EP0835111A2 - Procede de ralentissement dynamique de la cinetique du cycle cellulaire, destine a potentialiser les lesions cellulaires - Google Patents

Procede de ralentissement dynamique de la cinetique du cycle cellulaire, destine a potentialiser les lesions cellulaires

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Publication number
EP0835111A2
EP0835111A2 EP96923453A EP96923453A EP0835111A2 EP 0835111 A2 EP0835111 A2 EP 0835111A2 EP 96923453 A EP96923453 A EP 96923453A EP 96923453 A EP96923453 A EP 96923453A EP 0835111 A2 EP0835111 A2 EP 0835111A2
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Prior art keywords
cells
agent
cell
tci
dthd
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English (en)
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Philip M. Grimley
Sunil Mehta
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Henry M Jackson Foundation for Advancedment of Military Medicine Inc
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Henry M Jackson Foundation for Advancedment of Military Medicine Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention relates to a method of potentiating cell damage by administering an agent that retards the rate of movement of a target cell through some portion of the cell-division cycle and administering a cytotoxic agent that acts within a portion of the cell-division cycle through which movement has been slowed.
  • the method of the invention can be used in chemotherapy as well as in other medical and non-medical applications.
  • deoxythymidine (dThd) is the agent retarding the rate of movement of a target cell through a portion of the cell-division cycle and staurosporine is the cytotoxic agent.
  • the invention also relates to a method of using a microculture indicator system (MIS) and auxiliary data analysis procedures to determine the degree of interaction between agents.
  • MIS microculture indicator system
  • data are collected reflecting the effect on cell growth of two or more agents arrayed in serial bivariate dilutions, and a database is caused to process the data in a spreadsheet according to predetermined relationships with reference measurements of cell growth and to present, in graphical or tabular form, the spectrum of interaction of the agents with respect to reference measurements.
  • a vast array of physical, chemical, or biological agents are hazardous to living cells and can inflict damage upon biological systems such as tissues or organs. In many cases, however, the damage is not specifically targeted to events related to the cell-division cycle.
  • cell damage may be initiated in direct relation to the hierarchy of the cell-division cycle.
  • a cytotoxic agent that acts during some portion of the cell-division cycle, causing biologically significant or irreversible damage to a proliferating cell may serve as a "targeted cytotoxic insult" or "TCI", as defined herein.
  • TCI targeted cytotoxic insult
  • the portion of the cell-division cycle during which a given TCI initiates a relevant action is its "target interval.”
  • TCIs agents that can act as TCIs are diverse and include natural substances, products of microbial or other cellular origins, synthetic or semi-synthetic organic or inorganic chemical compounds, or simple inorganic reagents. Other factors that can act as a TCI are also known and may include deprivation of nutrients essential to cell growth or sustenance as well as changes in the physicochemical environment. Examples of the latter include temperature changes and exposure of the cells to radiant or particulate energies, vibrational waves, or various other physical forces. Cytotoxic effects of a TCI may not be immediate, so that cell damage initiated in one phase of the cell-division cycle may not become manifest until a later phase or a subsequent cell cycle.
  • cell cycle all growing cells must duplicate their genomic DNA and pass identical copies of this genetic information to their progeny.
  • proliferating somatic (non-reproductive) and germ (reproductive) cells of all living organisms undergo repetitive cell- division cycles (hereinafter "cell cycle” or "CC").
  • cell cycle each completed cell-division cycle results in the duplication of the cell's genetic information and the division of the parent cell into two daughter cells, with an equal division of the parental cell DNA.
  • the biochemical and biomolecular processes that comprise the cell cycle include, among other things, enzyme-dependent DNA replication, enzyme-dependent phosphorylation, signal cascades, association and dissociation of transcriptional activating molecular complexes, and formation and dissociation of macromolecular assemblies of cytostructural elements including cytomembranes and the cytoskeleton.
  • the processes characterizing the cell cycle form a regulated hierarchy and advance in a strict order dependence under the control of a cell cycle "engine” or “control system.”
  • the control system functions as a biomolecular “clock” or “oscillator” and includes critical controls at "checkpoints.”
  • LN Edmunds, Jr. Ann. NY Acad. Sci. 719:77-96 (1994); IA Carre et al., J Cell Sci. 104:1163-73 (1993); BG Gabrielli et al., J. Biol Chem 267:1969-75 (1992); A Goldbeter, Proc. Natl. Acad. Sci. (USA) 88:9107-11 (1991); Murray AW and Kirschner MW, Science 246:614-621 (1989).
  • the hierarchy of the eukaryotic cell cycle relates to four conserved functional landmarks (Fig. 1): S phase, in which nucleotides are synthesized and DNA is semi-conservatively replicated in double-stranded helixes of polynucleotides; G 2 phase, which follows completion of DNA synthesis and during which DNA associates with nucleoproteins; M phase, in which nuclear filaments condense as chromosomes and chromosomes segregate for mitosis; and G, phase, during which cells prepare for renewed division by replacement of depleted products and repair of any lesions in DNA. See Alberts, supra. Cells entering S phase normally are committed to completion of G 2 phase, M phase, and cytokinesis. B. Cell Cycle Checkpoints
  • Rb tumor suppressor gene product pRb
  • function of p53 and phosphorylation of the Rb tumor suppressor gene product are also associated with the G,/S transition.
  • the T DBL provides a yardstick for determining the "fractional duration" of major phases in the cell cycle phases, i.e., the time required for a fraction of the cell population to complete G. (T G1 ), S (T s ), or G 2 & M (T G2 & . .
  • T DBL can be determined from serial cell counts, or flow cytometry, AC Begg et al., Cytometry 6:620-626 (1985), while F s , F G2+M or F GI/G0 can be measured in DNA histograms obtained from flow cytometry.
  • G, phase When changes in physiological stimuli or ambient growth conditions slow down the growth of a cell population, the fraction of cells found in G, phase (the preparation phase) typically increases at the expense of cells in S phase or in the growth fraction, i.e., G 2 and M phases. Such changes or abnormal conditions may include hormonal, nutritional, or environmental changes. If abnormal conditions prevail, then cells in G, phase may retire temporarily from the cell cycle to become “quiescent” or non-active. Quiescent cells are commonly designated to be in G 0 phase. R Baserga, Cell Division, Molecular Biology, IN: Encyclopedia of Human Biology 2:253-266 (1991); AB Pardee, Science 246:609-613 (1989). The process of differentiation, or specialization of cells, is also associated with a retirement of cells to G 0 . In terminal differentiation, the transition out of the cell cycle, i.e., into G 0 , becomes irreversible. Examples of terminally differentiated cells are adult neurons, keratinized epithelia, and voluntary muscle cells.
  • Apoptosis is referred to as a process of "programmed cell death.”
  • cell populations in specific organs or tissues may be programmed for death as part of the developmental progression of tissue remodeling or obsolescence.
  • Apoptosis is internally triggered by biochemical or biomolecular mechanisms intrinsic to the cell cycle, resulting in an activation of endogenous endonucleases (enzymes that degrade DNA), leading to DNA strand breaks between nucleosomes and degradation of the genomic DNA by fragmentation.
  • endogenous endonucleases enzymes that degrade DNA
  • Apoptosis in mature tissues occurs in normal processes such as inflammation or rejuvenation. M Schmied et al., Am J Pathol 143:446-52 (1993). Abnormal clonal proliferations in immunologic diseases or malignancies may be related to a failure of normal apoptosis. J Marx, Science 259:760-1 (1993).
  • TCI tumor necrosis .
  • induction chemotherapy or as an adjunct to surgery or radiotherapy (adjuvant chemotherapy).
  • adjuvant chemotherapy or as an adjunct to surgery or radiotherapy.
  • DeVita, supra (1994) Local treatments have included infusion of TCI into body cavities to control the spread of malignancies such as breast or ovarian cancers.
  • Neoplastic (cancerous) populations are heterogeneous and a fraction of resistant cells typically escape death. The subpopulation of malignant cells with protective mechanisms eventually replaces the original populations of susceptible cells.
  • a TCI can inhibit enzymes, compete for substrates, inhibit the transcriptional, translational or post-translational steps in molecular biosynthesis, introduce transcriptional or translational errors, disrupt molecular conformational changes, inhibit molecular transport, compete for energy transfer molecules, interfere with macromolecular polymerization, form molecular crosslinks, alkylate or cause strand breaks in DNA, or intercalate into the DNA helix.
  • a TCI may impair cell cycle processes such as RNA transcription and translation, DNA strand elongation, replication, repair, supramolecular organization or separation, molecular transport, or macromolecular segregation. Alternatively, it may selectively injure any of the multiple cellular organelles associated with successful completion of specific subsets of the cell cycle hierarchy.
  • checkpoint control such as a cyclin-dependent kinase (cdk or cdc), which normally controls the cell cycle hierarchy.
  • cdk or cdc cyclin-dependent kinase
  • the role of checkpoint controls has been defined by observed effects of agents or mutations which relieve order dependence. See HA Crissman et al., Proc. Natl. Acad. Sci. (USA) 88:7580-84 (1991); Kastan, supra.: Murray, supra. (1992); Hartwell, supra.
  • the cell cycle of normally cycling cells must traverse the G, decision point (START) which commits a cell to continue through S phase resulting in DNA replication. Heichman, supra.
  • cells exposed to a TCI prior to START may be partially protected from DNA damage by a delay within G,.
  • This G, delay can be mediated by the tumor suppressor p53 and enables cells to repair damaged strands of DNA prior to replication.
  • Kastan, supra. Damage to DNA after START or DNA damage and bypass of START can be biologically deleterious, PM O'Connor et al., Cancer Res. 4776 (1994), possibly leading to DNA replication infidelity in S phase, with resulting genetic instability and ultimately premature cell death.
  • agents targeting S phase in eukaryotic cells are intrinsically complex due to the nature of DNA replication in S phase.
  • replication origins are discontinuous, chain elongation proceeds asynchronously, and progression at replication forks may be irregular.
  • CS Newlon Science 262:1830-31 (1993); V Levenson et al., Nucleic Acids Res. 21:3997-4004 (1993).
  • RNR ribonucieotide reductase
  • HU dThd or hydroxyurea
  • MTX methotrexate
  • Aph DNA polymerase inhibitors
  • each agent should be effective as a single agent in cell killing
  • agents should be combined from different classes of actions to allow maximum dose intensity; (iii) additive patient morbidity or mortality should be avoided; and (iv) schedules or intervals of agent administration should be optimized.
  • Efforts have been aimed at modulating the cell cycle as a means for increasing cell damage by combinations of chemotherapy agents.
  • the objective was to maintain malignant cells within the S phase of the cell cycle, where they may be most vulnerable to damage. See HO Klein et al., Semin. Hematol. 11 :203-27 (1974). RL Stolfi et al., Pharmac. Ther. 49:43-54 (1991), have referred to these strategies as "cytokinetic modulation".
  • Agents in combination may have additive, synergistic or antagonistic effects.
  • a combination of agents causing a cell cycle arrest or static synchronization of the malignant cells would circumvent the problem of first order kinetics, combined dosages can be increased to a level sufficient to fill all malignant cells without sacrificing the host.
  • Restriction of a malignant cell population to a limited set of the cell cycle hierarchy, where the cells were specifically vulnerable to damage by a successive TCI might be expected to shift the dynamics of cell killing toward greater efficiency, and reduce side-effects by diminishing the cumulative time of host exposure to a TCI.
  • This method can be compared to the arrest method of HR Zielke, supra and JL Littlefield et al., 1974, in which an arrest of the cell-division cycle at a specific detention point is reversed so that the cells then move in concert through the cell cycle at a normal or possibly an accelerated rate.
  • mice were moving in concert through the cell cycle.
  • Mean survival time of the mice was determined. Mice treated with Ara-C at zero time, just after the HU infusion ended, showed improved survival, but treatments with Ara-C at later times after stopping the HU infusion did not potentiate the effect of HU.
