Note: Descriptions are shown in the official language in which they were submitted.
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I
METHOD OF DYNAMIC RETARDATION OF CELL
CYCLE KINETICS TO POTENTIATE CELL DAMAGE
CROSS-~FFF.RFNCF TO RFT,~TF.T~ APPT,TCATION
The present invention is based on U. S. provisional application number 60/000,546, filed
June 27, 1995, incol~oldl~d herein by reference, in its entirety.
GOVF,Rl~l~FNT Il~TF,RF,~T
The invention described herein may be m~nllf~ hlred, licensed, and used for United
States of America govt?rnment~l purposes without the payment of any royalties to the inventors
or ~cci~n~e
FTFT,T) OF TT-TF~ INVENTION
This invention relates to a method of potenti~ting cell damage by a~lmini~t~nng an agent
that retards the rate of movement of a target cell through some portion of the cell-division
cycle and ~-iminictloring 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. In a specific
embodiment, deo~ylllylllidine (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 determin~ the degree of interaction between agents. In a specific
embodiment, data are collected reflecting the effect on cell growth of two or more agents
arrayed in serial bivariate dilutions, and a ~l~t~h~ce is caused to process the data in a
spr~lch~et according to predet~rmin~d relationships with reference measurements of cell
growth and to present, in graphical or tabular form, the spectrurn of interaction of the agents
with respect to reference measurements.
BACKGROUND OF THE~ INVENTION
The use of drugs or other agents for destroying or inflicting perm~n.ont darnage on living
cells serves a number of valuable and legitim~te objectives. A major clinical use is for the
ablation of m~ n~nt tumors or other abnormal tissue growths. VT DeVita, Jr., IN: Cancer,
Principles and Practice of Oncology, 4th ed., pp. 276-292, JB Lippincott Co. Phil~ lphi:~
(1994).
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Other valuable clinical uses have included (1) medical control of abnormal immnnnlogic
reactions, K Wilson et al., Rheumatol. 21:1674-7 (1994); CM Neuwelt et al., Am. J. Med.
98:32-41; (1995); (2) exfoliative ~erm~tological disease, GD Weinstein et al., J. Am. Acad.
Dermatol. 28:454-9 (1993), RJ Van Dooren-Greebe et al., Br. J. Dermatol. 130:204-10 (1994); ,,
(3) killing of cells infected by viruses, viral replicative elements, or prions, P Calabresi et al.,
Section XII--Chemotherapy of neoplastic ~ Ç~ce~ IN: Goodman and Gilman's The
ph~ cologicBasisofTherapeutics, 8thed., 1202-1263 (P~lg~~ onPress,NewYork 1990);
S Chou et al., Antiviral chemotherapy, Chapter 17 IN: Virology, pp. 323-348, ed. BN Fields
et al., Raven Press, New York (1985); (4) therapies for systemic or topical Plimin~tion of
infective agents including bacteria, mycobacteria, mycoplasma, rickettsia, fungi, yeast, or
parasitic organi~m~, HP Willett, The action of chemotherapeutic agents, Chapter l 0, IN: Zinsser
Microbiology, 17th ed., pp. 234-277, ed. Joklik et al., Appleton-Century-Crofts, NY (1980); V
Lorian, Antibiotics in Laboratory Medicine, 3d ed., Williams and Wilkins, Baltimore (1991);
S Sternberg, Science 266:1632-1634 (1994), (5) and fertility control. Nonclinical uses of
agents capable of inflicting permanent damage on living cells occur in agriculture, horticulture,
or public health, e.g., application of specific pesticides or herbicides.
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.
In other cases, cell damage may be initiated in direct relation to the hierarchy of the
cell-division cycle. A cvtotoxic 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. The portion of the cell-division cycle
during which a given TCI initi~t~.~ a relevant action is its "target interval."
Known 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.
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Cytotoxic effects of a TCI may not be immetli~te, so that cell damage initi~teci in one
phase of the cell-division cycle may not become manifest until a later phase or a subsequent
cell cycle. As just one example, in cisplatin tre~tment~ p~rrn~n~ntly injured progeny cells may
be sterile or exhibit a reduced capacity to proliferate or survive. M Sorenson, J Natl. Cancer
Inst. 82:749-55 (1990). Thus, an undçrstanding of the cell-division cycle hierarchy becomes
useful to filrther underst~n(lin~ of agents that can act as TCIs.
I. The Ccll-Divisiorl Cyclç
All growing cells must duplicate their genomic DNA and pass identical copies of this
genetic inforrnation to their progeny. In order to accomplish this task, proliferating somatic
(non-reproductive) and germ (reproductive) cells of all living or~ni~mc undergo repetitive cell-
division cycles (hereinafter "cell cycle" or "CC"). Each completed cell-division cycle results
in the duplication of the cell's genetic inforrnation and the division of the parent cell into two
d~llght~or 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
c~cc~-les, association and dissociation of L~ s~ tional activating molecular complexes, and
formation and dissociation of macromolecular assemblies of cytostructural elements including
cytomembranes and the cytoskeleton.
A. Cell Cycle Hierarchy
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 F-lmllntls, 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 ~irs~hner MW,
Science 246 :614-621 (1989).
In the norrnal cell cycle hierarchy, DNA replication is followed by mitosis and cy-
tokinesis. ~ç ~er~erally AW Murray, Nature 359:599-604 (1992); B Alberts et al., The
cell-division cycle, IN: Molecular Biologv of the Cell, 3d edition, Garland Publishing Inc., New
York (1994); BA Edgar et al., Genes Dev 8:440-52 (1994). A series of molecular processes,
each process functioning in an ~ o~liate order during the cell cycle, moves the cell in the
direction of cell division with a downstream momentnm In this context, the term
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"downstream" refers to events that occupy a "subordinate position" in the cell cycle hierarchy
as defined by Alberts, ~. Order depentlen~e in the cell cycle hierarchy ensures that DNA
replication proceeds with maximal fidelity. See LH Hartwell et al., Science 246:629-634
(1989); PM O'Connor et al., Semin. Cancer Biol. 3:409-416 (1992).
The hierarchy of the eukaryotic cell cycle relates to four conserved functional l~nrlm~rkc
(Fig. 1): S phase, in which nucleotides are syntht-~i7~d and DNA is semi-conservatively
replicated in double-stranded helixes of polynucleotides; G2 phase, which follows completion
of DNA synthesis and during which DNA associates with nucleoproteins; M phase, in which
nuclear fil~m~nt~ condense as chromosomes and chromosomes segregate for mito~i~; and G,
phase, during which cells prepare for renewed division by repl~l~ement of depleted products and
repair of any lesions in DNA. See Alberts, ~. Cells entering S phase normally are
cornmitted to completion of G2 phase, M phase, and cytokinesis.
B. Cell Cycle Checkpoints
Transitions between phases are the major checkpoints in the cell cycle. In normal cells,
they are tightly regulated by a decision point in G, (START) and checkpoint controls associated
with the boundaries between G, and S (G,/S) and G2 and M (G2/M). See KA Heichman et al.,
Cell 79:557-562 (1994); P Nurse, Cell 79:547-550 (1994); AW Murray, ~.; AW Murray
et al., Sci. Am. 264:56-63 (1991); Hartwell, ~. Controlled interactions of specific proteins
such as cyclins, cyclin-dependent kinases (cdk or cdc), and a series of accessory proteins
(including pl6, p21, p27, p45 or p53), which regulate cdk or cdc cyclin complexes, regulate
successive phases of the cell cycle. T Hunter et al., Cell 79:573-582 (1994); Heichman, ~.;
Nurse, ~.; RW King et al., Cell 79:563-571 (1994); LH Tsai et al., Oncogene 8: 1593-602
(1993); M Doree et al., FASEB J. 8:1114-1121 (1991). Moreover, function of p53 and
phosphorylation of the Rb turnor suppressor gene product (pRb) are also associated with the
G,/S transition. V Karantza et al., Mol. Cell Biol. 13:6640-52 (1993); ME Ewen et al., Cell
73:487-97 (1993), SJ Kuerbitz et al., Proc. Natl. Acad. Sci. USA 89:7491-95 (1992);
MB Kastan Cell, 71:587-597 (1992).
C. Cell Cycle Kinetics
In a population of cells, the mean duration of each cell cycle phase is proportional to J
the probability of finding a cell within a given phase. If it is assumed that no loss, quiescence,
or differentiation of progeny cells will occur during the continuous proliferation of a cell
population, then for any given population, i.e., cohort, the mean duration of a single cell cycle
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will equal the time to double; i.e., the time required for the starting cell population to double,
i.e., its generation time. ~, LA Perez et al., Cancer Res. 55:392-398 (1995). This concept
can be expressed m~them~tically. Thus, TDBL is defined as the time required for the present
population (Np) divided by the original population (No) to double (N/No = 2).
The TDBL provides a yardstick for dete~ninin~ 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
Gl (TGI)~ S (T5), or G2 & M (TG2.~ t~sl~min~ continuous proliferation of an ideal cohort
without any loss, quiescence, or differentiation of progeny, the fractional duration of each phase
of the cell cycle is directly proportional to the fraction of the cell population (F) that is cycling
through that phase at any moment in time, i.e., the FGi~ FS or FG2 'Y~ M; for example, Ts~ can be
calculated as Ts = Fs x TDB~. See Alberts, supra, and Perez, supra. Practically, this is an
important equation since TD8L can be determined from serial cell counts, or flow cytometry, AC
Begg et al., Cytometry 6:620-626 (1985), while Fs~ FG2+M or FGI/GO can be measured in DNA
histograms obtained from flow cytometry.
