fields of medicine, immunology, hybridoma technology, pharmacology, and / or related fields are intended to be within the scope of the invention, This is limited only by the scope of the appended claims.

All publications and patent applications in this specification are indicative of the level of technicians in the field to which this invention belongs. All documents and patent applications are herein incorporated by reference to the same extent as if each individual document or patent application was specifically and individually indicated to be incorporated by reference.

[Brief description of drawings]

FIG. 1 shows fatty acid synthesis pathways and the effect of various fatty acid synthase inhibitors on fatty acid synthesis and tumor cell growth.

FIG. 2 shows malonyl CoA levels under various conditions.

FIG. 3 shows the results of clonogenic and apoptotic assays on breast cancer cells treated with various inhibitors.

FIG. 4 shows various parameters in tumor cells and hepatocytes.

FIG. 5 shows malonyl CoA levels in tumor cells and hepatocytes.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE, TR), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, G M, KE, LS, MW, MZ, SD, SL, SZ, TZ , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, B Z, CA, CH, CN, CR, CU, CZ, DE, DK , DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, J P, KE, KG, KP, KR, KZ, LC, LK, LR , LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, R O, RU, SD, SE, SG, SI, SK, SL, TJ , TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Townsend, Craig A.             Bo, 21212, Maryland, USA             Lutimore, Midhurst Road             116 (72) Inventor Kuha Juda, Francis Pea             21209, Maryland, United States             -Cirville, Broadway Road             1211 F term (reference) 2G045 BB10 BB24 CB01 CB02 DA60                       FA29 FB03 FB06 FB08 GC07                       GC10 JA01                 4C084 AA17 AA20 BA44 CA53 CA56                       CA59 DC32 MA02 NA14 ZB262                       ZC202

Claims (21)

[Claims] 1. A method for inhibiting the growth of tumor cells in a living body,
The above method, which comprises administering to the living body a composition that induces a rapid decrease in intracellular free coenzyme A in the cancer cells in the living body. 2. The intracellular malonyl CoA in the cells of the living body sharply increases,
The method of claim 1. 3. The method according to claim 1, wherein the intracellular malonyl CoAg in the cells of the living body is increased within 3 hours of the administration. 4. The method of claim 1, wherein intracellular malonyl CoA is increased prior to cell growth inhibition. 5. The method of claim 1, wherein the increase in intracellular malonyl CoA correlates with a decrease in malonyl CoA consumption. 6. The method of claim 1, wherein said increase in intracellular malonyl CoA precedes any increase in malonyl CoA consumption rate. 7. The method of claim 1, wherein said increase in intracellular malonyl CoA correlates with decreased intracellular activity of malonyl CoA decarboxylase (MCD) or decreased intracellular activity of fatty acid synthase. 8. The method of claim 1, wherein the composition comprises an inhibitor of MCD. 9. The method of claim 1, wherein said increase in intracellular malonyl CoA correlates with an increase in malonyl CoA synthesis. 10. The method of claim 1, wherein said increase in intracellular malonyl CoA correlates with increased intracellular activity of acetyl CoA carboxylase (ACC). 11. The composition contains an activator of ACC, an activator of citrate synthase, an inhibitor of 5′-AMP dependent protein kinase (AMPK), and / or an inhibitor of acyl CoA synthase. The method according to claim 1. 12. The method according to claim 1, wherein the second chemotherapeutic agent is administered to the living body. 13. The method of claim 1, wherein intracellular malonyl CoA levels prior to administration of said composition are at least 2-fold higher than normal malonyl CoA levels in non-tumor cells. 14. The method of claim 1, wherein the intracellular level of malonyl CoA is increased and the intracellular levels of acetyl CoA and free CoA are decreased as compared to the level before treatment. 15. The method according to claim 1, wherein the rate of fatty acid synthesis in certain cells of the living body is at least 2-fold higher than the standard prior to administration of the composition, and administration of the composition is cytotoxic to the cells. the method of. 16. The method of claim 1, wherein reduced intracellular free coenzyme A levels correlate with apoptosis of cells with reduced coenzyme A. 17. The composition comprises an inhibitor of pantothenate kinase, an inhibitor of phosphopantothenoylcysteine synthetase, an inhibitor of phosphopantothenoylcysteine decarboxylase, and / or an inhibition of phosphopantothein adenylyl transferase. The method according to claim 1, further comprising an agent. 18. The method of claim 1, wherein the composition contains a substrate capable of esterifying with CoA. 19. The method according to claim 1, wherein the living body contains tumor cells having an increased rate of fatty acid synthesis, and the cell number of the tumor cells is decreased after administration of the composition. 20. A screening method for facilitating the detection of a composition having a cytotoxicity selective to tumor cells, which comprises administering a target composition to a target cell, and releasing and / or releasing in the cell after the administration. The screening method as described above, which comprises monitoring the intracellular level of derivative coenzyme A, wherein the rapid decrease in intracellular free coenzyme A indicates selective cytotoxicity. 21. A screening method that facilitates the classification of tumor cell-selective cytotoxic compositions in the absence of an ACC inhibitor in an amount sufficient to limit the production of malonyl CoA. , And at the same time administering the targeting composition to the target cells in the presence thereof, wherein the difference in cytotoxicity results from the cytotoxic activity resulting from the effect on the intracellular level of free and / or derivative coenzyme A. Shown above the screening method.