Rapid assay to test anti-cancer drugs under physiological conditions

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This invention relates to an assay that allows for the rapid determination of the activity of a given drug against leukemic cells either taken from a patient or derived from a cell line. The assay is performed in the presence of whole blood or serum.

The research leading to the present invention was supported, at least in part, by a grant from the National Institutes of Health (NIH R01DE16133). Accordingly , the Government may have certain rights in the invention.

This invention relates to an assay that allows for the rapid determination of the activity of a given drug against leukemic cells either taken from a patient or derived from a cell line.

Each year, more than 60,500 people die of hematologic malignancies (leukemia, lymphoma, myeloma) with more than 110,000 new annual diagnoses in the US alone. Current treatment for these cancers includes the use of synthetic compounds that target the cell division process of nearly all cells of the body, not just the cancerous ones. Furthermore, a significant percentage of patients eventually show resistance to many of the drugs, thus rendering treatment largely ineffective. Indeed, there is an effort to identify agents that induce cancer cell death by methods other than damage to DNA or cell division (20).

The initial identification and testing of novel anti-cancer agents relies on in vitro killing assays using relevant cancer cell lines. While in vitro assays performed under cell culture conditions prove useful and necessary for preclinical testing of new therapeutics, extrapolation to the physiological conditions of a living organism is often difficult or impossible (27). Because of the high cost of drug development ($800 million), new drug screens are constantly being sought to more efficiently eliminate or identify candidate therapeutic agents (27). Indeed, increasing the clinical success rate from ⅕ to ⅓ because of more effective preclinical drug screens would reduce drug development costs by more than $200 million (27).

The activity, specificity, or toxicity of a compound in the physiological environment can vary significantly from cell culture conditions. While no in vitro assay or screen can represent the complexity of the human body, several assays have been developed to more closely mimic in vivo conditions. Several of these assays include the colony forming cell assay using bone marrow cells (27,29), hepatic drug biotransformation assays (3), and assays in whole blood (4,45). Because most chemotherapeutic agents are administered intravenously and are therefore immediately affected by blood cell components, screening for potential drugs in the presence of whole blood would be expected to yield more meaningful results. Blood contains biological components, such as proteases, antibody, and blood cells, which can affect the nature of a compound. For example, red blood cells and plasma proteins are known to affect the pharmacokinetics of drugs such as the anti-cancer agents docetaxel and gemcitabine (8,9). Vaidyanathan et al. (43) also reported that the cardioprotective drug, dexrazoxane, inhibits binding of the anti-cancer agent, doxorubicin, to red blood cells and that this interaction alters the pharmacokinetics of doxorubicin, and Clarke et al. (4) used an in vitro whole blood assay to study the binding affinity of a surrogate anti-CD11a monoclonal antibody to blood components. In addition, leukocytes produce a cytochrome P450 isoform (CYP2E1) that is involved in drug biotransformation (3). Thus, identifying and studying drugs in the presence of whole blood or blood components can offer a unique advantage over assays using cells in monoculture.

For studies on leukemia therapeutics, the cell line HL-60 is used as a standard target cell line. HL-60 cells were originally isolated from a 36-year-old female patient with acute promyelocytic leukemia (13). Testing the efficacy of anti-leukemia therapeutics against HL-60 cells in whole blood or other biological material is currently a challenge due to the inefficiency in differentiating the viability of HL-60 cells from other cells. Thus there remains a need to develop an efficient screen for anti-leukemia therapeutics and facilitate preclinical studies on a highly specific bacterial leukotoxin as a novel anti-leukemia therapeutic agent.

Accordingly, a stable bioluminescent HL-60 cell line whose viability can be rapidly and effectively determined in the presence of whole blood and live animals has now been developed along with an assay that allows for the rapid determination of the activity of a given drug against a leukemic cells either taken from a patient or derived from a cell line. The assay is carried out in the presence of whole blood or serum. This quantitative assay can screen thousands of drugs at a time or multiple concentrations of a drug in a 96- or 384-well format.