In standard flow cytometry, nuclei are stained with propidium iodide, a dye which
intercalates into the minor groove of DNA. NM Shapiro, Practical Flow Cytometry, Alan R
Liss, NY (1988). Histograms of dye absorbance discriminate the fractions of cells with
different arnounts of DNA/nucleus. Thus cells in the process of synthet~i~ing DNA (S phase),
or with a complete duplication of DNA (G2 phase and M phase), are distinguished from cells
that have not begun to replicate their DNA (Gl or Go). FIOW cytometric DNA histograms are
reproducible and accurate under most testing conditions. ~ee Perez, ~; J Pierrez et al., Acta
Biotheor. 40: 131-7 (19g2). DNA synthesis is also measured by BudR incorporation (Begg,
~-
When changes in physiological stimuli or ambient growth conditions slow down thegrowth of a cell population, the fraction of cells found in G~ phase (the p~ Ut .~lion phase)
typically increases at the expense of cells in S phase or in the growth fraction, i.e., G2 and M
phases. Such changes or abnormal conditions may include hor~nonal, nutritional, or
environm~nt~l 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 tlesign~t~d to be in Go phase. R Baserga, Cell Division, Molecular Biology, IN:
Encyclopedia of Human Biology 2:253-266 (1991); AB Pardee, Science 246:609-613 (1989).
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The process of differentiation, or specialization of cells, is also associated with a
retirement of cells to Go. In te.nnin~l dirr~ nliation, the transition out of the cell cycle, i.e.,
into Go~ becomes irreversible. Examples of tennin~lly dirr~ t~l cells are adult neurons,
ker~tini7ecl epithelia, and voluntary muscle cells.
D. Apoptosis
Apoptosis is referred to as a process of "programmed cell death." During normal
somatic development, cell populations in specific organs or tissues may be prograrnmed for
death as part of the development~l progression of tissue remodeling or obsolesc~n-~e ~
JJ Cohen, Avd. Immunol 50:55-85 (1991); M Baringa, Science 259:762-3 (1993). Apoptosis
is internally triggered by biochemical or biomolecular mech~ni~mc 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
~gment~tion. AH Wyllie, Nature 284:555-6 (1980). Apoptosis in rnature tissues occurs in
normal processes such as infl~mm~tion or rejuvenation. M Schmied et al., Am J Pathol
143:446-52 (1993). Abnormal clonal proliferations in immunologic tlice~cec or m~lign~n~ies
may be related to a failure of normal apoptosis. J Marx, Science 259:760-1 (1993).
The relationship of apoptosis and/or cell damage to the cell cycle, including checkpoint
controls, during cancer chemotherapy is a subject of interest to oncologists and molecular
biologists. ~ T Shimizu et al., Cancer Res. 55:228-231 (1995); O'Connor, ~. (1992)
The expression of pS3 in damaged cells is one factor in determining the course of divergent
biochemical pathways, which can lead to either DNA repair or apoptosis. E Yonish-Rouach
et al., Mol Cell Biol 13:1415-23 (1993); DE Fisher, Cell 78:539-542 (1994).
Conflicting signals in the cell-division cycle may underlie the diversion of cell activities
from proliferation to apoptosis. Fisher, supra. Cells entering or traversing the cell cycle
transition boundaries or in the process of DNA replication or repair are most susceptible to
apoptosis. In cells treated with a TCI, or in neoplastic cells, checkpoint controls such as
cyclin-dependent kinases may be deregulated. This deregulation can release DNA replication
or cell division events from ST,4RT and homeostatic order dependence, intensifying cell
damage. Id.
II. The Role of Cytoto~ic Agents in Chemotherapy
Current models of cancer chemotherapy are based largely upon two central dogmas. "
First, any mass of tumor cells which is clinically detectable must include a significant number
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of cells which will exhibit some biologically significant level of re~ict~nre to any single
chemotherapeutic agent. JH Goldie et al., Cancer Treat Rep. 53:1727-1733 (1979). Second,
according to the accepted Cc,~ clkian model, tumor cell killing relates to the fraction of cells
in active growth. LA Norton, Cancer Res. 48:7067-71 (1988).
In chemotherapy for m~ n~ncy, treZ~tmentc with TCI have involved a nurnber clinical
considerations: they may be used in the primary effort to control cancer (indu~tion
chemotherapy), or as an adjunct to surgery or radiotherapy (adiuvant chemotherapy). DeVita,
~Pr_ (1994). Local tre~tment~ have included infusion of TCI into body cavities to control the
spread of m~lign~nries such as breast or ovarian cancers.
A. Single Agent Chemotherapy
In single agent induction, the usual objective is to atimini.~ter the highest safe and
tolerated dose to achieve maximal cancer cell killing or growth arrest. However, due to a
cancer patient's decreased ability to mount a cell m~ te-1 imrnune response against m~lign~nt
cells, single agent chemotherapies rarely prove sufficient to control cancer in the hurnan body.
Neoplastic (cancerous) populations are heterogeneous and a fraction of resistant cells typically
escape death. The subpopulation of m~lign~nt cells with protective merl ~ni~m~ eventually
replaces the original populations of susceptible cells.
The first of the single agents, folic acid antagonists, targeted DNA biosynthesis. See
VT DeVita, New Engl. J. Med. 298:907-910 (1978). Both replication of DNA and cell
division, associated with the S phase, have been targets of chemotherapy. Examples of TCI
targeting DNA synthesis have included antimetabolites, alkylating agents, natural toxins or
antibiotics, platinum coordination complexes, and substituted urea. Known actions of these
agents have been ~ cl~sed by Calabresi, ~_.
An agent that can act as a TCI can initiate cell damage during the cell cycle hierarchy
in various ways. For example, a TCI can inhibit enzymes, compete for substrates, inhibit the
Ll~ls~ Lional, translational or post-translational steps in molecular biosynthesis, introduce
transcriptional or translational errors, disrupt molecular conforrnational changes, inhibit
molecular transport, compete for energy transfer molecules, interfere with macromolecular
polymeri7~tion, form molecular crosslinks, alkylate or cause strand breaks in DNA, or
intercalate into the DNA helix. Thus, a TCI may impair cell cycle processes such as RNA
Lla,.s~ Lion and translation, DNA strand elongation, replication, repair, supramolecular
c"p~ ion or separation, molecular transport, or macromolecular segregation. Alternatively,
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it may selectively injure any of the multiple cellular organelles associated with s-lcc~fill
completion of specif1c subsets of the cell cycle hierarchy.
Another possible mode of selective damage by a TCI in neoplastic cells is a loss or
deficiency of a checkpoint control, such as a cyclin-dependent kinase (cdk or cdc), which
normally controls the cell cycle hierarchy. The role of checkpoint controls has been defined
by observed effects of agents or mutations which relieve order depenc1~llre~ HA Crissman
et al., Proc. Natl. Acad. Sci. (USA) 88:7~80-84(1991); Kastan, ~pl_.; Murray, ~pl_. (1992);
Hartwell, SUpra~ The cell cycle of normally cycling cells must traverse the G, decision point
(STARI) which commits a cell to continue through S phase resulting in DNA replication.
Heichman, ~. Thus, cells exposed to a TCI prior to ST~RT may be partially protected from
DNA darnage by a delay within G,. This G, delay can be mediated by the tumor suppressor
p~3 and enables cells to repair damaged strands of DNA prior to replication. Kastan, ~.
Damage to DNA after STA~T or DNA damage and bypass of ST~RT 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.
Hartwell, ~; Kuerbitz, ~..; Shaw et al., Proc. Natl. Acad. Sci. USA 89:4496-9 (1992);
T. Weinert, Semin. Cancer Biol. 4:129-140 (1993).
The actions of agents targeting S phase in eukaryotic cells are intrinsically complex due
to the nature of DNA replication in S phase. For example, 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). Even as the DNA strands replicate in parallel process hierarchies
within the overall cell cycle hierarchy, however, they share critical enzymes or metabolic
interm~ teS Murray, suPra (1992); Laskey et al., Science 246:609-613(1989); Nurse,
Heichman, ~.
Modem approaches to cancer chemotherapy developed during a time when knowledge
of the cell cycle was advancing rapidly. Thus, it was recognized that neoplastic cells are
vulnerable to agents that act during the S phase of the cell cycle. To better study the S phase,
anti-metabolic agents were used to inhibit enzymes associated with purine or pyrimidine
nucleotide biosynthesis affecting the ability of the DNA to replicate. These included
ribonucleotide re~ t~ce (RNR) inhibitors, such as dThd or hydroxyurea (HU); dihydrofolate
re~luct~e inhibitors, such as methotrexate (MTX); or DNA polymerase inhibitors such as
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aphidicolin (Aph) to completely arrest progress of the target cells through the cell cycle at
G,/S. G Galavazi et al., Exp. Cell Res. 41:428-51 (1966); D Thomas et al., Cell 5:57-32
(1975); T Ashihara et al., Methods Enzymol. 58:248-262 (1979); Levenson, ~.
It also was established that an excess of the normal metabolite dThd could reversibly
arrest DNA replication in many cell lines of m~lign~nt origin or other proliferating cells.