  • This procedure of cell cycle synchronization followed by a second agent relies on the nonsimultaneous action of the two agents.
  • the indirect results with respect to mean survival time of the animals cannot be directly translated to effects on tumor cell damage.
  • a major challenge in combination chemotherapy is to determine an optimal synergy in view of the multiple variables of dose, pharmacokinetics, sequence, and scheduling. Even for two agents, the most effective utilization is not necessarily clear from a simple combinational analysis of the optimum for each agent.
  • Empirical variables include the dose or effective concentration of each agent, sequence of agents, intervals between doses, i.e., schedule, duration of dosages and numbers of doses per course of therapy. See Capizzi, supra; Adel, supra. See also MC Berenbaum. Pharmacol. Rev. 41 :93-141 (1989).
  • protein kinases play an important role in neoplasia. Overexpression has been associated with hematologic malignancy. GQ Daley et al., Science 247:824-30 (1990). Neoplastic cells may be deficient in kinase-mediated control of progression through G, and commitment to DNA replication. High levels of several protein kinases in cancer cells have been associated with multidrug resistance to conventional chemotherapeutic agents which are targeted to S phase. Baltuch, supra: JA Posada et al., Cancer Commun. 1 :285-92 (1989); K Kawamura, Hokkaido Igaku Zasshi 69:354-71 (1994).
  • the potent protein kinase inhibitor staurosporine (STSP) and several functional analogues have been of interest since they can (1) reverse or modulate multidrug resistance, KE Sampson et al., J Cell Biochem 52:384-95 (1993); CH Versantvoort et al., Br. J. Cancer 68:939046 (1993); K Miyamoto et al., Cancer Res. 53:1555-9 (1993); I Utz et al., Int. J. Cancer 57:104-10 (1994); (2) arrest cell cycle progression, S Bruno et al., Cancer Res.
  • STSP is a product of Streptomyces staurosporous, Meksuriyen D and Cordell GA, J of Nat. Products 51 :893-899 (1988), and is one of the most powerful broad spectrum inhibitors of protein kinases, Tamaoki, Methods Enzym. 201 :340-347 (1991).
  • STSP inhibits cyclin-dependent kinases associated with the S/G 2 transition, DM Gadbois, Biochem. Biophys. Res. Comm. 189:80-85 (1993), and it can arrest neoplastic cells in G 2 phase of the cell cycle.
  • the kinase inhibitor agents K252A, KT5720, and KT5926 have different ranges of potency with regard to inhibition of protein kinases. They are described in a series of references: WE Payne et al., J. Biol Chem. 263:7190 (1988); RL Raynor, J. Biol Chem. 266:2753 (1993); C.
  • the first problem is non-specificity.
  • a TCI may not be sufficiently selective, resulting in the injury of cells not intended for damage. See IsL, EM Ross, Chapter 2, p. 33-48, Pharmacodynamics: Mechanisms of drug action and the relationship between drug concentration and effect, IN: Goodman and Gilman's The Pharmacologic Basis of Therapeutics, 8th ed., (AG Gilman et al., ed., Pergammon Press, New York, 1990).
  • a second problem is heterogeneous vulnerability where the inherent genetic variability of cells in a population intended for killing can frustrate efforts to achieve absolute and specific lethality. See Calabresi, supra.
  • a third problem is acquired resistance, where some fraction of the cells intended for damage by a TCI acquire resistance to the TCI by physiological or metabolic adaptation, or by genetic mutation. L
  • important objectives of therapeutic drug development or improvements in therapeutic drug application include efforts to increase the ratio of specific cytotoxic benefits, e.g., the intended killing or damage of a designated cell population, to non-specific side effects which produce host morbidity or environmental disruptions.
  • cytotoxic benefits e.g., the intended killing or damage of a designated cell population
  • non-specific side effects which produce host morbidity or environmental disruptions.
  • a major limitation of cancer chemotherapy has been perceived to be the inability to escalate doses of effective anticancer agents, such as TCIs, into the high end of dose-response curves due to intolerable side effects. DeVita, supra (1994). It is also a current concern that omission of one agent from a designed combination may allow overgrowth by a cell lineage susceptible to that agent, but resistant to other agents. Another concern is that the use of an effective agent in less than maximum strength may vitiate the objectives of a combined agent protocol.
  • Fig. 1 is a diagram of the cell-division cycle and associated major cyclins, cyclin kinases and other and regulatory proteins.
  • Fig. 2 depicts bar diagrams representing transit of cells through a target interval of a TCI (A) and the slowed transit of cells through such a target interval in the presence of an RA, resulting in "cell stacking" (B).
  • Fig. 3 depicts an algorithm to identify agent concentrations with operative characteristics of RA to determine their reference point cell cycle position.
  • Fig. 4 depicts the results of an MTT assay showing that during approximately one doubling time of 24 hours of human promonocytic lymphoma cells, the 40% inhibitory concentration of dThd exceeded 2 mM.
  • Fig. 5 depicts the results of a flow cytometric analysis showing how cell cycle dynamics of human promonocytic lymphoma cells changes upon treatment with various concentrations of dThd. Specifically Fig. 5 A shows increasing concentrations up to 3 mM dThd and Fig. 5 B shows concentrations well below the IC 40
  • Fig. 6 depicts a dose response curve of increasing concentrations of HU on human promonocytic lymphoma cells. This figure indicates that the IC 40 for HU exceeds 3 mM.
  • Fig. 7 depicts a flow cytometric analysis of human promonocytic lymphoma cells treated with varying concentration of HU.
  • Fig. 8 depicts the results of DNA gel electrophoresis experiments showing fragmentation of DNA extracted from human promonocytic lymphoma cells treated with > 0.5 mM dThd.
  • Fig. 9 is a series of flow cytometric DNA histograms showing the effect of dThd on cellcycle progression (a-c) and an immunoblot for pRb mobility reflecting phosphorylation status (d).
  • Fig. 10 is an algorithm for synergistically matching an RA and a TCI.
  • Fig. 11 is a schematic showing the actions of chemotherapeutic agents related to events in S phase.
  • Fig. 12 is a diagrammatic representation of the set-up of a multiwell microculture plate with bivariate two-fold serial dilutions of agents for the microculture indicator system.
  • Fig. 13 depicts the digitized reflectance image of an MIS assay, testing the interactive effects of dThd and STSP on human promonocytic lymphoma cells.
  • Fig. 14 shows a data processing system capable of carrying out the present invention.
  • Fig. 15 depicts the results of MTT tests with dThd and STSP plotted as line graphs of observed results and summation results against two-fold serial dilutions of dThd.
  • Fig. 16 depicts the results of MTT tests with dThd and STSP in a multiwell microculture plate graphed as the differences between the observed results and the summation results against two-fold serial dilutions of the dThd.
  • Fig. 17 depicts a digitized reflectance image of the LDH assay testing the interactive effects of dThd and STSP on human promonocytic lymphoma cells.
  • Fig. 18 depicts O/S plots of human promonocytic lymphoma cells treated with varying concentrations of dThd and STSP as measured in an LDH assay.
  • Fig. 19 depicts a graph and digitized reflectance image demonstrating the comparable linearity of the MTT and LDH assays.
  • Fig. 20 depicts cumulative growth curves for human promonocytic lymphoma cells treated with varying concentrations of dThd and staurosporine showing effects in a delayed proliferation assay.
  • Fig. 21 is a graph depicting percent population loss for HPLC treated with varying cocentrations of dThd and STSP in a delayed proliferation assay.
  • Fig. 22 depicts an algorithm for identifying a TCI in its target interval
  • Fig. 23 depicts the results of DNA gel electrophoresis showing a ladder pattern of DNA fragmentation in extracts from human promonocytic lymphoma cells exposed to dThd and/or STSP.
  • Fig. 24 depicts the results of flow cytometric analyses of human promonocytic lymphoma cells treated with STSP, showing accumulation in the G 2 and M phases of the cell cycle.
  • Fig. 25 is a series of bivariate flow cytometric DNA histograms (A-I) showing the results of analyses of human promonocytic lymphoma cells labelled with dUTP.
  • Panel A is untreated control cells;
  • panel B and C are cells treated with dThd;
  • panels D is cells treated with STSP only; and
  • panel E is cells treated with dThd prior to STSP,
  • panel F is cells treated with KT5926, alone,
  • panel G is cells treated with KT5926 and dThd;
  • panel H is cells treated with KT252a alone; and
  • panel I depicts cells treated with K252a and dThd.
  • Fig. 26 is a schematic depicting the effects of dynamic retardation on the cell cycle.
  • Fig. 27 depicts the results of a DNA gel electrophoresis experiment of DNA extracted from human promonocytic lymphoma cells incubated with TPA prior to addition of dThd and STSP.
  • Fig. 28 depicts the results of DNA gel electrophoresis experiments showing decreased fragmentation of DNA extracted from human promonocytic lymphoma cells exposed to dThd and STSP in the presence of dCyt.
  • Fig. 29 depicts a differential O/S plot of human promonocytic lymphoma cells treated with varying concentrations of STSP and bleomycin.
  • Fig. 30 depicts the results of a DNA gel electrophoresis of DNA extracted from human promonocytic lymphoma cells after treatments with dThd alons and as an RA and various indole carbazoles and as TCI in combinations with dThd.
  • Fig. 31 is a bar graph depicting the numbers of colony counts of HPLC treated with indicated concentrations of dThd and or STSP based upon combinations which were found to be synergisitc matches.
  • Fig. 32 depicts differential O/S plots of C33A cells treated with various concentrations of Aph and STSP.
  • Fig. 33 depicts differential O/S plots of C33A cells treated with various concentrations of U937 cells.
  • Fig. 34 depicts differential O/S plots of Jurkat cells treated with various concentrations of dThd and STSP.
  • Fig. 35 depicts O/S plots of Raji cells treated with various concentrations of HU and STSP.
  • Fig. 36 is a diagram depicting dephosphorylation of p34 CDa as a critical factor in regulating cell movement through G 2 phase into M phase.
  • Fig. 37A is an immunoblot showing the effect of STSP and ⁇ ATA on the phosphorylation of p34 CDC2 .
  • Fig. 37B is an immunoblot demonstrating that STSP induced a functional activation of cdc2 as shown both by the ability to phosphorylate histone protein (HI).
  • Fig. 37C is an immunoblot showing the effect of STSP and ⁇ ATA on c-myc expression.
  • Fig. 38 is an immunoblot showing the effect of STSP and ⁇ dThd on MAP kinases, JNK (A) and ERK(B) .
  • the present invention fulfills a need in the art for new and improved combinations of cell-killing agents and new methods of identifying, evaluating, and administering synergistic combinations of agents for inflicting cell damage on target populations.
  • the invention provides an improved method for inducing cell damage by administering a restraining agent (RA) to a target cell population at a concentration and under conditions sufficient to retard but not arrest the progress of the target cells through the cell cycle, and administering a targeted cytotoxic insult (TCI) concomitant with or subsequent to the application of the RA.
  • RA restraining agent
  • TCI targeted cytotoxic insult
  • the invention also relates to a microculture indicator system and auxiliary data analysis procedures for identifying, designing and using new agents as restraining agents or targeted cytotoxic insults, and for improving synergistic combinations of existing agents.
  • the RA can be a ribonucieotide reductase inhibitor, a dihydrofolate reductase inhibitor, a thymidylate synthase inhibitor, a DNA polymerase inhibitor, a protein kinase inhibitor or a topoisomerase inhibitor.
  • embodiments of the invention include, as TCI, indole carbazoles, such as staurosporine, K252a, KT5926, and KT5720.
  • the RA is thymidine and the TCI is staurosporine.
  • the RA is bromodeoxyuridine and and the TCI is staurosporine.
  • Other specific embodiments are disclosed in the working examples.
  • the method of inducing cell damage of the invention is a method of treating patients suffering from cancer.
