WO2002040702A2

WO2002040702A2 - Methods for the treatment of cancer and other diseases and methods of developing the same

Info

Publication number: WO2002040702A2
Application number: PCT/US2001/046194
Authority: WO
Inventors: Vladimir D. Kravtsov; Mark J. Koury; Mace Rothenberg
Original assignee: Vanderbilt University
Priority date: 2000-11-09
Filing date: 2001-11-08
Publication date: 2002-05-23
Also published as: AU2002239491A1; WO2002040702A3

Abstract

The invention relates to methods of determining drug combinations and administration protocols that can be used for the treatment of cancer and other diseases and conditions. The invention also relates to specific methods of treating diseases and conditions such as cancer, as well as to pharmaceutical compositions and kits that can be used in such methods.

Description

METHODS FORTHE TREATMENT OFCANCER AND OTHERDISEASESANDMETHODS OFDEVELOPINGTHE SAME

Priority is claimed to U.S. provisional application no. 60/246,717, filed November 9, 2000, the entirety of which is incorporated herein by reference.

1. FIELD OF THE INVENTION

2. BACKGROUND Traditionally, cancer drugs were discovered through large scale screening of synthetic chemicals and natural products against animal tumor systems, primarily murine leukemias. The agents discovered in the first two decades of cancer chemotherapy largely interacted with DNA or its precursors, inhibiting the synthesis of new genetic material or causing irreparable damage to DNA itself. In recent years, the discovery of new agents has extended from more conventional natural products such as paclitaxel and semisynthetics such as etoposide, both of which target the proliferative process, to entirely new fields of investigation that represent the harvest of new knowledge about cancer biology. Goodman & Gilman's The Pharmacological Basis of Therapeutics, Hardman, J. G., et al., Eds. pp. 1225-1232 (9^th ed., 1996). The first successful applications of this knowledge include diverse drugs. One- agent, interleukin-2, regulates the proliferation of tumor-killing T lymphocytes and so- called natural killer cells, and has proven capable of inducing remissions in a fraction of patients with malignant melanoma and renal cell carcinoma. Another drug, all-trans- retinoic acid, elicits differentiation and can be used to promote remission in acute promyelocytic leukemia, even after failure of standard chemotherapy. Initial success in characterizing unique tumor antigens on melanoma cells and oncogene products, such as mutated p53 or ras proteins, offers the real possibility of tumor vaccines. Id.

Generally, methods of determining the chemosensitivity of tumor cells can be divided into two groups: (a) long term assays based upon clonal growth of the tumor cells; and (b) short term assays of the total cell population relying on loss of cell viability. See, e.g., Preisler, E. D., "Prediction of response to chemotherapy in acute myelocytic leukemia." Blood 56:361 (1980); Park, C. E., et al, "Prediction of chemotherapy response in human leukemia using an in vitro chemotherapy sensitivity test on the leukemic colony-forming cells," Blood 55:595 (1980); Park, C. E., et al, "Clinical correlations of leukemic clonogenic cell chemosensitivity assessed by in vitro continuous exposure to drugs," Cancer Res. 43:2346 (1983); Pieters R., et al., "Comparison of a rapid automated tetrazolium based (MTT)-assay with a dye exclusion assay for chemosensitivity testing in childhood leukaemia," Br. J Cancer 59:2 17 (1989); Sargent, j. M., et al., "Appraisal of the MTT assay as a rapid test of chemosensitivity in acute myeloid leukaemia," Br. J. Cancer 60:206 (1989); Twentyman P. R., et al., "Chemosensitivity testing of fresh leukaemia cells using the MTT Colorimetric assay," Br. J. Hematol. 71:19 (1989). Technical and theoretical limitations of each of these methods have been reviewed in the literature with the common conclusion that development of a new chemosensitivity assays based on alternative methodologies is needed. See, e.g., Weisenthal, L. M., et al., "Laboratory detection of primary and acquired drug resistance in human lymphatic neoplasms," Cancer Treatment Reports 70:1283 (1986); Neerman A. P., and Pieters, R., "Drug sensitivity assays in leukaemia and lymphoma," Br. J. Hematol. 74:381 (1990); Weisenthal, L., and Lippman, M. "Clonogenic and nonclono genie in vitro chemosensitivity assays," Cancer Treat Rep 69:6 15 (1985); Fruehauf I. P. and Bosanquet, A. G. "In vitro determination of drug response: A discussion of clinical applications," Principles & Practice of Oncology 7:1 (1993).