D. Kufe et al., Cancer Treat. Rep. 64:1307-1317 (1980).
In other studies, the use of excess dThd was found to be less ~l~m~ginE than MTX or
HU and removal of dThd could be followed by a synchronous progression of cells through the
ren ~in~lPr of the cell cycle. HR Zielke et al., Methods in Cell Biology 8:107-121 (1974); RE
Meyn et al., Methods in Cell Biology 9: 103-113 (1975). As a result, repetitivesyncL~ ion of the cell cycle with dThd produced relatively pure cell populations in S
phase. Zielke, ~.
Use of RNR inhibitors such as dThd or HU as a single agent in high dosages was also
explored in cancer therapy. Many of the published reports conrerning use of dThd have been
critically reviewed. See Ellims, ~; O'Dwyer et al., Cancer Res 47:3911 (1987); SO Ooi et
al., Experientia 49:576-81 (1993). Some successful results in patients with lellkçmi~,
lymphomas, or solid tumors were reported. DW Kufe et al., Cancer 48:1513-6 (1981); A
Levya et al., J Cancer Res. Clin. Oncol. 107:211-216 (1984); RL Schilsky et al., Cancer Res.
46:4184-4188 (1986). Blood levels of up to 6 mM dThd could be achieved with oral doses.
MS Blumenreich et al., Cancer Res. 44:2203 (1984); O'Dwyer, ~pra. In general, however, the
use of dThd as a single agent chemotherapy was considered marginally potent for damage to
m~lign~nt cells. Toxic side-effects often were intolerable at the dosages required to produce
any therapeutic benefits.
B. Combination Chemotherapy
In cancer therapy, the survival of even a few m~ n~nt cells is more critical than in
anti-infective therapies, since host imrnune mech~ni~m~ for killing of m~lign~nt cells typically
are not effective. Therefore, exogenous cell killing plays a major role in prolonging clinical
remissions or achieving cure. In the context of conventional chemotherapy, however, cell
killing is described by first order kinetics: increasing doses of a single TCI will selectively
damage an increasing percentage of the rem~ining m~ n~nt cells, but cannot destroy every
potentially m~lign~nt cell without sacrificing the host. Calibresi, supra. M~them~tically, this
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is analogous to Zeno's paradox of fast and slow runners (WL McT .~ hlin, Sci. Amer. 271 :84-
89, 1994).
Since the 1960s, heavy reliance has been placed upon combinations of agents to produce
more durable clinical responses than are possible with single agents. RL Capizzi et al., Sem.
Oncol. 4:227-253 (1977); DeVita et al., Cancer 35:98-110 (1975). DeVita, ~ (1994),
c-lc~es the generally agreed objectives of agent combinations: to m~ximi7~ cell killing with
tolerable toxicity, to provide coverage of cancer cells with ~1ifferin~ levels of vulnerability in
a heterogeneous tumor population, and to prevent or slow the evolution of neoplastic clones
that develop increasing resistance.
DeVita also sets forth several principles in the current selection of agent combinations:
(i) each agent should be effective as a single agent in cell killing;
(ii) 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 ~rlmini~tration should be optimi7~A
As mentioned above, development of cell resistance to cytotoxic agents may involve
mutations in pS3 or other cell cycle control genes and may be accompanied by abnormalities
in the cell cycle o}der dependence or checkpoint controls. CS Morrow et al., Ann. NY Acad.
Sci. 698:289-312 (1993). Appropriate selection of multiple agents and achievement of high
dose intensity are currently perceived as the critical issues in the design of chemotherapeutic
protocols to avoid the development of that resistance.
Efforts have been aimed at mo~ tin~ the cell cycle as a means for increasing cell
damage by combinations of chemotherapy agents. The objective was to m~int~in m~ n~nt
cells within the S phase of the cell cycle, where they may be most vulnerable to damage. Sçe
HO Klein et al., Semin. Hematol. 11 :203-27 (1974). RL Stolfi et al., Phannac. Ther. 49:43-54
(1991), have referred to these strategies as "cytokinetic modulation".
Some uses of MTX, dThd, or pyrimidine analogs to arrest cell populations at a specific
point in the cell cycle to modulate synergistic killing by application of a successive cytotoxic
agent have been tested. B Bhutan et al., Cancer Res. 33:888-894 (1973); Ellims, supra;
SD Henderson et al., Invest. New Drugs 5:142-154 (1987); Stolfi, supra. This approach often
has been referred to as "synchronization" of the cell cycle. Capizi, ~. Cells exposed to
high concentrations of anti-metabolites are detained within a limited subset of the cell-division
cycle hierarchy, typically at a specific point within late G, or early S phase. Thus, few or no
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cells in the population can proceed beyond this point of detention. Therefore, this type of
effect is better described either as a cell cycle "arrest" or a "static synclllol~i;~Lion". W Vogel
et al., Hum. Genet. 45:193-8 (1978).
A number of other efforts to control cancer cell growth by manipulating the cell-division
cycle have been directed to altering the cell cycle distribution within the cell population tar-
geted for darnage. Other protocols were clesigne-l to stimulate m~ n~nt cells from Go phase
or G, phase into proliferative status and thus increase their vulnerability to anti-metabolic drugs
acting during DNA replication. HH Euler et al., Ann. Med. Interne. (Paris) 145:296-302
(1994); BC T ~mpkin et al., J. Clin. Invest. 50:2204-14 (1971); Alama et al., Anticancer Res.
10:853-8 (1990). Conversely, other protocols were ~ie~igned to prohibit normal cells from
entering S phase and thus protect them from l~ninten~ d damage by anti-metabolites. Capizi,
Agents in combination may have additive, synergistic or antagonistic effects.
Intuitively, it might be supposed that a combination of agents causing a cell cycle arrest or
static synchronization of the m~lign~nt cells would circumvent the problem of first order
kinetics, combined dosages can be increased to a level sufficient to fill all m~ n~nt cells
without sacrificing the host. Restriction of a m~lign~nt 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 rlimini~hing the cumulative time of host exposure to a TCI.
However, in actual trials, strategies of cell cycle arrest or static synchronization often
have been disappointing. Capizzi, ~. This is due to an essential incongruity of the
procedure. When the cell cycle is arrested, all cells are within a specific fraction of the cell
cycle. If this subset of the cell cycle does not completely overlap the subset of the cell cycle
where the TCI is most effective, the cell cycle arrest will not result in synergistic or even an
additive action of the successive TCI. Cell cycle arrest or static synchronization can be
advantageous only when the affected phase of the cell cycle actually encompasses a relevant
target interval of the successive TCI. The target interval of a successive TCI may be located
downstream and not within the kinetic boundaries of the arrested or statically synchronized
population. Since a high concentration of an agent effecting cell cycle arrest or static
synchronization can also act as a single agent TCI, some cell killing or darnage will occur even
prior to the addition of a successive TCI. Indeed, if a successive TCI were actually the more
CA 0222~682 1997-12-23
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potent killing agent and acted downstream from the point of cell cycle arrest, then cell cycle
arrest or static syncl.,oni~Lion would actually protect cells from the succes.~ive agent. This
problem was exemplified in previous trials with dThd used in high doses as a synch~ g
agent. Doses of dThd required to achieve effective blood concentrations for cell cycle arrest
(> 3 mM) often caused toxic side effects which were not well tolerated by patients, and could
not be justified in relation to the therapeutic effect. In one set of trials, the stated objective was
to deliver m~im~lly tolerable doses, and blood levels of up to 6 mM were achieved with oral
doses. Blumenreich, ~_. O'Dwyer, ~. This concentration range had been used
successfully in vitro to prohibit cells in G,-phase from entering S phase (i.e. a G,/S block). JH
Kim Biochem Ph~ rol 14:1821-9 (1965); Littlefield, supra.; Kufe, ~, but it did not prove
sufficiently potent to induce clinical inhibition of mz~lign~nt cell growth in the patients. Thus,
secondary toxicities proved difficult for patients to tolerate and the further use of dThd was
discouraged and its use is no longer advocated or reported. Blumenreich, ~; O'Dwyer,
~; Ooi, ~.
Most of the agents previously used in chemotherapy to effect cell cycle arrest or static
synchronization in chemotherapy have probably acted late in G, phase or early in S phase.
Capizi, ~. Therefore, they would be unlikely to potentiate TCI with actions beginning
later in the cell cycle hierarchy, e.g., later in S phase.
In modified approaches to static synchronization for cancer chemotherapy, a number of
investigators noted that the ~cheduling of particular drug combinations was critical for
production of synergistic lethal effects either in vitro or in vivo. Capizzi, ~; Stolfi, supra.
However, many of these combinations have only been tested on a trial and error basis without
a clear rationale for the agent sequence, cone.ontration ratios, schedule or duration employed.
AL Adel et al., Cancer Invest 11:15-24 (1993). In clinical parlance, therapeutic approaches
of combining cell cycle arrest with sequential applications of a second agent have been referred
to as "schedule dependent ~nh~nre~nent," e.g., Capizzi, ~., or "biochemical modulation."
FM Muggia et al., Semin. Oncol. (3 Suppl) 9:90-3 (1992).
One special strategy of cell cycle manipulation was referred to as "pulse dose
chemotherapy". RE Moran et al., Cancer Treat. Rep. 64:81-6 (1980). In this particular
approach, leukemic tumor cells in mice were detained in S phase of the cell cycle during
infusion treatment of the mice with hydroxyurea (HU). After the infusion of HU was ended,
the cells were "released" to continue transit of the cell cycle. At finite times after tPrrnin~tion
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of the HU infusion, ~ cnt~l animals were treated with a "pulse" of a seeond agent
(Ara-C). This method ean be eompared to the arrest method of HR Zielke, ~a and JL
T ittlefiel(l et al., 1974, in which an arrest of the eell-division eycle at a specific c7etPntiorl point
is reversed so that the eells then move in coneert through the cell cycle at a normal or possibly
an accelerated rate.