  • the method of the invention can also be applied to the treatment of malaria.
  • the invention can improve conventional chemotherapy or radiotherapy of neoplasms or diseases of the immune system, provide a basis for methods of selective delivery of an RA or TCI, and afford new applications of specific antisense molecules as an RA or TCI, uses of RAs or TCIs in conjunction with gene transfection therapies, and utilization of RAs or TCIs in conjunction with radiotherapies or other physical modalities of cell killing.
  • the invention may also be used for early destruction of cells infected by viruses or infectious nucleic acids, in anti-fungal or other anti-microbial therapies, and to aid in eradication of certain parasitic infestations.
  • This invention relates to a method for potentiating cell damage by identifying and/or administering a restraining agent (RA) and a targeted cytotoxic insult (TCI).
  • RA restraining agent
  • TCI targeted cytotoxic insult
  • a restraining agent refers to an agent administered under conditions that retard but do not arrest downstream progress of a target cell population through the cell cycle.
  • the role of an RA is to impose a dynamic retardation in cells designated for damage.
  • phrased processes Segregation of processes into phrases results from braking points in the forward momentum of the cell cycle due to a succession of regulatory checks.
  • the latter include START and checkpoint controls discussed by Hartwell and Weinert, supra (1989) and by Nurse, Supra.
  • START and checkpoint controls discussed by Hartwell and Weinert, supra (1989) and by Nurse, Supra.
  • the beginning point of phrased processes may only be detected during perturbations of the cell cycle or in cells with specific mutations altering cell cycle regulatory controls. For example, see Beach et al., Current Communications in Molecular Biology, pp. 1-211, Cold Spring Laboratory (1988).
  • phrased processes behave physicochemically as an "order dependent continuum,” i.e., once a regulatory checkpoint is cleared, (beginning of the phrase) successive processes are activated in order, like falling dominos, until the next regulatory checkpoint is reached (end of the phrase).
  • momentum changes can be transmitted through linked complexes such as biochemical reactions, biomolecular cascades, or macromolecular configurational changes.
  • the initiation of dynamic retardation by an RA is a negative change in the momentum (a slowing) of a subset of the cell cycle hierarchy.
  • the point within the cell cycle hierarchy at which an RA first acts to curb momentum through the cell cycle is its reference point.
  • An RA generates a physicochemical equilibrium shift in the biochemical reactions downstream from its reference point.
  • Dynamic retardation represents the downstream propagation of this slowing through one or more phrased processes.
  • the portion of the cell cycle slowed by an RA is a retardation field.
  • RAs can act at various points during the cell cycle. In those portions of the cell cycle where phrased processes are redundant, such as S phase, an RA may impose its effect at different times or at multiple reference points.
  • An agent acting as a TCI initiates cell damage after a reference point and during a specific portion of the cell cycle, known as its target interval.
  • a target interval is a subset of a retardation field consisting of phrased processes that are vulnerable to interaction with a TCI that results in initiation of cell damage.
  • an RA acts to retard movement through the cell cycle, it increases the time that cells of a target cell population are located within a target interval, thus increasing the extent to which the target population is vulnerable to the action of a given TCI.
  • the probability that the cell cycle of cells in a targeted population will be traversing a retardation field and a target interval within the retardation field increases as a result of dynamic retardation. This probability is depicted schematically in Fig. 2, and can be interpreted as a cell-cycle stacking.
  • Cell cycle stacking indicates a relative "compression" of the intervals separating cells in different positions of the cell cycle hierarchy.
  • a useful analogy is the stacking of jets entering the air space of a crowded airport.
  • Dynamic retardation potentiates the biologic damage inflicted by a TCI increasing the probability that target cells will traverse the target interval.
  • a synergistic match refers to a set of two or more agents acting as RAs and TCIs that function synergistically to inflict cell damage or retard cell growth as a result of dynamic retardation.
  • a target interval of the TCI must reside downstream of a reference point in order for the synergistic match to succeed.
  • a synergistic match of an RA and a TCI increases the effective damaging exposure (EZ ⁇ E), or the detrimental effect of a TCI, proportional to the effective strength or intensity of the TCI and to the operative duration of the TCI's relevant process interactions during the target interval.
  • the strength or intensity of an RA used in practicing the invention will be sufficient to retard, but not arrest, movement of target cells through the cell cycle at the point where the RA acts.
  • any agent or factor already known to cause cell-division cycle arrest or "static synchronization" has a potential to function in the role of RA at an appropriately reduced strength or for an appropriately limited duration.
  • the optimal strengths or intensities of an RA can be determined experimentally for a specific treatment, as discussed in more detail below.
  • Restraining agents can include natural products of microbial or other cellular origins, a range of synthetic or semi-synthetic compounds or antisense oligonucleotides designed to perturb the cell cycle.
  • Transfected genes could serve as direct modulators of genes controlling cell cycle kinetics.
  • multiple agents that can constrain momentum of the cell cycle could be used in a combination as the operative RA. The multiple agents may act either simultaneously or in a cascade of effects.
  • Environmental deprivation or physical changes can act as restraining agents.
  • Deprivations can include insufficiency of a nutritional factor essential to cell growth or sus ⁇ tenance.
  • Physical changes can include external temperature modulation or cell exposure to radiant or particulate energies, vibrational waves or other mechanical forces.
  • Fig. 3 shows a general algorithm for identifying concentrations at which a given agent can act as an RA and for determining a reference point of the agent.
  • the first step is to determine the population doubling time (T DBL ) of specified cells (i.e. a target population).
  • the target population includes proliferating cells or cells undergoing DNA repair in a population to be damaged. They can include, for example, neoplastic cells, hype ⁇ lastic cells, virus infected cells, parasite infected cells, free living parasites or fungi.
  • This step 1 is accomplished by a series of manual or automated cell counts or by flow cytometry. Alternatively, it can be accomplished by other methods known to those ordinarily skilled in the art of tissue culture (e.g. total DNA, new DNA synthesis, total protein or cell mass measured by radioisotope uptake, dye chromogenic metabolism, or dye staining).
  • the T DBL provides the time frame for determining the growth inhibitory effect of an RA.
  • the second step is to graph the dose response of the target population, showing growth inhibition in relation to the T DBL .
  • an RA will maximize synergy at a strength or intensity less than 40% of its inhibitory concentration (IC 40 ) at the T DBL .
  • An inhibitory concentration relates to growth inhibition as compared to the growth of untreated controls.
  • an IC 40 for a given RA is the concentration at which treated cells show 40% less growth than untreated controls at the T DBL .
  • the T DBL will likely be easiest to use as a consistent standard, a convenient time interval less than T DBL may also be used, provided it is consistently applied. Generally, the interval should be at least greater than 50 % T DBL to provide useful data.
  • Step 2 is performed as shown in Example 1. Fig.
  • dThd deoxythymidine
  • Example 2 shows the third step: performing flow cytometric analyses during the T DBL at, for instance five equal divisions, using different concentrations of the agent being tested at, for example, IC 20 30 40 J0 .
  • concentrations for instance, IC 20 30 40 J0 .
  • fewer analyses or concentrations may be used, for instance when pilot data, previous experience, or scientific literature provide good indication of expected results.
  • Step 4a The results of the flow cytometry, shown in Fig. 5, allow one to determine the IC at which the cell cycle is retarded (Step 4a in the algorithm). Step 4a is particularly well demonstrated with dThd. As shown in Fig. 5, concentrations of dThd less than IC 40 were sufficient to retard the cell cycle kinetics. In addition, Fig. 5 shows that the proportion of the cell population present within S phase (F s ) could become enormously increased with concentrations of less than 1 mM dThd.
  • Step 5a Any agent known to cause cell cycle arrest or static synchronization has the potential to operate in the role of RA when used at a concentration or intensity less than that necessary to arrest the cell cycle.
  • the locus of the arrest can be assumed to represent a reference point of the RA. In this perspective, cell cycle arrest represents the limiting effective strength of any agent as an RA.
  • Example 3 demonstrates step 4b, determining the inhibitory concentration at which the cell cycle is arrested.
  • Excess dThd has been used as a reversible means of cell cycle arrest in late G, or early S phase.
  • dThd human promonocytic lymphoma cells
  • U937 human promonocytic lymphoma cells
  • Fig. 5 shows that the cells treated with 3 mM dThd were detained in transition from G, phase to S phase at 8 and 16 h so that the proportion of cells in S phase and G 2 & M (F s & F G2 & M ) was stabilized or reduced.
  • This finding was consistent with previous reports that dThd arrested cells in close proximity to the transition from G, to S phase in the cell cycle.
  • the reference point for dThd is estimated to be the G, / S boundary of the cell cycle (Step 5b).
  • Example 4 The same methods were used to evaluate HU for use as an RA and are described in Example 4. As shown in Fig. 6, during approximately one population doubling, the IC 40 of HU exceeded 2 mM. As with dThd, concentrations of HU ⁇ IC 40 were sufficient to retard cell cycle kinetics. As shown in Fig. 7 and described in Example 5, HU treatment of a population of human malignant cells increased cell cycle transit times. Thus HU, an inhibitor of ribonucieotide reductase, applied in a range of concentrations less than its IC 40 , increased the T s of the proliferating malignant cells and is an RA in the claimed invention.
  • any agent already known to cause cell cycle arrest or static synchronization has a potential to function in the role of RA at an appropriately reduced strength or for an appropriately limited duration.
  • an agent that could function as an RA may cause apoptosis or other DNA-related damage and thereby behave as a TCI rather than as an RA.
  • the IC % at which the cell cycle was arrested by HU was determined with progressively increased concentrations.
  • Fig. 7 shows that the long term effects of high concentrations of HU and exposure over 16 hr were not clearly related to dynamic retardation. These concentrations of HU not only detained (i.e. arrested) cells in S phase, but also killed them. Thus, high concentrations of HU operated as a TCI.
  • Example 6 demonstrates that induction of apoptosis resulted from excess dThd for 24 hours.
  • Fig. 8 shows a DNA gel displaying an electrophoretic ladder pattern typical of DNA fragmentation in apoptosis.
  • dThd acting as an RA retarded progression of a targeted cell population through the cell-division cycle at a reference point near the G,/S transition.
  • concentrations of dThd of about IC 4 to about IC 40 with respect to a population generation time can be used as an RA.
  • concentrations from about IC 6 to about IC 30 can be used as the RA.
  • concentrations of dThd from about IC 10 to about IC 25 as the RA.
  • the RA retards progression of a targeted cell population through the cell-division cycle at a reference point during S phase, or near the S/G 2 transition.
  • Table 1 shows a number of agents that can act as RAs, categorized according to the portion of the cell cycle in which they are known or suspected to act. Not shown are possible secondary reference points that may be determined by practice of this invention. Most of the RAs shown in Table 1 are commercially available, for example from Sigma Chemical Co., St. Louis, MO or Calbiochem, San Diego, CA. Trimidox was recently synthesized, T. Szekeres et al., Cancer Chemother Pharmacol 34:63-66 (1994). HAG-IQ is synthesized as described by G. Weckbecker et al., J. Natl. Cancer Inst. 80:491-96 (1988).
  • an RA can be applied to the target cells, or administered, in various ways well-known to one of ordinary skill in the art.
  • an RA can be administered in an in vitro setting. In vitro testing may, for instance, be undertaken to rapidly establish synergy between an RA and TCI agents at various strengths or duration for a particular cell line.
  • the RA can be added to the target cells, appropriately diluted in standard biological buffer, such as RPMI 1640.
  • an RA can be delivered in solid, semisolid, liquid, or gaseous form and by various routes.
  • An RA can be introduced by oral, mucosal, topical, intravenous, intrathecal, intramuscular, subcutaneous, intravesicular, intrapleural, intrapelvic, intrauterine, intranasal, intraperitoneal, intraural, or intraocular routes, or by depot injections, or by aerosol, and by itself, or together with a suitable biological carrier.