Screens for compounds and proteins with anti-cancer activity employ viability assays using relevant cancer cell lines. For leukemia studies, the human leukemia cell line, HL-60, is often used as a model system. To facilitate the discovery and investigation of anti-leukemia therapeutics under physiological conditions, HL-60 cells have been engineered that stably express firefly luciferase and produce light. Bioluminescent HL-60luc cells could be rapidly detected in whole blood with a sensitivity of approximately 1000 viable cells. Treatment of HL-60luc cells with a bacterial leukocyte-specific toxin or the drug chlorambucil revealed that the bioluminescent viability assay is more sensitive than the trypan blue dye exclusion assay. HL-60luc cells administered intraperitoneally (i.p) or intravenously (i.v.) were visualized in living mice using an in vivo imaging system (IVIS). The rapidity and ease of detecting HL-60luc cells in biological fluid indicates that this cell line can be used in high throughput screens for the identification of drugs with anti-leukemia activity under physiological conditions.

In vivo bioluminescence imaging (BLI) is a technology that allows visualization of live bioluminescent cells (mammalian, bacterial, viruses) in complex biological material and living animals (24,31). Firefly luciferase has been used extensively in reporter systems and its expression can be measured quantitatively using a luminometer or highly sensitive charge coupled device (CCD) camera. Rocchetta et al. (32) found that the CCD camera was approximately 25 times more sensitive than a luminometer, and so the IVIS 50 imaging system (Xenogen, Alameda, Calif.) was used for the work presented here. Luciferase reacts with its substrate, luciferin, to produce oxyluciferin and light (11). Because ATP and oxygen are required for the reaction, photon production has been used as a quantitative measurement of cellular viability (14). Animal studies have demonstrated a strong correlation between the abundance of emitted photons and number of cells present in a given tissue or animal (5,11).

In general, the field of oncology has utilized BLI extensively to study the effects of anti-cancer therapy in vivo (15,23). However, application of BLI to study hematologic malignancies has been limited (6,22,44), and to date, there are no bioluminescent hematologic cell lines commercially available (Xenogen Corp., Alameda, Calif.). Validation of BLI in preclinical study models has been carried out using currently available methods and evidence indicates that BLI has excellent sensitivity and offers unique advantages (5,25,31,33). For example, non-invasive BLI allows visualization of cells temporally and spatially, thus permitting small changes in cell number and localization to be detected over time (24,31). In addition, animals need not be sacrificed at each sampling time point, decreasing the number of animals that are required for an experiment and minimizing inconsistency from animal-to-animal variations. A bioluminescent HL-60 cell line has been engineered that can be visualized in whole human blood and living mice and whose viability can be rapidly determined. A WBC-specific bacterial toxin has been shown to be active in blood. The engineered HL-60luc cells of the invention behave similar to the parental HL60 cell line. The BLI signal peaked approximately one hour following the addition of luciferin but remained relatively high for several hours. This type of in vitro kinetics where an early peak in luminescence is followed by a slow decline is consistent with other BLI cell lines. The detection limit of 1000 viable cells is also consistent with other reports (35,36). Because human blood contains plasma proteins, such as antibody and proteases, and other cells, that may affect the activity, availability, or stability of a drug, the anti-leukemia assays with HL-60luc cells in the presence of blood can yield more physiological results than with buffer or growth media alone.

There is a significant difference between the sensitivity of BLI and the trypan blue dye. exclusion assay. For a cell to be detected as nonviable with the trypan blue assay, the dye must enter the cytoplasm of the cell. Trypan blue is a relatively large molecule (mw 960.8) and while many cells may be metabolically dead, their membranes could be sufficiently intact to exclude the dye to appear viable. In contrast, BLI detects killing sooner because ATP is no longer available in a metabolically dead cell. The results are in strong agreement with Kuzmits et al. (17) who found that an ATP/bioluminescent assay with HL-60 cells indicated nearly complete killing after a 24 hour incubation with 5.7 μmol/l doxorubicin, while the trypan blue assay indicated almost no killing after 48 hours with the same drug concentration. Furthermore, Petty et al. (30) reported that a bioluminescent ATP assay could detect as few as 1500 viable cells/well while the MTT assay could not detect less than 25,000 cells/well.