The intention of "pulse dose chemotherapy" was to m~cimi7to impact of the secondagent as cells were moving in coneert through the cell cycle. Mean survival time of the miee
was det~rminerl- Mice treated with Ara-C at zero time, just after the HU infusion ended,
showed improved survival, but tre~tm~nt~ with Ara-C at later times after stopping the HU
infusion did not potentiate the effeet of HU. This procedure of cell cycle synchlolli~Lion
followed by a second agent relies on the nonsimultaneous action of the two agents. Moreover,
the indirect results with respect to mean survival time of the animals cannot be directly
tr~ncl~te~ to effects on tumor cell darnage.
A major challenge in combination chemotherapy is to determine an optimal synergy in
view of the multiple variables of dose, pharmacokinetics, sequence, and sch~ ing. Even for
two agents, the most effeetive utilization is not necessarily clear from a simple combinational
analysis of the optimum for each agent. Empirical variables include the dose or effective
eoneentration 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, ~; Adel,
. Sçe also MC Berenbaum, Pharmacol. Rev. 41:93-141 (1989).
In vitro testing using tissue cultures or testing in animal models can provide guidance
on proposed combinations prior to clinical application. J Plowman et al., Cancer Res. 55:862-7
(1995); ME Wall et al., Cancer Res. 55:753-60 (1995); J Hi~hih~ra et al., Gynecologic
Oncology 48:171-179 (1993); Berenbaum, ~. Rev. 41:93-141 (1989); PC Schroy III et al.,
Cancer Res. 48:3236-3244 (1988); RH Shoemaker et al., Cancer Res. 45:2145-53 (1985);
Capizzi, ~.
Technical methods of in vitro evaluation of chemotherapeutic agents have varied, but,
m~o~cl~ring inhibition of cell growth by staining or dye uptake is accepted by the National
Cancer Tnstitnte as a method for qual1LiL~Live analysis. Plowman, ~a. Uptake of
3H-thymidine to obtain the labelling index is another common method of analyzing tumor cell
., sensitivity to chemotherapeutic agents. GH Baltuch et al., Neurosurgery 33:495-501 (1993);
IP Hayward et al., Int. J. Cell Cloning 10:182-9 (1992); Schroy, Supra. Differences in nucleic
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acid salvage pool sizes or thymidine kinase activities can make this approach unreliable. A
similar approach is provided by the technique of flow cytometry in which antibodies against
halogenated pyrimidine analog or a Hoechst dye track cells in S phase. Perez, supra; JP Perras
et al., Cytometry 14:441-8 (1993); P Ubezio et al., Cytometry 12:119-126 (1991), M Poot et
al., Biochem. ph~ ol. 41:1903-9 (1991).
Classically, the synergistic interaction of two agents is ~cs~cse(l using a series of dose-
response curves from which fractional inhibitory concentrations can be calculated. GB Elion
et al., J. Biol. Chem. 208:477-88 (1954); Berenbaum, supra; GM Eliopoulos et al., Chapter 13,
pp. 432-492, IN: Antibiotics in Laboratory Medicine, 3d ed. V Lorian ed. The data can be
obtained by a checkerboard technique, ~Q Lorian, ~; Howard et al., Int. J. Cell Cloning
10:182-9 (1992). This method of analysis is known as an isobologram.
There are several practical problems with the isobologram method. First, the dose
response curve of many agents used in chemotherapy becomes nonlinear at very high
con~.ontrations. Second, if agents to be compared prove asymmetrically potent or weak in
single use, comparisons may be impossible. Third, results could be highly variable as
coefficients of synergy calculated from an isobologram can be different for each fixed level of
cytotoxicity.
Thus, collection of data for isobolograms can be laborious, and the isobologram method
has limited scope for demonstrating an optimal range of agent combinations for practical
effects. Berenbaum, ~. Efforts to resolve this shortcoming have proposed analyses of data
as a three dimensional surface construct. WR Greco et al., Cancer ~es. 50:5318-27 (1990) or
other special methods (RC Li et al. Antimicrobial Agents & Chemotherapy 37:523-531, 1993.
However, these solutions ultimately involve isobole data presentation. The problem becomes
inordinately complex for therapeutic strategies involving multivariate interactions of more than
two agents.
C. Other Cytotoxic Agents Affecting the Cell Cycle
Other recent efforts to relate the application of chemotherapeutic agents to cell cycle
events have focused upon the role of checkpoint controls, O'Connor, ~ (1992), or the
regulation of apoptosis by manipulation of the induction of pS3 gene product or related
products of p21W;'~C~P~, WS El-Deiry et al., Cell 75:817-825 (1993).
For instance, protein kinases play an important role in neoplasia. Overexpression has
been associated with hematologic m~lign~ncy. GQ Daley et al., Science 247:824-30 (1990).
,
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Neoplastic cells may be deficient in kinase-mediated control of progression through Gl and
commitm~nt 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, ~; JA Posada et al., Cancer Comrnun. 1:285-92 (1989);
K Kawamura, Hokkaido Igaku Zasshi 69:354-71 (1994).
Use of protein kinase inhibitors in cancer control appears pr~mi~ing, since some are
very potent toxins, but are less likely to be mutagenic than conventional agents which alkylate
or crosslink DNA. CA O'Brian et al., J. Natl. Cancer Inst. 82: 1734-5 (1990), S Akinaga et
al., Cancer Chemother. Pharmacol. 33:273-80 (1994); GK Schwartz et al., J. Natl. Cancer Inst.
85:402-7 (1993). 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.
51:470-473 (1992); Cric~m~n, ~.; or (3) induce apoptosis, Bertrand et al., Exp. Cell Res.
211:314-321 (1994); DW Jarvis et al., Cancer Res. 54:1707-14 (1994).
STSP is a product of Streptomyces staurosporoL~s, 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). In addition to actions on
protein kinase C and tyrosine kin~cPs, CD Smith et al., Biochem. Biophys. Res. Comm.
156:1250-1256 (1988), STSP inhibits cyclin-dependent kinases associated with the S/G2
transition, DM Gadbois, Biochem. Biophys. Res. Comm. 189:80-85 (1993), and it can arrest
neoplastic cells in G2 phase of the cell cycle.
Work within human glioma cell lines, see Baltuch, supra, and an appended editorial
comment by PL Komblith, as well as previous reports by Schwartz, ~, and studies of its
effects on multidrug resistance, discussed herein, indicated that STSP has potential in cancer
chemotherapy. However, there have been no reports to date of clinical trials in humans. The
work in rats and dogs by RA Buchholz et al., In Cellular and Molecular Meçhzlni~m.~ in
Hypertension, p. 199-204, Plenum Press, NY (1992) and Hypertension 17:91-100 (1991),
suggest that human plasma levels of 500 nM might be testable (see Table ~. However, this
is not yet certain.
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The kinase inhibitor agents K252A, KT5720, and KT5926, have dir~ 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. Schachtele et al., Biophys Biochem Res Comm 115:542 (1968); H Kase
et al., Biochem Biophys Res Comm 142:436 (1987); S N~k~ni~hi et al., Mol Ph~ ol 37:482
(1990); WH Fletcher et al., J Biol Chem 261:5504 (1986); and HC Chang et al., J Biol Chem
261:989 (1986). They have not been tested clinically as yet.
D. Problems with Chemotherapy
Several problems are widely recognized in the current use of TCI for chemotherapy and
the other purposes. 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, ph~ rodynamics: Mech~ni~mc of drug action and the relationship between drug
concentration and effect, IN: Goodman and Gilman's The ph~ ologic Basis of Therapeutics,
8th ed., (AG Gilman et al., ed., Pc~ "~lon Press, New York, 1990). A second problem is
heterogeneous vulnerability where the inherent genetic variability of cells in a population
int~n~ l for killing can frustrate efforts to achieve absolute and specific lethality. See
Calabresi, ~. A third problem is acquired resistance, where some fraction of the cells
int~nded for damage by a TCI acquire resistance to the TCI by physiological or metabolic
adaptation, or by genetic mutation. Id.
In single or multiple clinical chemotherapies, non-specific "side-effects" may become
noxious and intolerable, resulting in significant patient morbidity. Sçe M Pirisi et al., New
Engl. J. Med. 330:1279 (1994); SM Grunberg et al., New Engl. J. Med.329:1790-1796 (1993);
O'Dwyer, ~. In clinical ph~n~otherapeutics, it is considered beneficial to increase the
selectivity or "therapeutic index" of a cytotoxic agent. Gilman, ~. Thus, 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 inte~(lecl killing or
darnage of a (iecign~tPd cell population, to non-specific side effects which produce host
morbidity or environmental disruptions. In addition, there is also a need to develop more
potent ph~ e~lticals.
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, ~ (1994). It is also a current concern that
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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 m~imllm strength may vitiate the objectives of a combined agent
protocol.