  • An RA can be delivered as a component of, or in conjunction with another substance or molecule such as a ligand or an antibody or by a carrier such as a liposome or a microcapsule.
  • An RA can be delivered to effect a rapid or sustained release or as multiple intermittent doses.
  • an RA may be aided by gene or nucleic acid transfection, enzyme insertion into a cell membrane, or a virus infection, or any other agent that contributes toward transport or metabolism of an agent acting as an RA, or regulates an agent to act as an RA, including dominant negative regulation.
  • a virus infection or any other agent that contributes toward transport or metabolism of an agent acting as an RA, or regulates an agent to act as an RA, including dominant negative regulation.
  • a cytotoxic agent is any category of agent or circumstance that inflicts damage upon or inhibits growth of living cells, whether for medical, therapeutic or for any other purpose.
  • a TCI is a cytotoxic agent that initiates apoptosis or biologically significant damage during a target interval in the cell cycle hierarchy.
  • Various TCIs can be used in the context of this invention, however a target interval of the TCI should correspond to the portion of the cell cycle slowed by the RA. Damage inflicted by a TCI may be reversible, permanent, sublethal or lethal. In most practical applications, lethal damage with "total killing" of abnormal cells is preferred.
  • TCIs can damage or retard the growth of target cells in various ways.
  • Table 2 shows a number of TCIs, categorized by the estimated position of at least one of their target intervals. Estimation of the target intervals is based in part upon testing performed by the inventors (see for instance Example 12) and upon the known or suspected mechanisms of action as can be found in the scientific literature available to those ordinarily skilled in the art.
  • a TCI When appropriately delivered, a TCI can result in discrete damage to specific biochemical processes. However, when applied in excess strength or intensity, almost any TCI may inflict unexpected cell damage that is unrelated to the primary biochemical or molecular processes and thereby increase side effects. Therefore, a major objective in applying a TCI is to direct its effect to the appropriate subpopulation of cells, i.e., target population, in an optimal strength or intensity. Accordingly, discriminate targeting of specific cell populations for damage(s) inflicted by a TCI can be highly advantageous. Discriminate targeting can be achieved by an appropriate strategy of agent selection or design, first of the RA required to impose the limited restraint condition and second of the TCI. Discriminate targeting can also be achieved by an optimal strategy of agent deliveries to a target population.
  • TCIs can be applied to a target cell in an in vitro setting, for pre-clinical testing among other reasons, setting at an effective concentration after dilution in a suitable biological buffer.
  • a TCI can be delivered in solid, semisolid, liquid, or gaseous form and by various routes.
  • a TCI can be introduced by oral, mucosal, topical, intravenous, intrathecal, intramuscular, subcutaneous, intravesicular, intrapleural, intrapelvic, intrauterine, intranasal, intraperitoneal, intraural, or intraocular routes, or by depot injections, or by aerosol, and by itself, or together with a suitable biological carrier.
  • a TCI can be delivered as a component of, or in conjunction with another substance or molecule such as a ligand or an antibody or by a carrier such as a liposome or a microcapsule.
  • a TCI can be delivered to effect a rapid or sustained release or as multiple intermittent doses.
  • the delivery of a TCI may be aided by gene or nucleic acid transfection, enzyme insertion into a cell membrane, or a virus infection, or any other action or agent that contributes toward transport or metabolism of an agent acting as a TCI, or regulates an agent to act as a TCI, including dominant negative regulation.
  • RAs persons in the art would be able to determine the appropriate administrative route using routine skills.
  • RA or TCI deliveries may facilitate critical targeting to the appropriate cell subpopulation in human or other multicellular hosts (RC Juliano, Ann NY Acad. Sci. 507:89-103 (1987)) and are, therefore, within the invention.
  • the practice of the invention may also include specific antagonists to protect susceptible normal cells, tissues or organs from undesirable effects of an RA and/or a TCI that is targeted to malignant cells or infected cells.
  • aclarubicin, cardioprotective agent CRF-187, or chloroquine could antagonize the cytotoxicity of etoposide.
  • a TCI can be added to the target cells concomitant with or following the addition of an RA.
  • the TCI is added 0-8 hours after addition of the RA.
  • the TCI is added 4-6 hours after the addition of an RA.
  • An effective cytotoxic concentration (EC) of a TCI is an amount of TCI that, when matched with an RA, is sufficient to damage, inhibit the growth, or kill target cells, depending on the context.
  • a TCI is administered to the target cells in an amount sufficient to damage or inhibit the growth of target cells.
  • the TCI is added in amount sufficient to kill the target cell.
  • STSP can be added in an amount from IC 10 to IC 60 .
  • STSP can be added in an amount from IC 15 to IC 50 .
  • STSP can be added in an amount from IC 20 to IC 35 .
  • the amounts of other agents can be determined experimentally as described below. Synergistic Matching of an RA and a TCI
  • synergistic matches of RA and TCI is guided by the principles of dynamic retardation described herein.
  • the assay system and auxiliary data analysis provided herein can also be used to guide selection of synergistic matches.
  • Implementation of a synergistic match between an RA and a TCI uses a set of trial procedures: (1) pilot tests of the effects of a potential RA, in calibrated serial strengths, on the growth of proliferating cells; (2) pilot tests of the effects of a presumptive RA on the cell cycle perturbation and the re- equilibration of a proliferating cell population at serial points in time; (3) pilot tests of the effects of a potential TCI, in calibrated serial strengths, on the growth of proliferating cells; (4) pilot tests of the effects of a presumptive TCI in a specific portion of the cell cycle of a proliferating cell population; and (5) systematic tests of the synergy of an established RA and a proven TCI, at different intervals between applications, and at different levels
  • Fig. 10 is a flow diagram showing the selection of a synergistic match of RA and TCI.
  • the first step is selection of an agent with RA characteristics appropriate to the target population.
  • the second step is selection of a prospective TCI with a target interval downstream of the RA reference point.
  • the TCI may be selected from a complementary class according to the categories in Table 2. Selection can also be based upon a large body of extant pharmacologic, biochemical or molecular biologic data, such as that partially delineated in Fig. 11.
  • the methods of the invention provide an additional basis for which an agent may be selected for testing as a TCI.
  • An in vitro Microculture Indicator System Discerns and Quantitates Biological Synergy
  • the third step in Fig. 10 involves testing of potential RAs and TCIs for synergistic matching with an in vitro microculture indicator system (MIS) employing cultured eukaryotic cells.
  • MIS in vitro microculture indicator system
  • the MIS is a series of assays in which two parameters are varied by fixed multiples, i.e., bivariate serial dilutions (BVSD) of RA and TCI, in multiple well plates. For each bivariate combination, effects related to cell damage are quantitated by colorimetric or other measurable indicators.
  • Example 8 demonstrates a systematic series of tests of dThd, acting as an RA, and STSP, acting as a TCI.
  • the tests quantitate biologic damage to malignant cells using colorimetric assays, however, many other assays known to those skilled in the art could also be used to provide a measure of growth inhibition or cell killing.
  • Another aspect of the invention involves a method of analyzing the MIS data for the determination of agent synergy using comparisons of observed results to hypothetical sums.
  • the hypothetical sum for each data point is the expected value for the combination of each agent at a respective concentration if the results for each alone were simply added together.
  • Colorimetric readings or comparable data are imported into any relational spreadsheet, such as Borland Quattro Pro 4, that has been populated as shown in Table 3.
  • the data processor analyzes the data according to the predetermined relationships represented in the populated cells, comparing the results for wells containing both RA and TCI, with hypothetical results derived from the results for wells containing no agents and the results for wells containing each agent alone.
  • Fig. 14 shows a typical computer system 1400 for executing the procedure just described. Data can be entered either through disk 1410 via disk drive 1415, or through keyboard 1420 with or without mouse 1425. Processor 1430 would execute the spreadsheet program and the results can be displayed on monitor 1440.
  • the data processor may display, in a tabular form, combined results ratios (CRR), which reflect the ratios of the growth inhibition or cytotoxic effect observed in each well to that which would be expected in a hypothetical summation of the effects of the TCI and RA.
  • CRR combined results ratios
  • Table 4 shows a printout of percent growth inhibition (columns with %) and combined results ratios (interspersed columns) for bivariate strength combinations of dThd and STSP. Table 4 was generated through application of the formulas shown in Table 3A-E, to the data generated in Example 8.
  • the CRR equals 1 a summation effect is observed (i.e. hypothetical zero interaction); when the CRR is > 1, a synergistic effect is observed; and when ⁇ 1 an antagonistic effect is observed.
  • the data processor may display the results in graphical form as two superimposed plots showing, for each concentration of a TCI, the observed results ("O") as a function of an RA concentration in one graph and the hypothetical summation ("S") results as a function of an RA concentration in the other graph ("O/S" plots).
  • Fig. 15 shows O/S plots for two of the strength combinations of dThd and STSP. In each of the graphs shown in Fig. 15, the maximum difference between the plots for O and S ocurrs at approximately 0.1 mM dThd.
  • the data processor may also display in superimposed plots for each concentration of a TCI, the differences between the O and S as a function of RA concentration (differential O/S plot).
  • Fig. 16 shows differential O/S plots for various strength combinations of dThd and STSP.
  • the most synergistic concentrations of an RA for a given concentration of TCI are found along the parabolic maximum.
  • the most antagonistic concentrations of an RA for a given concentration of TCI are found along the parabolic maximum of a downwardly displaced parabola. While antagonistic interactions are not of direct interest in potentiating cell damage, they may be useful in other contexts, for example, protecting cells from damage.
  • the MIS and auxiliary data analysis procedures may provide: (i) an estimation of the potential operative range for specific RA and TCI interactions from a tabular presentation of the CRR in each well; (ii) a graphic comparison of potentially useful concentrations of the TCI by plotting line graphs of both the observed and summation results against serial concentrations of the RA; and (iii) a graphic display of bivariate synergy maxima or ranges (S M ⁇ ) by plotting line graphs of the differences between the observed results and summation results.
  • the results from these assays can therefore be supplemented by measuring another indicator of cell damage such as, for instance, a colorimetric assay for release of lactate dehydrogenase (LDH) into the tissue culture supernatant. Release of LDH is considered a useful indicator of cell membrane damage.
  • LDH lactate dehydrogenase
  • More assurance that a synergistic match produces cell killing may be obtained from the ratio of cell mass at completion of the test (N) to cell mass at the outset (N 0 ).
  • This ratio can be obtained by serial assays.
  • This ratio (N/N 0 ) also may be presented graphically as a function of RA concentration by populating the spreadsheet as shown in Table 3A-C, J-M. When less than 1 , this ratio reflects a net loss of cells in a target population.
  • Another presentation shows a ratio less than 1 as a percentage of population loss (1-N/N 0 ). This percentage also may be presented graphically as a function of RA concentration by populating the spreadsheet as shown in Table 3A-C, J-N.
  • Examples 8 and 9 detected effects on growth or damage of the U937 cells during an interval of 18 h, representing less than one mean generation time. Although the damage inflicted by STSP was potentiated by dThd during that time interval, delayed effects upon succeeding generations of progeny cells would also be significant in clinical chemotherapy.
  • An aspect of the invention is a "delayed proliferation assay" based upon the MIS shown in Example 8 to show that the rapid effect of STSP on DNA damage in cells treated with dThd affected cell growth inhibition for at least 48 hr.
  • Example 10 demonstrates such a delayed proliferation assay.
  • the results obtained proved that the dThd treatment potentiation of STSP shown in Example 8 by MTT assays for growth inhibition and in Example 9 by LDH release, caused a persistent growth suppression of the targeted population.
  • the effects of low concentrations of STSP and dThd which by themselves were reversible by washing, became irreversible once the agents were combined in synergistic matches. In comparison to CRR data, cumulative growth graphs are more revealing.
  • Step 4 of Fig. 10 recommends test scheduling increments within the T DBL to optimize any synergistic match.