Over the last 7 years, it has been recognized that chemotherapeutic agents exert their in vivo antitumor activity by triggering apoptosis in susceptible tumor cells. See, e.g., Gorczyca, W., et al, "Induction of DΝA strand breaks associated with apoptosis during treatment of leukemias," Eeu e..w^'« 7:659 (1993); Sachs, L., and Lotem, J., "Control of programed cell death in normal and leukemic cells: new implications for therapy," Blood 82:15 (1993); Kerr, j. F. R., et al, "Apoptosis: Its significance in cancer and cancer therapy," Cancer 73:2013 (1994); Hannun, Y. A., "Apoptosis and the dilemma of cancer chemotherapy," Blood 89:1845 (1997). When malignant cells are exposed to a chemotherapeutic agent in vitro, apoptosis in the population can be determined at a specific time by microscopic examination, electrophoretic separation of DΝA fragments, or flow cytometry. See, Wylie, A. H., "Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation," Nature 284:555 (1980); Darzynkievicz, Z., et al, "Features of apoptotic cells measured by flow cytometry," Cytometry 13:795 (1992). However, apoptotic cells exist in vitro for a limited time after which they promptly disintegrate. Wylie, A. H., et al, "Cell death: The significance of apoptosis," Tnt. Rev. Cytol 68:251 (1980). In addition, timing of apoptosis varies significantly depending on the drug, drug concentration and type of target cells. See Cejna, M., et al, "Kinetics of apoptosis and secondary necrosis in cultured rat thymocytes and mouse lymphocytes and human leukemia cells," Biochemistry & Cell Biology 12:611 (1994); Kravtsov, N. and Fabian, I., "Automated monitoring of apoptosis in suspension cell cultures," Lab. Invest. 74:557 (1996); Kravtsov,_.N., et al, "Use of the microculture kinetic (MICK) assay of apoptosis to determine chemosensitivities of leukemias," Blood 92:968-980 (1998); Kravtsov, N., et al, "Comparative analysis of different methodological approaches to the in vitro study of drug-induced apoptosis," Am. J. Path. 155:1327-1339 (1999). Thus, apoptosis in vitro occurs in a relatively broad temporal window such that an accurate determination of apoptosis in a population of cells requires either frequent determinations or continuous monitoring. The above mentioned tests for apoptosis require multiple steps and yet they permit only one time point per sample to be examined. These drawbacks make current assays of apoptosis cumbersome and impractical for chemosensitivity screening.

3. SUMMARY

This invention encompasses methods of discovering drug combinations and treatment protocols that can be used for the treatment of cancer and other diseases and conditions in animals (e.g., mammals such a humans, fish, and birds) and plants. The invention further encompasses specific methods of treating diseases and conditions such as cancer, and pharmaceutical compositions and kits that can be used in such methods.

The invention is based on a discovery that a kinetic assay can be used to determine drug combinations that induce apoptosis in particular types of cells. The kinetic assay can further be used to determine whether the order and relative timing with which drugs are contacted with particular cells affects the ability of those drugs to induce apoptosis. Consequently, a first embodiment of the invention encompasses a method of determining the ability of at least two substances to induce apoptosis, which comprises: contacting a first cell culture with a first substance at a first time; contacting the first cell culture with a second substance at a second time; measuring the optical density of the first cell culture at more than one time point; measuring the optical density of a second cell culture at more than one time point, wherein the second cell culture was not contacted with the first or second substances; and determining a net slope, which is the difference between the optical density change over time of the first cell culture and the optical density change over time of the second cell culture; wherein a positive net slope indicates the ability of the two substances to induce apoptosis.

In a particular method of this embodiment, the first and second times are the same; in another method, they are different. In another particular method, the optical density changes of the first and/or second cell cultures are measured before, at the same time as, or after the first and/or second times. another particular method of this embodiment, the net slope is determined by a method which comprises subtracting at each time point the optical density measurement of the second cell culture from the corresponding optical density measurement of the first cell culture. In another particular method of this embodiment, the net slope is determined by a method which comprises: calculating the rate at which the optical density of the first cell culture changes over time to provide a first rate of change; calculating the rate at which the optical density of the second cell culture changes over time to provide a second rate of change; and subtracting the second rate of change from the first rate of change.