An increase in therapeutic TCI effects would be valuable during both primary induction
or adjuvant chemotherapy, R Arriagada et al., New Engl. J. Med. 329:1848-52 (1993); WC
Wood et al., New Engl. J. Med. 33:1253-9 (1994); for post-remission chemotherapy, RJ Mayer
et al., New Engl. J. Med. 331:896-903 (1994); for high dose chemotherapy followed by
autologous hematopoietic rescue, WP Peters, et al., J. Clin. Oncol. 11:1132-43 (1993);
AM Marrnont, Lupus 2:151-6 (1993); for extracorporeal purging of m~lign~nt cells from tissues
intended for transplantation, F. Sieber and M. Sieber Blum 46:2072-6 (1986); F. Lin et al.,
Cancer Res. 52:5282-90 (1992); or for debulking of metastatic turnor in body cavities, MEL
van der Burg et al, New Engl. J. Med. 332:629-34 (1995); R. Arnold, Eur. J. Clin. Invest. 20
Suppl 1 :S82-S90 (1990).
Therefore, there is a need in the art for new combinations of cell killing agents,
including new combinations of dosage strategies, whereby the first agent modulates the cell
cycle so as to maximize the toxic effect of the second agent on target cells, while minimi7.ing
the toxic effect on non-target cells. This need in the art is particularly acute in the area of
cancer chemotherapy.
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BRIEF DESCRIPTION OF THE DRAWINGS
Fig. l is a diagram of the cell-division cycle and associated major eyelins, eyelin
kinases and other and regulatory proteins.
Fig. 2 depiets bar diagrams ~ ese~ g 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,
r~snlting in "eell staeking" (B).
Fig. 3 depicts an algorithm to identify agent concentrations with operative characteristics
of RA to determine their reference point eell eyele position.
Fig. 4 depiets the results of an MTT assay showing that during apl~oxiIllately one
doubling time of 24 hours of human promonoeytie lymphoma cells, the 40% inhibitory
concentration of dThd exceeded 2 mM.
Fig. 5 depiets the results of a flow eytometrie analysis showing how cell cyele dynamies
of human promonoeytie lymphoma cells changes upon tre~tm~nt with various coneentrations
of dThd. Speeifieally Fig. 5 A shows inereasing concentrations up to 3 mM dThd and Fig. 5
B shows concentrations well below the IC40
Fig. 6 depiets a dose response curve of increasing eoneentrations of HU on humanpromonoeytic lymphoma eells. This figure indieates that the IC40 for HU exeeeds 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 eleetrophoresis experiments showing fragmentation
of DNA extracted from human promonocytic Iymphoma cells treated with > 0.5 mM dThd.
Fig. 9 is a series of flow cytometric DNA histograms showing the effect of dThd on
celleycle progression (a-c) and an immunoblot for pRb mobility reflecting phosphorylation
status (d).
Fig. 10 is an algorithm for synergistically m~tching an RA and a TCI.
Fig. 11 is a schematic showing the actions of ehemotherapeutic 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 reflectanee image of an MIS assay, testing the interaetive
effeets of dThd and STSP on human promonoeytic lymphoma cells.
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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 surnmation 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 sllmm~ion
results against two-fold serial dilutions of the dThd.
Fig. 17 depicts a rli~iti7~d 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 Iymphoma 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
fr~gment~tion in extracts from human promonocytic Iymphoma cells exposed to dThd and/or
STSP.
Fig. 24 depicts the results of flow cytometric analyses of human promonocytic
lymphomzl cells treated with STSP, showing accumulation in the G~ 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 Iymphoma 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.
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Fig. 27 depicts the results of a DNA gel electrophoresis experi~nent of DNA ex~acted
from human promonocytic Iymphoma cells incubated with TPA prior to addition of dThd and
STSP.
Fig. 28 depicts the results of DNA gel electrophoresis expçrim~ntc showing decreased
fr~gm~nt~tion of DNA extracted from human promonocytic lymphom~ cells exposed to dThd
and STSP in the presence of dCyt.
Fig. 29 depicts a dirr~ie~l~ial 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 tr~tment~ 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 m~trhes
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 diagrarn depicting dephosphorylation of p34CDC2 as a critical factor in
regulating cell movement through G2 phase into M phase.
Fig. 37A is an immlmoblot showing the effect of STSP and ~ ATA on the
phosphorylation Of p34CDC2.
Fig. 37B is an immunoblot demonstrating that STSP induced a functional activation of
cdc2 as shown both by the ability to phosphorylate histone protein (Hl).
Fig. 37C is an immunoblot showing the effect of STSP and + ATA on c-myc
es~ion.
Fig. 38 is an immunoblot showing the effect of STSP and + dThd on MAP kin~e~,
JNK (A) and ERK(B) .
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SUMMARY OF THE; INVENTION
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 ~mini~t~ring synergistic
combinations of agents for inflicting cell damage on target populations. The invention provides
an improved method for inducing cell damage by ~-lrnini~teting a restrair~ing 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 ~lmini~t~ring a targeted cytotoxic
insult (TCI) concomitant with or subsequent to the application of the RA. The invention also
relates to a microculture indicator system and auxiliary data analysis procedures for identifying,
tl~signing and using new agents as restraining agents or targeted cytotoxic insults, and for
improving synergistic combinations of existing agents.
In embodiments of the invention, the RA can be a ribonucleotide redllct~ce inhibitor,
a dihydrofolate redl~ct~ce inhibitor, a thymidylate synthase inhibitor, a DNA polymerase
inhibitor, a protein kinase inhibitor or a topoisomerase inhibitor. In addition, embodiments of
the invention include, as TCI, indole carb~oles, such as staurosporine, K252a, KT~926, and
KT5720. In a specific embodiment, the RA is thymidine and the TCI is staurosporine. In
another specific embodiment, the RA is bromodeoxyuridine and and the TCI is ~Laulu~orine.
Other specific embodiments are disclosed in the working examples.
In specific applications, the method of intlllcing 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 trt ~tment 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.
Other objects and advantages of the present invention are set forth in the following
description. The accompanying drawings and tables, which constitute a part of the disclosure,
illustrate and, together with the description, explain the principle of the invention.
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DETAI~ED DESCRIPTIO N OF THE IN~VENTION
This invention relates to a method for potenti~ting cell damage by identifying and/or
lmini.ct~ring a restraining agent (RA) and a targeted cytotoxic insult (TCI). A lc~lldini~lg
agent refers to an agent ~lminictered under conditions that retard but do not arrest downstream
progress of a target cell population through the cell cycle. Thus, the role of an RA is to
impose a dynamic retardation in cells llccign~t~d for damage.
The concept of dynamic retardation rests on key axioms regarding the cell cycle. First,
the processes of the cell cycle are segregated into linked subsets of processes ("phrased
processes"). Segregation of processes into phrases results from braking points in the forward
moment~m of the cell cycle due to a s~-cce~cion of regulatory checks. The latter include
STARTand checkpoint controls discussed by Hartwell and Weinert, suPra (1989) and by Nurse,
. T}te beginning point of phrased processes may only be cletected during perturbations
of the cell cycle or in cells with specific mutations altering cell cycle regulatory controls. For
exarnple, ~ç~ Beach et al., Current Communications in Molecular Biology, pp. 1-211, Cold
Spring Laboratory (1988). Second, the "phrased processes" behave physicochemically as an
"order dependent contintll-rn," 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). Finally, for each set of phrased processes,
momentt~n changes can be transmitted through linked complexes such as biochemical reactions,
biomolecular c~c~cle~, 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 momentttm through the cell cycle is its reference point. An
RA generates a physicochemical equilibrium shift in the biochemical reactions downstrearn
from its reference point. Thus, an RA initiates a slowing of the processes in a subset of the
cell cycle hierarchy. 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 re~ n~l~n~7 such as S phase, an RA may impose its effect at
dirrel~;llt times or at multiple reference points.
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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 con.cicting of phrased processes that are vulnerable to interaction with a TCI
that results in initiation of cell damage.
Since 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 s~h~-m~tically in Fig. 2, and can be illle~ d as a cell-cycle stack~ng.
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 potenti~t~s 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 e~ective damaging exposure (EDE), 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.
After an RA has been applied, cells remain free to move through the cell cycle during
dynamic retardation. In a cycling population, this helps ensure that all of the cells int~n-l~cl for
darnage by a selected TCI, are likely to cycle into the relevant target interval. In terms
~liccucce-l above, these are the circumct~nces that create the potential for a synergistic match
of RA and TCI.
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. In this context, 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 ~rol..iately
reduced strength or for an a~p.ol).iately limited duration. The optimal strengths or intensities
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of an RA ean be deterrnint~l experim~nt~lly for a specifie tre~tment, as cli~cucsed in more detail
below.
Restraining agents ean inelude natural produets of mierobial or other eellular origins,
a range of synthetie or semi-synthetie eompounds or ~nticçn~e oligonucleotides ~le~ign-~l to
perturb the cell cyele. Transfeeted genes could serve as direct modulators of genes controlling
cell cycle kinetics. In principle, multiple agents that can eonstrain momenh-m of the eell eyele
could be used in a combination as the operative RA. The multiple agents may act either
simultaneously or in a cascade of effeets.
E~nvironmental deprivation or physical changes can act as restraining agents.
Deprivations ean inelude insuffieiency of a nutritional factor essenti~l to cell growth or sus-
t~n~nce Physieal changes can include external temperature modulation or cell exposure to
radiant or partieulate energies, vibrational waves or other meehanieal forees.