  • MIS and data analysis are performed for simultaneous administration of the RA and TCI and for gradually staggered application where the second agent is given after a fixed increment of delay for each test schedule.
  • the order of application may also be varied. For instance, the TCI could be given first, followed by the RA and vice versa.
  • Example 11 demonstrates that the effect of dThd in potentiating cell damage by STSP was schedule-dependent, see Table 7.
  • the MIS and auxiliary data analysis can be used to estimate optimal times for RA and TCI deliveries.
  • Fig. 22 The identification of an unknown agent or prospective TCI in appropriate strength and duration both for function in synergistic match with an RA and for the determination of the target interval in S or G 2 phases is charted in Fig. 22.
  • Examples with STSP provide a prototype for identification and successful application of a TCI complementary to an RA.
  • the first step of determining T DBL and the second step of determining the dose/growth inhibition graph at T DBL parallel the first steps for identifying an RA. (See Example 1.)
  • the next steps assess whether the prospective agent alone can damage a proliferating cell population and localize the position of a target interval in the cell cycle.
  • Many TCIs such as high concentrations of dThd or STSP, cause DNA fragmentation when used a single agents.
  • Assessment of the relationship of DNA damage to a target interval ideally involves a combination of techniques which may include conventional flow cytometry for cell cycle analysis shown in Example 2 and DNA gel electrophoresis shown in Example 6.
  • Example 12 demonstrates methods for evaluating the DNA damage initiated by a prospective TCI using the measurement of fractional population loss shown in Example 10.
  • Example 12 shows the localization of a target interval for a TCI, confirmed by both DNA gel electrophoresis and flow cytometry. Interaction of an RA and a TCI
  • Fig. 26 shows the basic principles of dynamic retardation and the importance of the relative hierarchical positions of the reference point of the RA and the target interval of the TCI.
  • the RA are shown acting during S phase, with the arrows indicating possible different reference points of an RA in relation to the cell cycle.
  • the resultant retardation fields ("RFl" and "RF2", respectively) are represented as distortions in different portions of the S phase, and are intended to reflect a local increase in the S phase transit time.
  • the reference point for RA I (Fig. 26(b)) is shown close to the G,/S boundary, while the reference point for RA II is shown closer to the S/G, boundary.
  • a target interval in early S phase would be included in the retardation field of RA I, whereas a target interval further downstream in late S phase (“B") might not.
  • the reference point of RA II (Fig. 26(c)) is shown to be located downstream of an early S phase target interval ("A"). Therefore, only a later target interval (“B") might be included in the retardation field.
  • Fig. 26 is a simplification of the real events.
  • macromolecular replication or other critical biochemical or biomolecular processes simultaneously occur at different positions.
  • dynamic retardation in S phase presumes the possibility that certain RA will impose effects at multiple reference points.
  • retardation fields also may occur simultaneously at different positions and may encompass portions of multiple target intervals with differing vulnerabilities to various TCI. The possibility of retardation fields being generated upstream of an RA is not theoretically excluded.
  • Example 12 STSP was shown to induce apoptosis during S phase, yet dThd, which acts in G,./S was able to act as a potent RA.
  • dThd uptake and metabolic pool sizes could be time related factors, potentiation of STSP by dThd clearly is not based upon the stoichiometry expected in a simple metabolic competition.
  • Enzymes involved in pyrimidine biosynthesis are associated with the initiation of DNA synthesis in all forms of living cells. Elledge, supra.
  • the biologically active R2 subunit of RNR contains a coupled iron center and tyrosyl free radical. Transcription of the messenger RNA for RNR, or an active subunit of RNR, increases during S phase in mammalian cells. J L; Bjorklund S et al., Biochem 29:5452- 58 (1990). Excess dThd inhibits RNR and deoxycytidylic deaminase by a metabolic feedback inhibition.
  • dThd deoxythymidine 5'-triphosphate
  • RNR ribonucieotide reductase
  • dCTP deoxycytidylic deaminase
  • RNR inhibitors were compared for their ability to act as RAs and potentiate STSP. These included dAde and dGuo which also inhibit reduction of nucleoside diphosphate substrates, and HU, which is a direct inactivator of RNR by scavenging the tyrosyl free radical. JW Yarbo, Semin Oncol 19:1-10 (1992).
  • the halogenated pyrimidine analog bromodeoxyuridine (BrdU) acted almost identically to dThd both in its effects on S phase prolongation as well as on STSP potentiation.
  • BrdU produced a slowing of S phase similar to that caused by dThd. This was shown by bivariate flow cytometry using PI staining of total DNA and a fluorescein-labelled antibody to BrdU.
  • each agent that was effective as an RA worked in a range of concentrations less than the IC 40 (similar to results with dThd).
  • each of the other RNR inhibitors caused cell cycle arrest and induced apoptosis when used alone at sufficiently high concentrations.
  • the concentrations of RNR effective in potentiating STSP consistently were up to several-fold lower than concentrations most active in direct growth inhibition.
  • Each RNR inhibitor used at reduced concentrations proved similar to dThd in potentiating cell damages by STSP.
  • Table 8 lists the results of tests of these agents as RA. CRR data for such testing appears as Tables 9-12.
  • the RA con ⁇ centration for STSP potentiation was at least one log lower than the concentration that caused cell cycle arrest.
  • Choices of RA in groups other than RNR inhibitors stemmed from initial observations with dThd and a general knowledge of other chemotherapeutic agents known to be capable of arresting the cell cycle when used in high concentrations.
  • dThd a general knowledge of other chemotherapeutic agents known to be capable of arresting the cell cycle when used in high concentrations.
  • replitase a deoxynucleotide synthesis and polymerization complex
  • RNR inhibitors such as, in particular, aphidicolin (Aph) and 1- ⁇ -D- arabinofuranosylcytosine, (commonly designated cytarabine, cytosine arabinoside or ara-C) are well known to inhibit polymerases involved in DNA synthesis, C. Sessa et al., J. Natl. Cancer Inst. 83:1160-4 (1991).
  • cytarabine cytosine arabinoside or ara-C
  • a dihydrofolate reductase inhibitor (MTX); a thymidylate synthase inhibitor (floxuridine); and two inhibitors of DNA polymerase ⁇ (Aph and ara-C) were selected for testing as RA with STSP as the TCI, generally following the algorithm set forth in Fig. 10 and Example 8.
  • MTX dihydrofolate reductase inhibitor
  • floxuridine thymidylate synthase inhibitor
  • ara-C two inhibitors of DNA polymerase ⁇
  • Fig. 30 is a DNA gel showing the synergistic interaction of dThd with indole carbazoles other than STSP, as evidenced by DNA ladder formation.
  • TCI including STSP, KT5926, and Cisplatin
  • the S ⁇ x for dThd was very similar.
  • the choice of potential RAs and TCIs should be made so as to maximize the damage to cancer cells or infected cells, while minimizing the damage to normal cells.
  • the combination of the MIS and auxiliary data analysis procedures affords flexibility for the individualization of clinical chemotherapy or radiotherapy.
  • in vitro testing using either established tumor cell lines or direct cultures of patient tumor cells as the indicator cells can provide a relatively rapid and clinically focused testing tool to individualize treatment parameters for specific neoplasms in particular patients.
  • the invention may be most promising in treating malignant cells that seem resistant to chemotherapy.
  • Such resistance of malignant cells to chemotherapy is often associated with deletions or mutations in the p53 gene.
  • Large cell lymphomas have been occurring with increasing frequency in patients with immunodeficiency conditions in the United States, and mutation or absence of p53 has been associated with resistance to chemotherapy or radiotherapy.
  • the U937 cells tested in many embodiments of this invention lacked p53, as reported by Calabresse C. et al., Biophys Biochem Res Commun 201 :266-83 (1994).
  • the present invention can help to circumvent the dilemma posed by malignant cells that have evolved drug resistance mechanisms both by intensifying the damaging effects of a TCI and by identifying strategic RA and TCI combinations specifically effective in resistant cell lines, delivered by techniques directed to achieving optimal results. Intensification of TCI effects can also be valuable in treatments of malignancy involving bone marrow transplantation, where either the patient or the extraco ⁇ oreal tissue can be subjected to a more rigorous regimen of malignant cell eradication.
  • Fig. 32 This cell line (C33A) originated from a human cervical carcinoma, Auersperg N et al., J Natl. Cancer Inst 32:135-148 (1964) and is negative for p53. Shivastrava, supra.
  • the O/S differential plots in Fig. 32 represent effects of Aph on STSP-treated human cervical carcinoma cells (C33A), which can be compared to effects on STSP-treated human promonocytic lymphoma cells (U937) shown in Fig. 33.
  • the O/S differential plots in Fig. 34 represent effects of dThd on STSP-treated Jurkat leukemic T cells which can be compared to effects on STSP-treated human promonocytic lymphoma cells (U937) shown in Fig. 15.
  • the O/S differential plots in Fig. 35 represent effects of HU on STSP-treated Raji cells.
  • dTTP deoxythymidine triphosphate
  • Tk thymidine kinase
  • Dynamic retardation also proved successful in potentiating STSP damage to cells of malignant epithelial origin.
  • the C33A cells and ZR-75-1 cells grow on the surfaces of microwells rather than in suspension, and cell damage results in detachment of cells from the substrate.
  • an MIS assay using crystal violet as a stain for attached cells proved informative and in some cases preferable to the MTT. (See Grando SA et al. Skin Pharmacol 6:135-147 (1993)).
  • cells susceptible to the effects of dynamic retardation may be of any type, including eukaryotic, prokaryotic and archaebacterial; organized, free-living, and parasitic, or growing in living hosts of the animal or plant or growing in manufactured environments.
  • This invention can be expected to increase the damaging effects of TCI which are calibrated for delivery to various sizes of populations of normal, abnormal, atypical, neoplastic or infected cells in living hosts or in manufactured environments.
  • the invention may potentiate damage inflicted upon an entire population of living cells or upon an entire plant, animal, or other living organism, or upon a discrete population or clone of living cells within any organism of the animal or plant kingdoms, or upon any population or clone of free-living cells or upon any population or clone of infected cells within a delineated environment.
  • TCI for medical therapeutic pu ⁇ oses requires that its damaging effect be inflicted upon the appropriate cell population in a patient. Discriminate targeting of specific cell populations is highly advantageous, since side-effects may threaten survival of the host or cause severe morbidity.
  • the in vitro MIS system in conjunction with data analyses provides a useful surrogate to living hosts for the identification of synergistic matches.
  • a relatively simple strategy for delivery of an RA in an intact host involves using a relatively high dose of the chosen agent. Blood levels are monitored at intervals to determine the appropriate point for introduction of the TCI. See O'Dwyer, supra: Schilsky RL et al., Cancer Res 46:4184-88 (1986); Donehower RC, Hydroxyurea, In Chabner BA (ed.) Pharmacologic Principles of Cancer Treatment, pp. 269-75, Philadelphia, PA, Saunders (1982); Sessa, supra- In this approach, the initial level of RA can exceed the optimal range of synergy. This system offers maximum clinical advantages if the RA is minimally cytotoxic and long act ⁇ ing while the TCI is highly cytotoxic and short acting.
  • the RA is delivered orally, or by depot injection, while the TCI is injected or infused intravascularly for a period of several hours defined by MIS.
  • Blood levels of the RA can be manipulated by auxiliary strategies affecting the pharmacokinetics, including an appropriately scheduled multiple dose regimen.
  • a "pharmacokinetic elimination strategy" would work very effectively with either dThd, Schilsky, supra. HU, Donehower, supra, or Aph, Sessa, supra, as the RA.
  • Significant blood and cerebrospinal fluid levels of dThd or HU have been achieved with continuous intravenous infusions and levels can be maintained for several days.
  • a major advantage of HU is that it is readily absorbed after oral ingestion and blood levels peak in 2-4 hours. Donehower, supra. It also distributes into the cerebrospinal fluid.