A second embodiment of the invention encompasses a method of determining a combination of drugs useful in the treatment of a disease or condition caused by the proliferation of a type of cell or microorganism, which comprises: contacting a first culture of the type of cell or microorganism with a first substance at a first time; contacting the first culture with a second substance at a second time; measuring the optical density of the first culture at more than one time point; measuring the optical density of a second culture of the type of cell or microorganism at more than one time point, wherein the second culture was not contacted with the first or second substances; and determining a net slope, which is the difference between the optical density change over time of the first culture and the optical density change over time of the second culture; wherein a positive net slope indicates the ability of the two substances to treat the disease or condition.

In a particular embodiment of the invention, the disease or condition is cancer. Examples of cancers include, but are not limited to, primary and metastatic cancer of the head, neck, eye, mouth, throat, esophagus, chest, bone, lung, colon, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, and brain. A specific cancer is colorectal cancer.

In another particular method of this embodiment, the first and second times are the same; in another method, they are different. In another particular method, the optical density changes of the first and/or second cultures are measured before, at the same time as, or after the first and/or second times. another particular method of this embodiment, the net slope is determined by a method which comprises subtracting at each time point the optical density measurement of the second culture from the corresponding optical density measurement of the first culture. In another particular method of this embodiment, the net slope is determined by a method which comprises: calculating the rate at which the optical density of the first culture changes over time to provide a first rate of change; calculating the rate at which the optical density of the second culture changes over time to provide a second rate of change; and subtracting the second rate of change from the first rate of change. Methods of this invention can be used to determine novel combinations of drugs that can be used to treat diseases and conditions such as cancer, as well as novel treatment protocols (e.g., routes of administering drugs, and/or the relative timing of such administrations) that can be used to treat such diseases and conditions. Consequently, this invention encompasses novel methods of treating diseases and conditions, as well as novel pharmaceutical compositions and kits that can be used in such methods.

A third embodiment of the invention therefore encompasses a method of treating cancer in a patient (e.g. , a human) in need of such treatment, which comprises the combined administration of at least two of: a platin-based compound; a folate inhibitor; and deoxycytidine or an analogue thereof. A preferred method of the invention comprises the administration of a platin-based compound, a folate inhibitor, and deoxycytidine or an analogue thereof. This method is particularly useful in the treatment of colorectal cancer. Examples of platin-based compounds include, but are not limited to, cisplatin and oxoplatin. Examples of folate inhibitor include, but are not limited to, MTA or LY231514, and methatrexate. Examples of analogues of deoxycytidine include, but are not limited to, cytarabin and gemcytabine.

A specific preferred method encompassed by this embodiment is a method of treating colorectal cancer in a patient in need thereof, which comprises the combined administration of oxoplatin, MTA, and gemcytabine. Preferably, the gemcytabine and MTA are administered to the patient prior to the administration of oxoplatin. More preferably, the gemcytabine and MTA are administered together or separately at least about 6, 12, or 24 hours before the oxoplatin is administered. The invention encompasses a kit useful in the treatment of colorectal cancer, which comprises at least two of: a pharmaceutical dosage form of a platin-based compound; a pharmaceutical dosage form of a folate inhibitor; and a pharmaceutical dosage form of deoxycytidine or an analogue thereof. A preferred kit of the invention comprises a pharmaceutical dosage form of oxoplatin, a pharmaceutical dosage form of MTA, and a pharmaceutical dosage form of gemcytabine.

3.1. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the correlation between KU and the percentages of morphologically apoptotic cells. FIG. 2 is a graph of apoptosis in treating cells with gemcitabine.

FIG. 3 is a graph of apoptosis in treating cells with MTA. FIG. 4 is a graph of apoptosis in treating cells with oxaliplatin. FIG. 5 is a graph of comparisons between the maximum apoptosis-inducing activities in all Protocols. FIG. 6 shows comparative graphs of a combination of different concentrations of gemcitabine and oxaliplatin compared with oxaliplatin and gemcitabine alone. FIG. 7 shows comparative graphs of a combination of different concentrations of MTA and 6h oxaliplatin compared with oxaliplatin and MTA alone.

FIG. 8 shows comparative graphs of a combination of different concentrations of MTA and 6h gemcitabine compared with MTA and gemcitabine alone. FIG. 9 shows comparative graphs of a combination of different concentrations of gemcitabine and 6h MTA compared with MTA and gemcitabine alone.