It should be elear, however, that not every agent that detains population transit through
the eell eyele has potential to operate in the capacity of an RA. Deregulation of a checkpoint
eontrol, for example, eould abort the eell cycle or short cireuit a portion of the eell cyele, e.g.,
Powell SN et al., Caneer Res 55:1643-48(1995); Fan S et al., Caneer Res 55:1649- 54 (1995),
yet this type of event would not necess~rily ehange the physieochemical kinetics of engaged
biochemical processes in the manner of an RA.
Identifying Operative Characteristics of an RA
Fig. 3 shows a general algorithm for identifying coneentrations at whieh a given agent
ean aet as an RA and for determining a reference point of the agent. The first step is to
determine the population doubling time (TDBL) of speeified cells (i.e. a target population). The
target population ineludes proliferating cells or eells undergoing DNA repair in a population
to be damaged. They ean include, for example, neoplastic cells, hyperplastic cells, virus
infected cells, parasite infected cells, free living parasites or fungi. This step I is accomplished
by a series of manual or automated cell counts or by flow cytometry. Alternatively, it can be
~compli~hed 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 TDBL provides the time frame for ~.
detPrmining 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 TDBL. Generally, an RA will maximize synergy at a strength or
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intensity less than 40% of its inhibitory concentration (IC40) at the TDBL. An inhibitory
conePntration relates to growth inhibition as compared to the growth of u~ c~L~d controls.
Thus, an IC40 for a given RA is the co~cerltration at which treated cells show 40% less growth
than untreated controls at the TD,3L. Although the TDBL will likely be easiest to use as a
consistent standard, a convenient time interval less than TD8L may also be used, provided it is
con~ ten~ly applied. Generally, the interval should be at least greater than 50 % TDBL to
provide useful data.. Step 2 is perforrned as shown in Example 1. Fig. 4 shows the
relationship of progressively increased concentrations of an agent p.~rel~d for use as an RA,
deoxythymidine (dThd), on growth inhibition of a population of hurnan m~lign~nt cells during
the mean time of a single cycle of cell-division. As shown with one cell line in Fig. 4, during
the time for approximately one population doubling, the 40% inhibitory concentration of dThd
occurred at a concentration range of approximately 2 mM.
E~ample 2 shows the third step: p~lrol,lling flow cytometric analyses during the TDBL
at, for instance five equal divisions, using different concentrations of the agent being tested at,
for example, IC20 30,40,50. In practice, fewer analyses or concentrations may be used, for
instance when pilot data, previous experience, or scientific literature provide good indication
of expected results.
The results of the flow cytometry, shown in Fig. S, 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. ~, concentrations of dThd less than IC40 were
sufficient to retard the cell cycle kinetics. In addition, Fig. 5 shows that the proportion of the
cell population present within S phase (Fs) could become enormously increased with
concentrations of less than 1 mM dThd. In principle, these dramatic effects on the cell cycle
S phase or G2 & M phase (FG2.,kM) could be explained either by a static expansion of S phase
or G2 & M phase due to the arrest or stationary trapping of cells, or by a real increase in the
time required for each viable cell to transit each phase (i.e. a real increase of the Ts or TG2,Y~M)
As set forth in Fig. 5, these cells continued in flux through S phase, so that transit into G2 and
M phases was persistent at times up to 40 hours. These effects were interpreted as a dynamic
retardation of the cell cycling, in contrast to the cell cycle arrest or static synchronization,
which occurred at higher concentrations of dThd. (Example 7 provides additional support for
such dynamic retardation. See also Fig. 9) Thus, these steps indicated that dThd in an
~plo~liate concentration range is a restraining agent in the terrns of the invention (Step Sa).
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Any agent known to cause cell cycle arrest or static syncl.,ul~izaLion has the potential
to operate in the role of RA when used at a concentration or intensity less than that n~cP~ry
to arrest the cell cycle. When a concentration of any agent serving as an RA does produce cell
cycle arrest, then the locus of the arrest can be assumed to represent a reference point of the
RA. In this perspective, cell cycle arrest ~ selll, the limiting effective skength 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. Zielke, ~; Kufe, ~, Krek W, DeCaprio JA, Methods Enzymol.
254:114-124 1995. Based upon earlier experience with cell cycle arrest in established lines of
human Iymphoma cells, Grimley PM et al., Cancer Res 144:3480-88 (I984); Hulanicka B et
al., Cancer Res. 37:2105-2113 (1 ~77), the inventors exposed human promonocytic Iymphoma
cells (U937) to up to 3 mM dThd at intervals of up to 24 h and analyzed changes in the
dynamics of the cell cycle. Fig. S shows that the cells treated with 3 mM dThd were ~l~tz~in~(l
in transition from G, phase to S phase at 8 and 16 h so that the proportion of cells in S phase
and G2 & M (F5 & FG2,S~ M) was stabilized or reduced. This finding was con.ci~tent with
previous reports that dThd arrested cells in close proximity to the transition from G, to S phase
in the cell cycle. W Vogel et al., Hum. Genet. 45:193-8 (1978). Thus, the reference point for
dThd is estim~t~cl to be the G, / S boundary of the cell cycle (Step Sb).
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 IC40 of
HU exceeded 2 mM. As with dThd, concentrations of HU < IC40 were sufficient to retard cell
cycle kinetics. As shown in Fig. 7 and described in Example 5, HU treatment of a population
of human m~lign~nt cells increased cell cycle transit times. Thus HU, an inhibitor of
ribonucleotide re~ rt~ applied in a range of concentrations less than its IC40, increased the
Ts of the proliferating m~lign~nt cells and is an RA in the claimed invention.
As noted, any agent already known to cause cell cycle arrest or static synchronization
has a potential to function in the role of RA at an ap~lopliately reduced strength or for an
a~lo~liately limited duration. By the same token, at excessive concentrations, 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.
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For example, the IC% at which the cell cycle was arrested by HU was ~1e~. " ,i~ l 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 EIU not only detained (i.e. arrested) cells in S phase, but also killed
them. Thus, high concentrations of HU operated as a TCI.
Excess dThd, beyond levels associated with dynamic retardation, also produced
apoptosis in m~lign~nt hurnan cells. Example 6 demonctrates that induction of a~o~lo~is
resulted from excess dThd for 24 hours. Fig. 8 shows a DNA gel displaying an electrophoretic
ladder pattern typical of DNA fr~Em~nt~tion in apoptosis. Thus, differences in the actions of
low and high concentrations of these inhibitors of ribonucleotide reduct~ce, and the flow
cytometric evidence of increased Ts in cells treated with relatively low concelltldlions indic~t.-d
that a slowing of forward momentum through the S phase was involved in the process by
which each agent, acting as an RA, was potenti~ting cell damage by a TCI.
Since cells can be effectively killed by growth arrest, it is not surprising that several of
the agents that operate as RA in the present invention previously have been used as single
agents for chemotherapeutic purposes, e.g., dThd and HU. This reiterates the point that
discrimination of RAs from TCIs in the context of this invention is functional and not based
merely upon the chemical structure or physical nature of a specific agent or factor. The range
of operative agent strength and duration a~plol)liate to an RA for dynarnic retardation in a
given tre~tm~.nt regime is dettorrnin~d for each particular target cell type and TCI.
In a specific embodiment of the invention, 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. For example, as ~liccncced in more detail in the working examples, conc~ Lions
of dThd of about IC4 to about IC40 with respect to a population generation time can be used
as an RA. In pief~ d embodimentc of this invention, concentrations from about IC6 to about
IC30 can be used as the RA. Especially preferred ernbodiments of this invention use
concentrations of dThd from about ICIo to about IC25, as the R~. In other embodiments of this
invention 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/G2 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 clet~rmined by practice of this invention.
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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 Ph~rm~ol 34:63-66 (1994). HL~G-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 ~rlminictered, in various ways well-known
to one of ordinary skill in the art. For example, in various embodiments of this invention, an
RA can be ~t1mini~t~red in an in vitro setting. In vitro testing may, for inct~nrr~ be undertaken
to rapidly establish synergy between an RA and TCI agents at various strengths or duration for
a particular cell line. In an in vitro setting, the RA can be added to the target cells,
appropriately diluted in standard biological buffer, such as RPMI 1640. In an in vivo setting,
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,
subc~lt~nPous, intravesicular, intrapleural, intrapelvic, intrauterine, intranasal, hll.d~eliloneal,
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 sl-ct~ined
release or as multiple intermittent doses. In addition, the delivery of 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. Given the
disclosed invention, persons of ordinary skill in the art can Aet~rmine the most effective
aAmini~tration route of the RA dependent on the needs and reaction of the patient as well as
other factors known in the art.
Targeted Cytoxic Insult
As noted above, a cytotoxic agent is any category of agent or circ-~m~t~nce that inflicts
damage upon or inhibits growth of living cells, whether for medical, therapeutic or for any
other purpose. As defined herein, 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.
-
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Damage inflicted by a TCI may be reversible, perm~n~nt, sublethal or lethal. In most
practical applications, lethal damage with "total killing" of abnormal cells is preferred.
However, practical problems of application, including agent delivery, ph~ rokinetic factors,
and biologic limits of tolerable side-effects may dictate effective dosages that are sublethal.
Nevertheless, limited or reversible biologic darnages by a TCI can be advantageous, particularly
when the host imml-ne system can target, ~ l~r~ lLially kill, and remove damaged or abnormal
cells.
TCIs can damage or retard the growth of target cells in various ways. Table 2 shows
a number of TCIs, categorized by the estim~ttod position of at least one of their target intervals.
F.stim~tion of the target intervals is based in part upon testing performed by the inventors (see
for inct~nce Example 12) and upon the known or suspected mech~ni~Jnc of action as can be
found in the scientific literature available to those ordinarily skilled in the art.