  • Peak plasma levels of 3 ⁇ g/ml can be achieved with minimal toxicity. Sessa, supra.
  • Timing and routes of administration of an RA and TCI depends upon specific pharmacodynamic characteristics of abso ⁇ tion or metabolism of each agent in a particular biologic system.
  • a major advantage of the present invention is that such knowledge is not essential for some degree of synergy to be achieved, even with simultaneous application of the RA and TCI.
  • catabolism or elimination of any agent must be gradual.
  • the optimal blood concentration level of an agent might be reached at some point during the pharmacokinetics of elimination.
  • the observed interaction of RNR inhibitors with STSP or K252A and the interaction of STSP with cisplatin or alkylating agents may provide significant new chemotherapeutic utilization or development of STSP and homologues.
  • STSP and homologues or analogues have been considered as anti-neoplastic agents, Schwartz, supra, clinical use has thus far been circumscribed.
  • the invention should permit their use and development.
  • This invention can also be used to control microbial or parasitic infections where the cell cycle of each infectious organism is much shorter than the cell cycle in human cells. Thus, even the brief application of a limited restraint condition and TCI might prove clinically significant. See Examples 17 and 22.
  • This invention is also useful in the application of herbicides, insecticides or other pesticides designed for the killing of a complex organism, extermination of agricultural or domestic pests, selective poisoning of any number of living unicellular or multicellular organisms including any member of the animal and plant kingdoms, or cells infected by mycoplasma, viruses, prions or other infectious agents may be possible.
  • the TCI when the RA is ara-C, the TCI is not dGuo. In other embodiments, when the RA is dThd or BrdU, the TCI is not ara-C. In still other embodiments, when the RA is dGuo, the TCI is not camptothecin; when the RA is ara-C, the TCI is not cisplatin; and when the RA is dipyridamole, the TCI is not cisplatin.
  • the TCI when the RA is bryostatin, the TCI is not cisplatin; when the RA is quercetin, the TCI is not cisplatin; when the RA is STSP, the TCI is not cisplatin; and when the RA is tamoxifen, the TCI is not cisplatin.
  • the following other uses are also contemplated:
  • This example shows the relationship of progressively increased concentrations of dThd to growth inhibition of a population of human malignant cells during the mean time of a single cycle of cell-division.
  • U937 cells human promonocytic lymphoma cells
  • CRL 1593 American Type Culture Collection
  • Dr. K. Zoon at the FDA in Bethesda, MD were set up in a multiwell plate with serial two ⁇ fold dilutions of dThd in tissue culture growth medium (RPMI 1640 plus 10% Nurserum and antibiotics).
  • the volume of medium per well was 100 ⁇ l and the cell number per well was about 1 x 10 5 .
  • the plate was incubated at 37° C for 24 hr.
  • a dye 3-[4, 5-dimethylthiazol-2-yl]-2 ,5-diphenyl-tetrazolium bromide (MTT) was used as a chromogenic indicator of the metabolically active cell mass. Viable cells metabolize the dye and accumulate a reduced formazan product (blue color) which is solubilized for colorimetry.
  • Mossman T J. Immunol. Methods 5:55-63 (1983); Li L and Lau BHS, In Vitro Cell Dev. Biol. 29A:531-536 (1993).
  • the MTT assay has been established as useful for measuring either growth inhibition or population killing. Plowman, supra.
  • the MTT method is ideal for cells tested in suspension growth, and can also be used for some cells growing in monolayers.
  • An alternative method for cells growing in monolayers utilizes a crystal violet (CV) stain for cell proteins. Damaged cells detach from the substrate so that wells in which cells have been depleted or where cell growth is inhibited stain relatively weakly in comparison to controls.
  • CV crystal violet
  • cells adherent to microwells are fixed by addition of 25 ⁇ l of 4% formaldehyde to the well medium. After 30 min. at room temperature, adherent cells are washed and stained for 15 min. with a solution of 0.5% crystal violet. Excess stain is washed off by repeated and gentle aqueous rinses and the plates are dried overnight in the dark.
  • Fig. 4 shows a plot of percent of growth inhibition for U937 cells as a function of treatment with increasing concentrations of dThd.
  • This Example shows flow cytometric analysis of the U937 cells exposed to a range of concentrations of dThd below the IC 40 .
  • Flow cytometric analyses were performed with a Coulter Epics System (purchased from Coulter Co ⁇ , Hialeah, FL). Cells were disrupted in buffer with 0.1% Triton-X 100 (or 0.6 % NP-40), so that the nuclei could be stained with 0.05%) propidium iodide (PI). See Shapiro NM, Practical Flow Cytometry, Man R Liss, NY (1988); Nicoletti I et al., J Immunol Methods 139:271-9 (1991).
  • Example 4 shows flow cytometric analysis of the U937 cells exposed to up to 3 mM dThd at intervals of up to 24 hr, in order to demonstrate the relationship of progressively increased concentrations of dThd to detention or static synchronization of human lymphoma cells in the cell cycle hierarchy. Flow cytometric analyses were performed as in Example 2. The results are shown in plot A of Fig. 5 from Example 2. Example 4
  • Example 5 shows the relationship of progressively increased concentrations of HU to growth inhibition of a population of the U937 cells during the mean time of a single cycle of cell-division.
  • An MTT assay was performed as in Example 1 except that the agent tested was HU.
  • Example 5 shows that the IC 40 for HU exceeded 2 mM.
  • Example 2 shows that HU treatment increased cell cycle transit times, according to flow cytometric analysis.
  • the U937 cells were exposed to a range of concentrations below the IC 40 and sampled for flow cytometry at intervals of 8, 16, and 24 hours.
  • Flow cytometric analysis was performed as described in Example 2.
  • Spectrophotometric quantitation of the DNA in the supernatant was quantitated by absorbance spectrophotometry at 260 nm. Volumes adjusted to contain equal amounts of DNA were mixed with a loading buffer of 40% (w/v) sucrose, 0.1M EDTA pH 8.0, 0.5% (w/v) sodium lauryl sulphate and 0.05% (w/v) bromophenol blue. Samples were applied to separate lanes of a 1.2% agarose gel and electrophoresed in a horizontal apparatus (BRL, Bethesda, MD) with TE buffer for 3 h at 5 volts/cm. Separated bands of DNA were stained with ethidium bromide and photographed in UV light (Fotodyne Inc.).
  • Fig. 8 shows a negative image of photographed results, showing multiple bands of low molecular weight oligonucleotides in cells treated with dThd (bracketed portion of the image).
  • the lowest molecular weight (about 180 bp) was determined with a standard 123 bp ladder from Sigma Chemical Co. (Cat. #D5042).
  • the result shown is a classical "DNA ladder," characteristic of the fragmentation of DNA macromolecules in apoptosis. See Tomei, supra: Obeid, supra: Gold, supra.
  • the immunoblot (d) shows protein mobility differences of pRb, specifically an accumulation of pRb p (the hypophosphorylated form) in the sample treated with 3 mM dThd for 4 or 11 hours, in comparison with either the control sample or the 0.2 mM dThd sample treated for 4 hours or 11 hours.
  • Phosphorylation of pRb is essential to progression of the cell cycle from G, into S phase. Wiman KG FASEB J 7:841-5 (1993). In a hypophosphorylated state, pRb fails to release the transcriptional activation factor E 2 F required for initiation of S phase (see Fig. 1). Therefore, hypophosphorylated pRb p is associated with G, phase, while phosphorylated pRbTM 9 is associated with S phase transition. At the relatively low concentration of 0.2 mM dThd, shown by flow cytometry to be associated with S phase retardation, the phosphorylation o ⁇ pRb did not appear to be grossly inhibited compared to control cells. In contrast, the more concentrated 3 mM dThd, shown by flow cytometry to be associated with G,/S phase arrest, pRb phosphorylation was inhibited.
  • This Example shows how the biological interactions of dThd and STSP were quantitated by an in vitro microculture indicator system.
  • the range of serial concentrations of dThd was 3 mM through 0.01 mM and the range of serial concentrations of STSP was 100 nM through 3 nM.
  • Cells were added after serial dilutions of the dThd had been dispensed. Cells were dispensed at about 10 X 10 4 cells per microwell to provide optimal sensitivity of detection of cell damage in subsequent colorimetric assays.
  • the plate was incubated at 37°C for 4 h before the addition of serial dilutions of the STSP.
  • the final volume of medium was 100 ⁇ l/well.
  • the plates were further incubated at 37°C for a total duration of 18-24 h.
  • the dye MTT was used as a chromogenic indicator of the residual and metabolically active cell mass. (Described in Example 1.)
  • Fig. 13 shows a digitized reflectance image of the actual plate used for this Example of BVSD as captured by a Scanmaker 2 (Microtek) with Adobe photoshop software in a Mackintosh Quadra 800 and transferred to Aldus Persuasion 3.0 for labelling and printing at 300 dpi.
  • Table 4 shows data for dThd and STSP mathematically translated into a percent inhibition of cell growth (columns with %) by comparison to the averaged absorbance in microcultures of untreated control cells (mean values from four wells in lower right corner of microplate, see Fig. 12).
  • This Example demonstrates a correspondence of cell depletion, or growth inhibition, as quantitated by MTT assays with cell damage quantitated by an assay for lactic dehydrogenase (LDH) enzyme release.
  • LDH lactic dehydrogenase
  • the MIS set up was identical to that described in Example 8.
  • the relative activity of LDH released into supernatant from each microculture well was detected by a coupled enzymatic assay, Decker et al., J. Immunol. Methods 15:61 (1988), in which the chromogen 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) is converted to a red INT-formazan product by NADH in the presence of diaphorase.
  • INT 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride
  • Table 5 shows the CRR for an LDH assay performed as described herein. This CRR was generated using the formulas shown in Table 3A-C, O and P.
  • Fig. 18 is an O/S plot for the data shown in Table 5, for one concentration of STSP. These formulas use useful forum assay in which positive numbers are obtained as compared to % inhibition used above.
  • Fig. 19 demonstrates the comparable linearity and accuracy of MTT and LDH assays, as a function of U937 cell numbers from 2-14 X 10 4 /cell in a test plate. Particularly for the latter pu ⁇ ose, all of the cells in each microculture well were totally lysed with Promega lysing reagent prior to the colorimetric measurement.
  • Example 10 Example 10
  • This Example shows a delayed proliferation assay to verify that the effect of a synergistic match between dThd and STSP on cell killing extended beyond a single mean doubling time of the targeted population.
  • Microcultures treated with an agent or agents for a defined period were washed. Seventy-five percent of the well volume was removed without disturbing the sedimented cells, and medium was added to yield a 1/5 dilution. In three additional washes and cell sedimentations, 50% of the medium was replaced such that the original concentration of agents was reduced to ⁇ 5%. The metabolic mass of cells surviving or proliferating at 48 hr after the washings, including the untreated control cells, was then assayed by the MTT method (total elapsed time of 70 hours). CRR are shown in Table 6.
  • Fig. 21 is a plot of the cell population loss in various concentrations of STSP as a function of dThd concentration in relation to the mean original cell mass (N 0 ) as determined in an "immediate plate". Taking the mean final cell mass at 70 hours as N, positive percentages represent the % effective cytotoxicity (EC % ) expressed as 1-N/N 0 and converted to a percentage, in relation to concentration. Thus, EC obtained only for ratios of N/N 0 ⁇ 1 (i.e. positive percentages). Fig. 21 shows that at 70 hours after treatment the maximum effective cytotoxicity for cells treated with 175 nM STSP was about 85%.
  • This Example shows scheduled testing of STSP as a TCI with dThd as the RA.
  • An MIS was performed as described in Example 8, except that results were compared when STSP was added to each microculture well at 0 or 4 h after the beginning of treatment with dThd. The total duration of STSP treatment in each plate was identical. Consistent with a dynamic retardation of S phase by dThd, potentiation of STSP by dThd was greater when cells were treated with dThd for 4 hr prior to STSP (top) rather than coincidentally at zero time (bottom). Time for dThd uptake and equilibration with metabolic pools could explain an action lag.