FIG. 10 shows comparative graphs of a combination of different concentrations of gemcitabine and 24h oxaliplatin compared with oxaliplatin and gemcitabine alone.

FIG. 11 shows comparative graphs of a combination of different concentrations of MTA and 24h oxaliplatin compared with oxaliplatin and MTA alone.

FIG. 12 shows comparative graphs of a combination of different concentrations of MTA and 24h gemcitabine compared with gemcitabine and MTA alone.

FIG. 13 shows comparative graphs of a combination of different concentrations of gemcitabine and 24h MTA compared with MTA and gemcitabine alone. FIG. 14 shows comparative graphs of a combination of different concentrations of gemcitabine, MTA and 6h oxaliplatin compared with oxaliplatin and (gemcitabine with MTA) 48h.

FIG. 15 shows comparative graphs of a combination of different concentrations of gemcitabine, MTA and 24h oxaliplatin compared with oxaliplatin and (gemcitabine with MTA) 48h.

4. DETAILED DESCRIPTION

The kinetic assay used in the methods of the invention is disclosed by U.S. Patent Nos. 6,077,684 and 6,258,553, both of which are incorporated herein by reference. It has been discovered that this assay (referred to herein as the "MiCK assay") can be used to discover novel combinations of drugs useful in the treatment of diseases such as, but not limited to, cancer. It has further been discovered that the assay can be used to determine specific treatment protocols that are particularly safe and efficacious when used to treat diseases and conditions such as cancer. Examples of cancers that can be treated by methods (e.g. , treatment protocols) and compositions of the invention include, but are not limited to, primary and metastatic cancer of the head, neck, eye, mouth, throat, esophagus, chest, bone, lung, colon, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, and brain. Specific examples of cancers include, but are not limited to: AIDS associated leukemia and adult T-cell leukemia lymphoma; anal carcinoma; astrocytoma; biliary tract cancer; cancer of the bladder, including bladder carcinoma; brain cancer, including glioblastomas and medulloblastomas; breast cancer, including breast carcinoma; cervical cancer; choriocarcinoma; colon cancer including colorectal carcinoma; endometrial cancer; esophageal cancer; Ewing's sarcoma; gastric cancer; gestational trophoblastic carcinoma; glioma; hairy cell leukemia; head and neck carcinoma; hematological neoplasms, including acute and chronic lymphocytic and myelogeneous leukemia; hepatocellular carcinoma; Kaposi's sarcoma; kidney cancer; multiple myeloma; intraepithelial neoplasms, including Bowen's disease and Paget's disease; liver cancer; lung cancer including small cell carcinoma; lymphomas, including Hodgkin's disease, lymphocytic lymphomas, non-Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large cell lymphoma, follicular mixed lymphoma, and lymphoblastic lymphoma; lymphocytic leukemia; neuroblastomas; oral cancer, including squamous cell carcinoma; ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas, including soft tissue sarcomas, leiomyosarcoma, rhabdomyosarcoma, liposcarcoma, fibrosarcoma, and osteosarcoma; skin cancer, including melanoma, Kaposi's sarcoma, basal cell cancer and squamous cell cancer; testicular cancer, including testicular carcinoma and germinal tumors (e.g., semicoma, non-seminoma[teratomas, choriocarcinomas]), stromal tumors and germ cell tumors; thyroid cancer, including thyroid adenocarcinonia and medullar carcinoma; and renal cancer including adenocarcinoma and Wilm's tumor. A preferred method of the invention is a method of treating colorectal cancer.

Compounds administered to patients according to methods of the invention maybe administered by any suitable routes known to those of skill in the art. For example, each of the drugs administered to a patient can be administered orally, mucosally (e.g., nasally, sublingually, vaginally, buccally, or rectally), parenterally (e.g., subcutaneously, intravenously, by bolus injection, intramuscularly, or intraarterially), or transdermally.