When ~ ,- iately 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 ~p.opl;ate 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 apylo~ iate 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.
In an in vivo setting, a TCI can be delivered in solid, semisolid, liquid, or gaseous form and
by various routes. A TCI can be int}oduced by oral, mucosal, topical, intravenous, intrathecal,
intrarnuscular, 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 interrnittent doses. In addition, the delivery of a TCI may be
=
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aided by gene or nucleic acid transfection, enzyme insertion into a cell memhr~n~, 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. As with RAs, persons in the art would be able to cletermine the a~plo~liate
~lmini~trative route using routine skills.
Special means of agent (RA or TCI) deliveries, including receptor or ligand targeting
or techniques using liposome or antibody carriers, may facilitate critical targeting to the
pliate cell subpopulation in human or other multicellular hosts (RC Juliano, Ann NY
Acad. Sci. 507:89-103(1987)) and are, therefore, within the invention. In addition, 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 m~lign~nt
cells or infected cells. For example, aclarubicin, cardioprotective agent CRF-187, or
chloroquine could antagonize the cytotoxicity of etoposide. PB Jensen, Cancer Res.
54:2959-2963 (1994).
A TCI can be added to the target cells concomitant with or following the addition of
an RA. In a preferred embodiment of this invention, the TCI is added 0-8 hours after addition
of the RA. In particularly preferred embodiments of this invention, the TCI is added 4-6 hours
after the addition of an RA. However, the exact time at which the TCI is added will depend
upon the exact conditions of treatment including the nature of the RA and TCI employed and
the characteristics of the target cell population. 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.
In embodiments of this invention, before, after, or with the ~1mini~tration of an RA, a
TCI is ~rlmini~tered to the target cells in an amount sufficient to damage or inhibit the growth
of target cells. In other embodiments of this invention, before, after, or with the ~1mini~tration
of an RA, the TCI is added in arnount sufficient to kill the target cell. For example, STSP can
be added in an amount from IC,o to IC60. In preferred embodiments of this invention, STSP
can be added in an arnount from IC,5 to IC50. In especially preferred embodiments of this
invention, STSP can be added in an amount from IC20 to IC35. The amounts of other agents
can be determined experiment~lly as described below.
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Synei~;~lic ~trhing ofan R~ and a TCI
The rational selection of synergistic m~tch~c of RA and TCI is guided by the pnnrirles
of dynarnic retardation described herein. The assay system and auxiliary data analysis provided
herein can also be used to guide selection of synergistic m~tc.ht~s. Imple.,.e"~ ion 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 ~fe~ e RA on the cell cycle pc~ ion 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 presurnptive 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 of absolute and
relative strengths, in inflicting damage upon cells of the proliferating cell population int~-nrlf d
for damage.
Fig. 10 is a flow diagram showing the selection of a synergistic match of RA and TCI.
As shown, the first step is selection of an agent with RA characteristics ~ liate 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 ph~nn~rologic, 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
m~tf~.hing with an in vitro microculture indicator system (MIS) employing cultured eukaryotic
cells. 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 darnage 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 m~lign~nt cells using
colorimetric assays, however, many other assays known to those skilled in the art could also
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be used to provide a measure of growth inhibition or cell killing. ~xi~ conrentr~tions of
RA=IC40 and TCI=IC>50 are recommtonlle~l for use in the assays.
Analysis of the MIS data using new algorithms
Another aspect of the invention involves a method of analyzing the MIS data for the
~letermin~tion of agent synergy using comparisons of observed results to hypothetical sums.
Seç Berenbaum MC, Pharm Rev 41:93-141 (1989). Briefly, 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 spr~-lch~et, 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
cont~inine both ~A and TCI, with hypothetical results derived from the results for wells
cont~inine no agents and the results for wells cont~inine 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 prograrn and the results can be displayed
on monitor 1440.
The data processor may display, in a tabular forrn, 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 s~mm~tion of the effects of the TCI and RA. For
example, 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. ~i'hen the CRR equals 1 a sumrnation effect is observed (i.e.
hypothetical zero interaction); when the CRR is > 1, a synergistic effect is obser~ed; and when
< 1 an antagonistic effect is observed. Therefore, in Table 4 a synergistic effect is observed,
for example, for the concentration of 25 nM STSP and 0.19 mM dThd (CRR 1.7, at IC,4).
Both the degree of synergy and the amount of cell depletion or damage are considered in
devising a therapeutic regimen for an RA and a TCI.
In addition, using the formulas shown in Table 3A-G, 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
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hypothetical sl~mn~tion ("S") results as a function of an RA concentration in the other graph
("O/S" plots). For example, 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 m~imum difference b~lw~l~ the
plots for O and S ocurrs at approximately O.l rnM dThd.
Applying the formulas shown in Table 3A-H, the data processor may also display in
superimposed plots for each concentration of a TCI, the differences bet~,veen the O and S as
a function of ~A 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. Conversely, 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 darnage, they may be useful in other contexts, for
example, protecting cells from damage.
It is preferred to repeat these experiments with progressively shorter exposure periods
suggested because, at a hypothetical s~lmm~tion cytotoxicity of l 00%, the analysis may become
inaccurate for those concentration combinations. In addition, although the CRR table contains
all the information of differential O/S plots and O/S plots, it does not provide exact
representation of the data trends. Consequently, obtaining a concrete synergistic interaction
range and optimal synergistic match with the CRR alone might involve extrapolations of data
beyond reasonable ranges or many additional plates testing each agent in different ranges of
concentrations .
In sum, 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
~lcse-lt~lion 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 (SMA~) by plotting line graphs of the differences between the observed results and
surnmation results.
Interpretation and Verification of MIS and Data Analysis Results
In examples of this invention showing synergistic matches of dThd and STSP, for
instance Example 8, an assay for growth inhibition was used. For those combinations, the
combined cell ~l~m~ging effect was sufficiently rapid that a depletion (killing) of treated cells
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rather than just a growth inhibition could be pr~s~-mçrl Nevertheless, in general, growth
inhibition cannot be completely assessed from cell depletion in a single assay for growth
inhibition; and growth inhibition is not necessa~.-ily synonymous with structural damage to cells
by a DNA ~l~m~ging agent. ,,
The results from these assays can therefore be supplemented by me~ ring another
indicator of cell damage such as, for instance, a colorimetric assay for release of lactate
dehydrogenase (LDH) into the tissue culture sup~"l,.l;"~t Release of LDH is considered a
useful indicator of cell membrane darnage. See Li L and Lau BHS, In Vitro Cell Devel Biol
29A:53 1-536 (1993); Mitchell DB et al., J. Tissue Cult. Meth. 6:113 (1980). (See Example 9).
In performing the auxiliary data analysis step for this type of assay, instead of percent growth
inhibition, absolute amount of cell damage is shown in the CRR.
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 (No) This ratio can
be obtained by serial assays. This ratio (N/No) also may be p,~sel.l~d 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 (I-N/No). 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.
The MTT and LDH procedures demonstrated in Examples 8 and 9 detected effects on
growth or damage of the U937 cells during an interval of 18 h, repres~nting 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 obtainedproved that the dThd tr~o~tm~nt 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. In addition, as shown in Figs. 20 and 21 the effects of low
concentrations of STSP and dThd, which by themselves were reversible by washing, becarne
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irreversible once the agents were combined in synergistic m~tr.h~os In CO~.~p~ cn to CRR data,
cllm~ tive growth graphs are more revealing.
Spread sheet formulations and graphic analyses are quick, facile, and çffici~nt as
co~ cd to isobolograrn analysis in h~ tillg experimental results during the practice ofthis
invention. The results are highly reproducible and provide the optimal synergistic range for
combinations of RA and TCI. In addition, this analysis is minim~lly time con~llmin~, no
special ~ lhnental design is required, and analysis can be made even if one of the drugs is
less toxic than the other agent.
Step 4 of Fig. 10 recommends test scheduling increments within the TDBL to c,~Li,l,ize
any synergistic match. In this step, MIS and data analysis are performed for simultaneous
~mini~tration 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 potenti~ting cell damage by STSP
was 5rh~d~ dependent, see Table 7. Thus, the MIS and auxilia~y data analysis can be used
to estimate optimal times for RA and TCI deliveries.
Use of MIS to Identify Agent or Agent Concentration as TCI
The identification of an unknown agent or prospective TCI in a~lupl;ate strength and
duration both for function in synergistic match with an RA and for the determination of the
target interval in S or G2 phases is charted in Fig. 22. Examples with STSP provide a
prototype for identification and successful application of a TCI complementary to an RA. In
reference to Fig. 22, the first step of dete~nining TDBL and the second step of deterrnining the
dose/growth inhibition graph at TDBL 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.
~ses~m~nt of the relationship ûf DNA damage to a target interval ideally involves a
combination of techniques which may include conventional flow cytometry for cell cycle
analysis shown in ~xample 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. In addition, Example 12
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shows the loc~li7~tion of a target interval for a TCI, confinne~ by both DNA gelelectrophoresis and flow cytometry.
Interaction of an RA and a TCI
In S phase, the strand replication of eukaryotic DNA origin~tes at multiple and dirr~
time points. However, the replication hierarchies share important enzymes or metabolic
interme~ tes Murray AW and Kirschner MW, Science 246:614-621 (1989); Laskey et al.,
Science 246:609-613(1989). Therefore, an RA acting by initi~ting dynarnic retardation during
S phase likely imposes effects at multiple reference points.