  • Fig. 23 is a DNA gel demonstrating that 50 nM STSP could induce some "ladder pattern" DNA fragmentation in U937 cells in 8 hours. The effect became more conspicuous in cells treated for longer times (not shown) or with higher concentrations of STSP, and was accentuated by dThd.
  • Biotin-labelled 16-dUTP and terminal transferase were added to provide the final concentration of 0.5 nM and 500 units/ml, respectively, and reacted with the cells at 37°C for 30 minutes.
  • Cells were rinsed in PBS, resuspended in a 4X saline-citrate buffer with 0.1% triton XI 00, 5 % (w/v) non-fat powdered milk and 2.5 ⁇ g/ml FITC-labelled avidin, Boehringer Mannheim, supra, and incubated at room temperature for 30 minutes. Excess avidin-FITC was removed by washing the cells with PBS containing 0.1 % triton X-100. In the cells with DNA damage, biotin-labelled dUTP was localized by the avidin-FITC: avidin and biotin form a strong linkage and reveal the extent of dUTP homopolymer tailing.
  • Fig. 25 shows flow cytometric cell cycle bivariate analyses concurrent with a test for DNA fragmentation to establish that S phase is a target interval of STSP.
  • Each histogram shows a 3-dimensional view of cell DNA content and quantity.
  • the x axis moving in the direction of the arrow, shows increasing amounts of DNA content per cell as measured by propidium iodide staining.
  • the z axis moving in the direction of the arrow, shows increasing number of cells.
  • the y axis, moving in the direction of the arrow shows increasing amounts of dUTP inco ⁇ oration at sites of DNA fragmentation (evidence of apoptosis).
  • the amount of DNA per cell reflects the cell cycle position of each cell.
  • Fig. 25(a) shows an untreated U937 cell population in a normal distribution of cell cycle positions.
  • Fig. 25(b) shows the appearance of cells with dUTP labelling, indicating DNA fragmentation, after 11 hours of treatment with 3 mM dThd. The position of these cells and the reduction of PI stained cells in S phase, suggests that the DNA damage occurred during S phase.
  • Fig.25(c and d) show very few cells labelled with dUTP, indicating minimal apoptosis. The large increase in cells positioned in G 2 M phase shown in Fig. 25(d) is characteristic of STSP and other indole carbazoles.
  • Fig. 25(e) shows a marked increase in the number of dUTP labelled cells after 4 hours of treatment of 0.2 M dThd followed by addition of 25 nM STSP lasting an additional 7 hours.
  • U937 human promonocytic lymphoma cells (U937) with 12-0- tetradecanoyl-phorbol-13 -acetate (TPA) causes them to undergo macrophage-like differentiation and adhere to the surface of the plastic. Kurcz, supra.
  • TPA 12-0- tetradecanoyl-phorbol-13 -acetate
  • F s fraction of cells in S phase
  • Example 14 After washing, the cells were placed in fresh growth medium, and treated ⁇ dThd (0.19 mM) or ⁇ STSP (25 nM) for 7 more hours. DNA extraction and gel electrophoresis was performed as in Example 6. Fig. 27 shows that TPA differentiation significantly reduced dThd potentiation of DNA fragmentation during STSP treatment.
  • Example 14
  • Fig. 28 shows decreased DNA fragmentation in samples subjected to both dThd and STSP when they also were treated with dCyt.
  • This Example shows, by means of a clonogenic assay, that exposure of cells to a synergistic match of an RA (dThd) and a TCI (STSP) for less than a mean generation time produced long term biologic damage to a targeted population of human malignant lymphoma cells (U937).
  • stock cell suspension was prepared in fresh growth medium (8 x 10 5 cells/ml), and two-fold serial dilutions of dThd and STSP were made up to 40 X final concentration in bivariate serial dilutions as described in Example 8.
  • U937 cell suspensions in fresh growth medium were dispensed to a 24-well plate at 1 ml per well, and 50 ⁇ l of selected concentrations of dThd and/or STSP were transferred to designated wells and incubated at 37°C (same protocol as Example 2A, Table 7).
  • Methylcellulose (MC) solution 2.1-2.3% was obtained from Stem Cell Technologies in Vancouver, B.C.
  • Fig. 31 shows results of a clonogenic assay at 96 hours.
  • This Example shows, by means of a tumorigenic assay, that exposure of cells to a synergistic match of an RA (dThd) and a TCI (STSP) for less than a mean generation time produced long term biologic damage to a targeted population of human malignant lymphoma cells (U937).
  • the biologic behavior of xenografts of human malignant cells in immunosuppressed mice can approximate natural growth conditions in a patient, since the cells receive a local supply of plasma nutrients and can survive for several weeks. Thus, inoculated cells may survive in a latent state prior to growth, a condition not readily duplicated in vitro. Inoculum size is critical for observation of tumor growth within a practical time frame. Although immunocompromised mice do not mount an effective cell-mediated immune response to xenografted tissue, small inocula may be damaged by macrophages or natural killer cells before developing an effective vascular support.
  • the IC 40 for an HU or other RA and the IC 50 for STSP or other TCI are determined with respect to growth inhibition of a yeast or fungal population during a predetermined generation time.
  • Each microwell is inoculated with an equal number of the yeast or fungal organisms (100-2000 colony forming units/well).
  • Samples from plates with semi-solid medium are obtained by mixing each well contents in 100 ⁇ l of saline and macerating the agar with thorough mixing. From either plate, an aliquot of 0.5 ml then is plated on semi-solid growth medium in a petri dish; the petri dishes are incubated at 37°C overnight and the yeast or fungal colonies are counted on each plate according to standard microbiologic procedures. Lorian, supra, pp. 53-197. The percentage inhibition of proliferation is calculated by taking the ratio of number of colonies formed by treated organisms to the number of colonies formed by untreated control organisms. Results of the data analysis by data algorithms show a potentiating action of the RA on colony growth inhibition by a TCI in a range of concentrations below the RA IC 40 .
  • This Example demonstrates a use of the invention in potentiating damage to cells infected by a virus and selectivity due to changes in thymidine kinase (Tk) or pRb
  • Tk thymidine kinase
  • cells are infected with a strain of cytomegalovirus according to procedure described by Berezesky IK et al., Exp and Mol Pathol. 14:337-49 (1971) and seeded into 96-well plates.
  • the IC 40 of dThd is determined for uninfected cells.
  • the virus infection proceeds over a period of several days with visible changes in infected foci: cells become enlarged and rounded with increases in both nuclear and cytoplasmic volume.
  • RA dThd
  • Aph IC 40
  • Cell damage is assessed using either the the LDH, MTT, or crystal violet assays.
  • Results are compared to uninfected control cells treated identically with the bivariate combinations of dThd and STSP and data are analyzed using the appropriate set of formulas for CRR or O/S plots as described in Example 8.
  • cells in the infected culture show increased rates of cell damage as determined by increased LDH release at serial time points for each combination of RA and STSP.
  • human malignant lymphoma cells are infected by he ⁇ es simplex virus as described by Bedoya V et al., J. Natl. Cancer Inst. 41 :635-52 (1968).
  • the IC 40 of dThd is determined for uninfected cells. Beginning at times from 0 to 2 h after infection the potentiation of cell damage by bivariate combinations of RA (IC 40 maximum) and STSP (50 nM maximum) are applied in 96-well plates as described above, and data are analyzed after MTT, LDH or crystal violet methods. Cells in the infected culture show increased rates of cell damage at serial time points during the combined agent treatments. Analysis of the data and calculations of S max show action of dThd as an RA and potentiation of STSP in a range of dThd concentrations below the IC 40 .
  • Virus transformed cells such as cells that antecede development of human cervical carcinoma are particularly vulnerable to induction of apoptosis by STSP since they are not impeded from moving through G,/S into the target interval for STSP. Thus, they are selectively vulnerable to dynamic retardation by an RA and killing with STSP.
  • transfection of cells with oncogenic virus proteins serves as an RA to synergize with effects of STSP or other TCI.
  • This Example shows that the MIS and auxiliary data algorithms may be used to test for antagonistic interactions of drugs, or other agents used in medical therapeutics.
  • This Example shows that the MIS and auxiliary data algorithms may be used to test for synergistic interactions of drugs, toxic substances or other environmental hazards for genotoxicty.
  • the agent tested was DEET (N,N-Diethyl-m-toluamide) which is commonly applied in minimal dilutions as a topical insect repellant and has been a subject of safety investigations Osimitz TG and Grothaus RH J Am Mosq Control Assoc; 11 :274-8 (1995).
  • DEET N,N-Diethyl-m-toluamide
  • Osimitz TG and Grothaus RH J Am Mosq Control Assoc 11 :274-8 (1995).
  • applicants identified induction of apoptosis in the U937 cells as well as in Jurkat T cells and Daudi B cells.
  • preliminary results with dThd or HU as prospective RA in ranges of about 0.05 to 3 mM showed no significant enhancement of the DEET-induced apoptosis. Additional tests of this and other agents using the MIS and auxiliary data analysis are thus possible.
  • Example 21 Example 21
  • This Example shows that the MIS and auxiliary data algorithms can be used to measure effects of a radiation source as a TCI or an RA for use in synergistic matches.
  • tritiated water as a low energy (beta ray) radiation source, tests are conducted for radiation effects on growth inhibition and % population loss at times up to 24 hours using the MTT assay. Serial two-fold changes in calibrated radioactive dosages are combined with two ⁇ fold serial dilutions of an agent being tested as an RA or a TCI. Data are analyzed by the auxiliary analytic methods shown in Examples 8 and 10.
  • the tritiated water is obtained at specific activity of 100 mCi/gm or 5 Ci/gm (DuPont) is dispensed by serial dilution from a working stock solution (no less than 1 :25) into usual cell culture growth medium.
  • the IC 40 and EC j o are determined by MTT assay as shown in Example 1 and in Example 10 at approximately 24 hr.
  • the specific range of activity required is based upon dosimetry calculations.
  • pilot data the U937 cells are exposed to gamma radiation in an irradiator and cumulative exposures in the range of 2.5-20 Grey produce apoptosis.
  • the equivalent tritium required is estimated to be in the range of 1 to 4 mCi/ml of medium.
  • the pilot data also suggest that the beta radiation should have a target interval during S or G 2 phases. This is anticipated from extensive published literature (See for example Kuerbitz. supra. Giocanti N et al. Cancer Res 53:2105-1 1(1993)
  • dThd, HU, aphidicolin or other RA becomes evident if it is administered to the cell cultures at 2-6 hr prior to the radiation source; and the radiation exposure is continued for up to 24 hours. It is possible; however, that administration of the RA at some time after the radiation may be advantageous if secondary effects of the radiation within the cell are the indirect cause of the radiation effect and require a time interval for manifestation. Therefore various doseages of the radiation and of a prospective agent as RA or TCI must be tested pragmatically. In pilot data with STSP as an RA in a range of approximately 25 nM, we discerned an enhancing effect upon damage produced by a single exposure of the U937 cells to 2.5 to 10 Gy of gamma radiation. Thus, STSP is a candidate for testing either as RA or TCI.
  • tritiated water in these experiments is a matter of convenience and safety.
  • the radioactive water will equilibrate uniformly through cells without respect to DNA synthesis in contrast to other isotopes which might be selectively inco ⁇ orated into replicating DNA and bias results.
  • the tritiated water is a relatively low energy beta emitter which produces damage in short ranges. Nevertheless it can produce DNA breaks analogous to those produced by higher energy gamma rays used in medical therapeutics.
  • Example 22 A closer approximation of medical conditions can be achieved with a gamma radiator or other physical device, such as a series of radiolabelled beads or platens, in which rows of wells are exposed to progressively incremental doseages of radiation for defined periods using the general approach of the MIS with BVSD so that formulas described in Example 8 can be applied.