The magnitude of a prophylactic or therapeutic dose of an active ingredient in the acute or chronic management of a disorder or condition will vary with the severity of the disorder or condition to be treated and the route of administration. The dose, and perhaps the dose frequency, will also vary according to age, body weight, response, and the past medical history of the patient. Suitable dosing regimens can be readily selected by those skilled in the art with due consideration of such factors. Compounds administered to patients according to methods of the invention may be orally administered by various techniques which include, but are not limited to, tablets, coated tablets, caplets, troches, lozenges, dispersions, suspensions, suppositories, ointments, cataplasms (poultices), pastes, powders, dressings, creams, plasters, solutions, capsules, soft elastic gelatin capsules, sustained release formulations, and patches. Pharmaceutical compositions may also be administered parenterally as a sterile, injectable solution, in a sterile lyophilized powder suitable for reconstitution into a solution, or in sterile, hermitically sealed ampules. In practical use, an active ingredient can be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. In preparing the compositions for an oral dosage form, any of the usual pharmaceutical media can be employed as carriers, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (such as suspensions, solutions, and elixirs) or aerosols; or carriers such as starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents can be used in the case of oral solid preparations. For example, suitable carriers include powders, capsules, and tablets, with the solid oral preparations being preferred over the liquid preparations.

Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid pharmaceutical carriers are employed. If desired, tablets can be coated by standard aqueous or nonaqueous techniques. Compounds administered to patients according to methods of the invention are suitable for oral administration and can be presented as discrete dosage forms, such as capsules, cachets, or tablets, or aerosol sprays each containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion. Such dosage forms can be prepared by any of the methods of pharmacy, but all methods include the step of bringing the active ingredient into association with the carrier, which constitutes one or more necessary ingredients, h general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

5. EXAMPLES

Various chemotherapeutic agents (e.g., gemcitabine, MTA, and oxaliplatin) were evaluated, singly and in selected combination and sequence, for their ability to induce apoptosis in three well characterized human colorectal cancer cell lines. The MiCK assay provided an efficient method to screen a large number of 1, 2, and 3 drug permutations against a panel of human tumor cell lines.

Single agent data from the MiCK assay corresponds to the known patterns of clinical activity for each of these agents against colorectal cancer. Gemcitabine and MTA demonstrated minimal ability to induce apoptosis in any of the three colorectal cancer cell lines screened. These drugs have little clinical activity in patients with advanced colorectal cancer as first-line therapy in patients with locally advanced or metastatic colorectal cancer. In contrast, oxaliplatin demonstrated significant activity in 2 of the 3 cell lines, which is quite consistent with clinical experience that has identified oxaliplatin as one of the most active single agents against this disease.

The single agent inactivity of gemcitabine and MTA against human colon cancer cell lines is not altered by combining the agents with each other in any sequence for any duration of time.

Unexpectedly, however, the use of the MiCK assay revealed that high apoptotic responses were observed with the sequence of gemcitabine, MTA, or both (for either 6 hours or 24 hours) followed by oxaliplatin. From a mechanistic perspective, and without being limited by theory, this might be the result of gemcitabine or MTA reducing the expression or activity of DNA repair enzymes that might be involved in the recognition and repair of oxaliplatin-DNA adducts. Alternatively, gemcitabine or MTA might down- regulate cellular detoxification proteins, such as glutathione or metallothionine, that might "trap" oxaliplatin before it can platinate DNA.

5.1. PROTOCOL

Eighteen different protocols of drug treatment were evaluated in each of the three cell lines. These protocols included cell exposure to a single agent (See, for example protocols 1-3) and to various drug combinations (See, for example, protocols 4-18). Table 1 provides a description of the protocols:

Table 1 Protocol Design

Protocol Drug 1 and Duration Drug 2

1 Gemcitabine x 48 hours -

2 MTA x 48 hours -

3 Oxaliplatin x 48 hours -

4 Gemcitabine x 6 hours Oxaliplatin

5 MTA x 6 hours Oxaliplatin

6 MTA x 6 hours Gemcitabine

7 Gemcitabine x 6 hours MTA

8 Oxaliplatin x 6 hours MTA

9 Oxaliplatin x 6 hours Gemcitabine

10 Gemcitabine x 24 hours Oxaliplatin

11 MTA x 24 hours Oxaliplatin

12 MTA x 24 hours Gemcitabine Protocol Drug 1 and Duration Drug 2

13 Gemcitabine x 24 hours MTA

14 Oxaliplatin x 24 hours MTA

15 Oxaliplatin x 24 hours Gemcitabine

16 Gemcitabine + MTA x 6 hours Oxaliplatin

17 Gemcitabine + MTA x 24 hours Oxaliplatin

18 Gemcitabine + MTA x 48 hours -

the single-drug protocols, the MiCK assay was initiated within 30 min after addition to the cells of a drug at eight increasing concentrations from about O.OlμM to about 400μM. In the drug-combination protocols, cells were exposed to the eight concentrations of the first compound for about 6 or about 24h after which time the second compound was added at eight concentrations and the MiCK assay was initiated within 30 min. h two protocols (16 and 17), the cells were exposed for about 6h or about 24h to the mixture of gemcitabine and MTA after which time oxaliplatin was added and the MiCK assay was initiated, h protocol 18, the MiCK assay was initiated within about 30 min after addition to the cells of the two compounds, i.e., gemcitabine and MTA. h all protocols, the MiCK assay, once initiated, monitored apoptotic responses in non-disturbed cell cultures for about 48h.

5.2. METHODS AND MATERIALS

HT29, LoNo, and COLO human colon adenocarcinoma cell lines were obtained from ATCC (Rockville, MD). Cells were maintained in Iscove's Modified Dulbecco medium (IMDM) without phenol red supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan,UT), 100 μg/mL penicillin and 100 μg/mL streptomycin (complete medium) in completely humidified air with 5% CO₂ at 37°C. Before use, exponentially growing cells were harvested, washed with prewarmed medium and resuspended in complete medium. Cell counts and viability were determined using a hemocytometer and trypan blue dye exclusion.

5.2.1. MiCK ASSAY

Cells were suspended in complete medium and plated in 96-well microliter plates (Corning-Costar, Cambridge, MA) at 1.5xl0⁵ cells/mL. Cells were allowed to accommodate in cultures for 18h before adding drugs. At the concentration used, cells of all of the three cell lines were distributed sparsely on the well bottom and never achieved confluency. Appropriate dilutions of oxaliplatin (Ox), gemcitabine (Gem) or MTA were added to wells in 5 μL aliquots to yield final concentrations of 0.01, 0.1, 1.0, 10, 50, 100, 200 and 400 μM. The microtiter plates were incubated at 37 °C for 30 min in a completely humidified atmosphere of 5% CO₂in air. Next, 30 μL of sterile mineral oil (Sigma, St. Louis, MO) was layered on the top of each microculture. The microtiter plate was placed in the incubated chamber of a spectrophotometer (Power Wave 340, BioTek.), incubated at 37 °C and the OD at 600 nm was read every 5 minutes for a period of 48 h. The reader was calibrated to zero absorbance using wells containing only complete medium without cells. Data acquisition and computing of cell apoptotic responses were performed using a proprietary KORsoft 1.1 software (patent pending). All tests were performed in triplicate and the results shown are the mean values for those three replicate tests.

5.2.2. CELL MORPHOLOGY

Percentages of cells with morphological evidence of apoptosis were counted on Giemsa-stained cytosine preparation of control and drug-treated cultures. A total of 200 cells were counted in each preparation. Apoptotic cells were identified by plasma membrane blebbing, aggregated chromatin, fragmented nuclei, and condensed basophilic cytoplasm.

Correlations between KU and the percentages of morphologically apoptotic cells for the three cell lines were determined as described previously. Resulting curves are shown in Fig. 1 and serve as nomograms to convert KU into the percentages of apoptotic cells.

5.2.3. DRUGS USED AND PREPARATION

Drags were dissolved in water to get 10 mM stock solutions. Appropriate serial dilutions of the stock solutions of oxaliplatin (Ox), gemcitabine (Gem) or MTA were added to wells in 5 μL aliquots to yield final concentrations of 0.01, 0.1, 1.0, 10, 50, 100, 200 and 400 μM.

Each drug was evaluated over a 4-log range of concentrations, from 0.01 μM to 400 μM. This range encompassed the peak plasma concentrations achieved in Phase I clinical trials for each of the agents:

Agent Dose C max Terminal t 1/2-

Gemcitabine lOOO mg/rn² 125 μm 8.2 minutes (plasma) MTA 600 mg/rn² 290 μm 3.08 hours Oxaliplatin

130 mg/rn² 12.8 μm 38.7 hours (Total platinum) 5.3 μm 24.2 hours (Ultrafiltrable platinum)

Tests concerning comparisons between protocols were completed using the restricted/residual maximum likelihood (REML)-based repeated measure model (mixed model analysis) with various covariance structure. Comparisons were performed between the maximal responses for each of the protocols. The mean apoptotic response (expressed in KU) difference between protocols was estimated using the generalized least-squares method with adjusted dose levels as well as the interaction terms.