  • a gamma radiator or other physical device such as a series of radiolabelled beads or platens, in which rows of wells are exposed to progressively incremental doseages of radiation for defined periods using the general approach of the MIS with BVSD so that formulas described in Example 8 can be applied.
  • Example 22 A closer approximation of medical conditions can be achieved with a gamma radiator or other physical device, such as a series of radiolabelled beads or platens, in which rows of wells are exposed to progressively incremental doseages of radiation for defined periods using the general approach of the MIS with BVSD so that formulas described in Example 8 can be applied.
  • This Example demonstrates a use of the invention in the treatment of a falciparum malaria infection.
  • the malaria plasmodium falciparum (p. falciparum) is a eukaryotic organism that grows by division within human erythrocytes of mammalian hosts in cycles of 44 hours from ring to trophozoite to schizont stage. There is also a tissue phase during which parasites replicate in the human liver. In humans, p. falciparum is particularly dangerous because of its rapid and uncontrolled proliferation and clogging of cerebral microvasculature.
  • the malarial parasite is susceptible to certain agents which we have shown can act as class I of RA.
  • Genes for ribonucieotide reductase have been characterized for the malarial parasite. Chakarabarti et al., Proc. Natl. Acad. Sci. 90:12020-4 (1993). Susceptibility to iron chelators which inhibit RNR has been shown, Lytton SD et al., Blood 84:910-15 (1994), and the parasites can take up dThd or fluorodeoxyuridine. Rathod PK and Reshmi S, Antimicro Agents and Chemother. 38:476-80 (1994); Wright M and Tollon Y, J. Cell Physiol. 139:346-53 (1989). Parasites are also susceptible to effects of STSP as a single agent. Ward GE et al., Exp. Parasitol. 79:480-7 (1994).
  • the inhibitory concentrations (IC 50 ) in strain W2/D6 were staurosporine, 0.15 ⁇ M/0.19 ⁇ M, hydroxyurea, 219 ⁇ M/175.2 ⁇ M and aphidicolin 0.123 ⁇ M/0,40 ⁇ M respectively.
  • inbred Swiss Albino mice of either sex weighing 25-30 g in groups of 6-8 are infected with Plasmodium yoelii or Plasmodium berghii.
  • the infection is transmitted by sacrificing an infected animal when the percentage parasitemia is approximately 40%: 0.5 ml of blood is aspirated from the heart of an anesthetized mouse, diluted into 5 ml with phosphate-buffered sodium citrate anticoagulant and injected intraperitoneal ly into a fresh animal (0.5 ml of the diluted sample).
  • HU is administered orally, beginning on day zero or at 24 hours after an infection, to achieve a plasma concentration in a range up to 150 ⁇ M.
  • STSP at 700 ⁇ g/kg (Buchholz, supra)
  • Parasitemia is monitored in groups of 6 mice at subsequent intervals of 12 hr: untreated mice; mice treated with HU only; mice treated with STSP only; and mice treated with both agents. Survival of the mice is recorded, and treatments may be repeated as indicated by initial results.
  • Thin blood smears are prepared with drops of blood from tail veins.
  • Example human malignant lymphoma cells (U937) were treated with STSP or a synergistic match of dThd and STSP.
  • samples of 5 x 10 6 cells were extracted in SDS sample buffer and subjected to 12% SDS-PAGE and immunoblorting as described in Example 4.
  • the cyclin-dependent kinase p34 cdc2 was detected with antibody clone #1 from Signal Transduction Laboratories.
  • p34 cdc2 was immunoprecipitated with monoclonal antibody (Santa Cruz, #17) using protein A sepharose and reacted with HI substrate (Boehringer Mannheim) in the presence of ⁇ 32 P-ATP (Amersham). Phosphorylated product was separated and resolved by 12% SDS-PAGE and visualized by autoradiography with X-Omat film (Kodak).
  • MAP kinases were detected by immunoblotting with monoclonal antibodies from Pharmingen (San Diego, CA).
  • MAP kinases were immunoprecipitated as above with polyclonal antibody to erk2 or with a GST-JNK substrate for JNK.
  • the substrate for erk2 was myelin basic protein substrate and for JNK was GST-JNK (Pharmingen).
  • the protooncogene product c-myc was detected by immunoblotting with an antibody from Oncogene Sciences.
  • Fig. 36 shows that dephosphorylation of p34 cdc2 on p-Y-15 and p-T-14 is a critical factor in regulating cell movement through G 2 phase into M phase.
  • the ratio of MAP kinases JunK (JNK) and Erk-2 were of interest in relation to the molecular mechanism of apoptosis due to evidence that STSP altered the balance of activity of the MAP kinases JNK and ERK2 (Xia Z. et al. Science 270:1326-1331.
  • ATA aurintricarboxylic acid
  • Fig. 37A is an immunoblot showing the effect of STSP and ⁇ ATA on the phosphorylation of p34 CDC2 .
  • Fig. 37B is an immunoblot demonstrating that STSP induced a functional activation of cdc2 as shown both by the ability to phosphorylate histone protein (HI).
  • Fig. 37C is an immunoblot showing the effect of STSP and ⁇ ATA on c-myc expression.
  • Fig. 38 is an immunoblot showing the effect of STSP and ⁇ dThd on MAP kinases.
  • Table 2 Functional categories and examples of proven or potential TCI * .
  • Table 3A-P Example 8 Relational formulas used by us for calculation of MIS-MTT combined results ratios from tabular format.
  • Table 7 Example 11 Combined results ratios from MTT data showing schedule testing of dThd potentiation of STSP cell damage.
  • Table 10 Combined results ratios from MTT data for dAde and STSP Table 11: Combined results ratios from MTT data for dGuo and STSP Table 12: Combined results ratios from MTT data for HU and STSP Table 13: Combined results ratios from MTT data for MTX and STSP Table 14: Combined results ratios from MTT data for floxuridine (flox) and STSP Table 15: Combined results ratios from MTT data for Aph and STSP Table 16: Combined results ratios from MTT data for ara-C and STSP Table 17: Combined results ratios from MTT data forSTSP and bleomycin Table 18: Combined results ratios from MTT data for STSP and mitomycin C Table 19: Combined results ratios from MTT data forSTSP and cisplatin Table 20: Combined results ratios from MTT data for STSP and daunorubicin Table 21: Combined results ratios from MTT data forSTSP and etoposide Table 22: Combined results ratios from MTT data
  • Table 36 Characteristics of human malignant cells lines successfully treated by dThd or other RA to potentiate the action of STSP or other TCI.
  • Table 38 Combined results ratios from MTT data for dThd and STSP in HL-60 cells.
  • Table 39 Combined results ratios from MTT data for dThd and STSP in Jurkat cells.
  • Table 40 Combined results ratios from MTT data for Aph and STSP in Daudi cells.
  • Table 41 Combined results ratios from MTT data for Aph and STSP in C33A cells.
  • Table 42 Combined results ratios from MTT data for HU and STSP in Raji cells.
  • TCI Cytotoxic Insults
  • DNA intercalation (dactinomycin, daunorubicin.
  • Actions of agents underlined are demonstrated in present examples. Actions of other agents have been inferred from previously published data. Some TCI such as STSP may function in more than one category of action depending upon the cell type or strength and duration of application. Agents marked with ® have not yet been tested clinically.
  • Numbers in C81 to LU indicate positions of columns in the 96-well plate
  • SUBSTTTUTE SHEET RULE 26 Mean data from columns of wells in immediate assay plate for use as N-. (MTT assay in Examples)
  • Numbers in C132 to L132 indicate positions J136 (F2) [W32] 1-J103 of the columns in the 96-well plate K136 (F2)[W32]1-K103

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Steroid Compounds (AREA)
  • Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)

Abstract

L'invention se rapporte à un procédé amélioré destiné à induire des lésions cellulaires, par l'administration d'un agent de ralentissement (RA) à une population cellulaire cible en une concentration suffisante et dans des conditions appropriées pour retarder, mais non interrompre la progression des cellules cibles dans le cycle cellulaire, et l'administration simultanée ou ultérieure à celle de l'agent de ralentissement, d'un agent d'agression cytotoxique ciblé (TCI). L'invention se rapporte également à un système indicateur en microculture et à des procédures d'analyse de données auxiliaires visant à identifier, concevoir et utiliser de nouveaux agents comme agents de ralentissement ou agents d'agression cytotoxiques ciblés, ainsi qu'à améliorer les associations synergiques des agents existants. Dans des modes de réalisation de l'invention, l'agent de ralentissement peut être un inhibiteur de ribonucléotide réductase, de dihydrofolate réductase, de thymidylate synthase, d'ADN polymérase, de protéine kinase ou de topoisomérase. En outre, selon des variantes de l'invention on utilise, comme agent TCI, les indole carbazoles tels que la staurosporine, K252a, KT5926, et KT5720. Dans une variante spécifique, l'agent de ralentissement est la thymidine et l'agent TCI est la staurosporine.
EP96923453A 1995-06-27 1996-06-26 Procede de ralentissement dynamique de la cinetique du cycle cellulaire, destine a potentialiser les lesions cellulaires Withdrawn EP0835111A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US54695P 1995-06-27 1995-06-27
US546P 1995-06-27
US66893296A 1996-06-24 1996-06-24
US668932 1996-06-24
PCT/US1996/010921 WO1997001344A2 (fr) 1995-06-27 1996-06-26 Procede de ralentissement dynamique de la cinetique du cycle cellulaire, destine a potentialiser les lesions cellulaires

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EP0835111A2 true EP0835111A2 (fr) 1998-04-15

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EP (1) EP0835111A2 (fr)
JP (1) JPH11509193A (fr)
AU (1) AU715527B2 (fr)
CA (1) CA2225682A1 (fr)
WO (1) WO1997001344A2 (fr)

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MX2009005300A (es) * 2006-11-17 2009-06-08 Schering Corp Combinacion de un inhibidor de acido desoxirribonucleico polimerasa-alfa y un inhibidor de una cinasa de punto de verificacion para trastornos proliferativos.

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US4448534A (en) * 1978-03-30 1984-05-15 American Hospital Corporation Antibiotic susceptibility testing
US5166140A (en) * 1987-05-05 1992-11-24 City Of Hope Use of certain nucleoside analogs to attenuate cancer cell resistance to DNA damaging chemotherapy
DE3827974A1 (de) * 1988-08-18 1990-02-22 Boehringer Mannheim Gmbh Kombinationspraeparate von proteinkinase-c-inhibitoren mit lipiden, lipid-analoga, cytostatica oder inhibitoren von phospholipasen
WO1992019765A1 (fr) * 1991-05-08 1992-11-12 The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Procede de conception de traitements du cancer, procedes et compositions pharmaceutiques de traitements du cancer
EP0593663A4 (en) * 1991-07-03 1996-10-30 Regeneron Pharma Method and assay system for neurotrophin activity
EP0612248B1 (fr) * 1991-11-15 2003-08-20 Smithkline Beecham Corporation composition contenant le cisplatin et le topotécane comme agent antitumoral
JPH08500112A (ja) * 1992-08-12 1996-01-09 ジ・アップジョン・カンパニー タキソールと組み合わせるプロテインキナーゼ阻害剤および関連化合物
ZA941290B (en) * 1993-02-26 1995-08-25 Res Dev Foundation Combination cisplatin/tamoxifen therapy for human cancers
EP0703917A1 (fr) * 1993-06-17 1996-04-03 Novartis AG Compose d'indolocarbazole utilise comme inhibiteur de proteine-kinase c

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See references of WO9701344A2 *

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Publication number Publication date
AU715527B2 (en) 2000-02-03
WO1997001344A3 (fr) 1997-03-27
CA2225682A1 (fr) 1997-01-16
WO1997001344A2 (fr) 1997-01-16
JPH11509193A (ja) 1999-08-17
AU6396096A (en) 1997-01-30

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