All tests of significance were two-sided, and differences were considered statistically significant when p-value was <0.05. All data was expressed as means ±SD.

5.3. RESULTS

5.3.1. PROTOCOLS 1-3: SINGLE AGENTS In all 3 cells lines studied, treatment with single agent gemcitabine (Protocol 1,

Fig.2) or single agent MTA (Protocol 2, Fig.3) induced minimal apoptosis (<1 KU). In contrast, single agent oxaliplatin (Protocol 3, Fig.4) induced significant apoptosis in 2 of the 3 cell lines (active against HT29 (8.5KU) and LoNo (5.5 KU), while demonstrating moderate activity against COLO cells (3.5 KU). It is important to note that these results are quite consistent with clinical observations of inactivity for single agent gemcitabine and single agent MTA in patients with advanced colorectal cancer and substantial clinical activity for single agent oxaliplatin (RR= 10-15% in patients with recurrent or refractory disease and 20-35% in patients with chemotherapy-naive colorectal cancer).

Comparisons between the maximum apoptosis-inducing activities of all Protocols are shown in Fig.5.

An ample database generated during this study allows a great variety of comparisons between different protocols and their combination to be made. For selected combinations of protocols, the full range apoptotic response comparisons are shown in Fig. 6 through Fig. 15. All figures were built using the attached sets of raw data.

5.3.2. PROTOCOLS 4-9: COMBINATIONS: 6 HOUR EXPOSURE OF DRUG 1 FOLLOWED BY 48 HOUR EXPOSURE TO DRUG 2

The sequence of gemcitabine followed by oxaliplatin (Protocol 4) induced similar levels of apoptosis in COLO and HT29 cells as single agent oxaliplatin (Fig.6). However, this sequence was significantly more active than single agent oxaliplatin in the LoNo model

(p = .03 17) (Fig.6). The sequence of MTA followed by oxaliplatin (Protocol 5) was not significantly more active than single agent oxaliplatin in inducing apoptosis in any of the three cell lines (Fig.7). Protocol 6, in which MTA was followed by gemcitabine was significantly more active than either agent given alone in the COLO cell line, but was no more effective than single agent gemcitabine in the HT29 or LoNo cell lines (Fig.8).

Suprisingly, the reverse sequence (Protocol 7) was completely inactive in the COLO and

LoNo cell lines, and less effective than single agent gemcitabine in the HT29 model (p < .0001) (Fig.9). This suggests antagonism when the nucleoside analog, gemcitabine, precedes the antimetabolite, MTA. Protocols 8 (oxaliplatin followed by MTA) and 9 (oxaliplatin followed by gemcitabine) did not produce interpretable results due to the substantial effect of single agent oxaliplatin on inducing apoptosis prior to the addition of the second agent.

5.3.3. PROTOCOLS 10-15: COMBINATIONS: 24 HOUR EXPOSURE OF DRUG 1 FOLLOWED BY 48 HOUR EXPOSURE TO DRUG 2 24 hour exposure to gemcitabine followed by oxaliplatin (Protocol 10) and 24 hour exposure to MTA followed by oxaliplatin (Protocol 11) produced the highest levels of apoptosis observed in this series of experiments in the HT29 and LoNo models. Both combinations were significantly more active than any of the single agents in the HT29 model (all comparisons significant at the p = .0001 level or less). Figures 10 and 11 depict this comparative data in graphical form. Little enhancement in activity is observed at the two lowest concentration of oxaliplatin (0.01 and 0.1 μM) when added to either gemcitabine (Fig. 10, A-H) or MTA (Fig. 11, A-H). However, at concentrations of oxaliplatin ranging from 1 - 400 μM, significant augmentation of apoptosis is for a wide range of concentrations of either gemcitabine or MTA. Importantly, this enhancement occurs at clinically-achievable concentrations for all three drugs. 24 hour exposure to MTA followed by gemcitabine (Protocol 12) was more active than single agent gemcitabine in the LoNo cell line (p = .0003) (Figure 12) and more active than single agent MTA in the HT29 cell line (p = .0008) (Figure 12). Similarly, the reverse sequence of 24 hour exposure to gemcitabine followed by MTA (Protocol 13) also proved to be more active than single agent gemcitabine in both the HT29 and LoNo models (p = .007 and .0013, respectively; Fig. 13). No apoptosis was observed with either of these combinations in the COLO model (Fig. 13). As in the previous series of experiments, no useful data was obtainable from Protocols 14 and 15 due to the high degree of apoptosis induced by prolonged exposure of the cells to oxaliplatin before the addition of the second agent.

5.3.4. PROTOCOLS 16-18: COMBINATIONS:

SIMULTANEOUS EXPOSURE TO GEMCITABINE AND MTA WITH OR WITHOUT SUBSEQUENT EXPOSURE TO OXALIPLATIN Simultaneous exposure of cells to MTA and gemcitabine for 6 hours followed by oxaliplatin (Protocol 16) was significantly more active than 48 hour exposure to gemcitabine alone (Protocol 1) (p = .0507) in the LoNo model (Fig. 14). Simultaneous exposure of cells to MTA and gemcitabine for 24 hours followed by oxaliplatin (Protocol

17) was significantly more effective than either agent alone in the LoNo model, but failed to demonstrate any increase in apoptosis in the HT29 or COLO models (Fig. 15). Simultaneous exposure of HT29 cells to 48 hours of MTA and gemcitabine (Protocol 18) was no more effective than single agent gemcitabine (Fig. 14 and Fig. 15).

It should be understood that variations and modifications within the spirit and scope of the invention may occur to those skilled in the art to which the invention pertains. Accordingly, all expedient modifications readily attainable by one versed in the art from the disclosure set forth herein that are within the scope and spirit of the present invention are to be included as further embodiments of the present invention. The scope of the present invention accordingly is to be defined as set forth in the appended claims.

Claims

THE CLAIMSWhat is claimed is:

1. A method of determining a combination of drugs useful in the treatment of a disease or condition caused by the proliferation of a type of cell or microorganism, which comprises: contacting a first culture of the type of cell or microorganism with a first substance at a first time; contacting the first culture with a second substance at a second time; measuring the optical density of the first culture at more than one time point; measuring the optical density of a second culture of the type of cell or microorganism at more than one time point, wherein the second culture was not contacted with the first or second substances; and determining a net slope, which is the difference between the optical density change over time of the first culture and the optical density change over time of the second culture; wherein a positive net slope indicates the ability of the two substances to treat the disease or condition.

2. The method of claim 1 , wherein the disease or condition is cancer.

3. The method of claim 2, wherein the cancer is primary or metastatic cancer of the head, neck, eye, mouth, throat, esophagus, chest, bone, lung, colon, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, or brain.

The method of claim 3, wherein the cancer is colorectal cancer.

The method of claim 1, wherein the first time and the second times are the same.

6. The method of claim 1 , wherein the first time and the second times are different.

7. The method of claim 1 , wherein the optical density changes of the first and/or second cultures are measured before the first and/or second times.

8. The method of claim 1, wherein the optical density changes of the first and/or second cultures are measured at the same time as the first and/or second times.

9. The method of claim 1, wherein the optical density changes of the first and/or second cultures are measured after the first and/or second times.

10. The method of claim 1 , wherein the net slope is determined by a method which comprises subtracting at each time point the optical density measurement of the second culture from the corresponding optical density measurement of the first culture.

11. The method of claim 1 , wherein the net slope is determined by a method which comprises: calculating the rate at which the optical density of the first culture changes over time to provide a first rate of change; calculating the rate at which the optical density of the second culture changes over time to provide a second rate of change; and subtracting the second rate of change from the first rate of change.

12. A method of determimng a dosing schedule which comprises running at least two experiments according to the method of claim 1, wherein the difference between the first and second times for each of the at least two experiments are different, wherein the dosing schedule corresponds to the difference between the first and second times that killed the largest percentage of cells or microorganisms in the first cultures.

13. A method of determining a drug combination which comprises running at least two experiments according to the method of claim 1, wherein the first and second substances of each of the at least two experiments are different, wherein the drug combination corresponds to the first and second substances that killed the largest percentage of cells or microorganisms in the first cultures.

14. A method of treating a disease or condition caused by the proliferation of a type of cell or microorganism, which comprises administering to a patient in need of such treatment at least two drugs that cause apoptosis of the cell or microorganism when administered in sufficient amounts at on a dosing schedule determined by the method of claim 12.

15. A method of treating a disease or condition caused by the proliferation of a type of cell or microorganism, which comprises administering to a patient in need of such treatment the drug combination determined by method of claim 13.