As an illustration, Fig. 26 shows the basic principles of dynamic lt:~.laLion and the
importance of the relative hierarchical positions of the reference point of the RA and the target
interval of the TCI. In Fig. 26(b and c), 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 intt-n~ 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. Thus, a target
interval in early S phase ("A") would be included in the retardation field of RA I, whereas a
target interval further downstream in late S phase ("B") might not. In contrast, the reference
point of RA Il (Fig. 26(c)) is shown to be located downstrearn 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. In certain portions of the eukaryotic cell
cycle, particularly S phase, macromolecular replication or other critical biochemical or
biomolecular processes simultaneously occur at different positions. Thus, dynamic retardation
in S phase presumes the possibility that certain RA will impose effects at multiple reference
points. In consequence, 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.
As noted, dynamic retardation also requires movements through some portion of the cell
cycle and will not occur in non-cycling cells. In experiments with TPA, cells forced into G~/Go
phase or in a state of t~rmin~l differentiation escaped the (1~m~ging effects associated with
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dynamic retardation in the presence of a TCI. This was evident in DNA gel analyses. (See
Example 13 and Fig. 27.) This protective effect also was observed in the MIS assay.
Addition of TPA together with STSP rather than prior to the dThd was significantly less
effective than the TPA pre-tre~tment This excluded a direct biochemical inhibition of STSP
by TPA. Results thus were consistent with a physicochemical effect of dThd related to kinetic
changes during S phase.
Potentiation by dThd of STSP cell is due to dThd inhihition of RNR
As shown in Example 12, STSP was shown to induce apoptosis during S phase, yet
dThd, which acts in Gl./S was able to act as a potent RA. Concentrations of dThd > 3 mM,
which almost totally arrested U937 cells in progression from G, phase to S phase of the cell
cycle (Fig. 5), proved to be less synergistic with STSP than log lower dThd concentrations
which pF~rmitt~l continuous cell cycle progression. Although 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, particularly RNR and deoxycytidylicde~rnin~ce, are associated with the initiation of DNA synthesis in all forrns of living cells.
Elledge, ~. The biologically active R2 subunit of RNR contains a coupled iron center and
tyrosyl free radical. Tld-ls~ Lion of the messenger RNA for RNR, or an active subunit of
RNR, increases during S phase in m~mm~ n cells. ~; Bjorklund S et al., Biochem 29:5452-
58 (1990). Excess dThd inhibits RNR and deoxycytidylic de~min~e by a metabolic feedback
inhibition. Xu YZ et al., Biochem Pharmacol 44:1819-27 (1992) and Ellims PH, Cancer
Chemother ph~rm~col 10:1-6 (1982). The particular effect of dThd was ascribed tointracellular ~ec-lm~ tion of deoxythymidine 5'-triphosphate (dTTP), feedback inhibition of
ribonucleotide reductase (RNR) and deoxycytidylic ~le~rnin~e7 and a resultant depletion of the
intracellular pool of deoxycytidine 5'-triphosphate (dCTP). Elledge, ~.; Xu, ~; and
Ellims, ~. This effect of excess dThd or BrdU can be mitigated by equimolar
deoxycytidine (dCyt). Kim et al., Biochem Ph~rrn~rol 14:1821-9 (1965); Hulanicka, ~.
In the present work, the role of RNR inhibition in the potentiation of STSP by dThd
was shown by adding dCyt prior to or concomitantly with dThd. This significantiy decreased
the effect of dThd in its potentiation of DNA fragmentation by STSP (Example 14, Fig. 28).
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Inhibition of RNR by other agents also potentiates STSP
Additional known 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. In additional exp~rim~nt~, BrdU produced a
slowing of S phase similar to that caused by dThd. This was shown by bivariate flow
cytometry using PI s~ining 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 IC40 (similar to results with dThd). In a fashion similar to excess 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 con.cictently
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 pot~nti~ting 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. In the specific examples of dThd, BrdU and HU, the RA con-
centration for STSP potentiation was at least one log lower than the concentration that caused
cell cycle arrest.
The potentiation of cell damage by STSP in the presence of RNR inhibitors could not
be explained by stoichiometry and is not explained by any known pathway of metabolic or
molecular interactions. Indeed, STSP and related compounds and the inhibitors of RNA
evidently act upon enzymes with different substrates and temporal functions in the cell cycle
hierarchy. The targeted enzymes are not known to be cooperatively involved in the
biosynthesis of common metabolic int~rrn~ t~s, in the biosynthesis of nucleotides, or in the
replication or repair of DNA. Flow cytometric analyses demonstrated that a prolongation of
S phase occurred during the action of each of the additional RNR inhibitors tested, and in the
range of concentrations useful for potentiating STSP.
The specific mechanism(s) by which an RA induces retardation of cell cycle momentum
and potenti~tes the TCI activity is not fully understood for all combinations. However, for HU
action, it has been shown, based on a combination of isotopic labelling and two dimensional
gel mapping of DNA during replication, that even when HU maximally inhibited incorporation
-
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of radiolabelled dThd into DNA, by inhibition of RNR, it did not completely prevent DNA
chain initiation or elongation and allowed continued molecular action at replication forks. V
Levenson et al., Nucleic Acids Res. 21:3997-4004 (1993).
These data are consistent with dynarnic retardation during S phase prolonging a
vulnerable physicochemical or configurational state that otherwise would occur only transiently
during the processes of replication or repair of DNA macromolecules. A closely related
.~xrl~n .ti.~n, from a kinetic perspective, would be that dynamic ,. ~-l~lion shifts the
biochemical equilibrium, leading to accelerated interaction between a transient interrr.eAi~
product and a TCI.
These theoretical explanations are not e~senti,.l to the use or operation of this invention
but they are int~rn~lly consistent with the idea that the cell cycle hierarchy is comprised of
"phrased processes." They serve as a utilitarian first order approximation of events that can
aid in prediction of synergistic m~trh~c or exclude useless m~fl hes
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. (See Fig. 11.) GPV Reddy and A
Pardee, Proc. Natl Acad. Sci. 77:3312-16 (1980), postulated a close functional interrelationship
between the enzymes involved in nucleic acid metabolism and the polymerases required for
DNA strand replication, and the presence of a deoxynucleotide synthesis and polymerization
complex called "replitase" has been s~lbst~nti~t.~ Plucinski TM et al., Mol. Pharmacol.
38:114-20 (1990). For in~t~n~e, cross inhibition of thymidylate synthase activity was reported
during the use of RN~ inhibitors, such as, in particular, aphidicolin (Aph) and l-,B-D-
arabinofuranosylcytosine, (comrnonly ~lesign~.~ted 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). The action of ara-C is complex, since it may substitute for dCyt in
replicating DNA and have other effects. M. Tanaka et al., Jpn. J. Cancer Res. 76:729-735
(1985). Based on these considerations, a dihydrofolate reductase inhibitor (MTX); a
thymidylate synthase inhibitor (floxuridine); and two inhibitors of DNA polymerase cc (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. Each agent tested at concentrations less than IC40 proved
successful in potenti~ting DNA damage by STSP in the U937 cell. The CRR for each of these
agents are shown as Tables 13-16.
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In addition, low concentrations of STSP were tested as RA with the following agents
acting as TCI: Bleomycin; mitomycin C; Cisplatin (CDDP); etoposide; and daunorubicin. CRR
for these tests appear as Tables 17-21. In addition, Fig. 29 depicts O/S dirr~ lLial plots for
combinations of STSP and bleomycin.
Using the RA previously identified described herein, other chemotherapeutic agents
were tested in the combinations shown in Table 33. CRR for these combinations appear in
Table 9-32. In addition, Fig. 30 is a DNA gel showing the synergistic int~raction of dThd
with indole carbazoles other than STSP, as evidenced by DNA ladder formation. With
different TCI, including STSP, KT5926, and Cisplatin, the SM~X for dThd was very similar.
Role of Target Cell Type
When the method of the invention is used in the context of treating a patient, for
example one suffering from cancer or infection, the choice of potential RAs and TCIs should
be made so as to maximize the damage to cancer cells or infected cells, while minimi7ing the
damage to norrnal cells. The combination of the MIS and auxiliary data analysis procedures
affords flexibility for the individ-l~li7~tion of clinical chemotherapy or radiotherapy. In
addition, 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 tr~tmt~nt parameters for specific neoplasms in particular patients.
The invention may be most promising in treating mz~lign~3nt cells that seem resistant to
chemotherapy. Such r~ci~t~nce of m~lign~nt cells to chemotherapy is often associated with
deletions or mutations in the pS3 gene. Rouach E. et al., Mol Cell Biol 13:1415-23 (1993);
Fisher, supra; Lowe, ~; Lotem, ~; Fan, ~. Large cell Iymphomas have been
occ-lrring with increasing frequency in patients with immunodeficiency conditions in the United
States, and mutation or absl?nt e 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 Cornmun 201 :266-83 (1994). This was
confirmed by the inventors using a comparable immunoblot methodology and a pantropic
antibody to p53 that was obtained from Oncogene Sciences. The U937 cells, therefore, were
useful to test agents as RA and TCI because the cells are of histiocytic (large cell) m~lign~nt
Iymphoma origin, Sundstrom, supra, and negative for p53. See Calabresse, ~.
In synergistic matches of dThd and STSP, effective killing of U937 cells occurred.
Delayed proliferation assay results supporting such effective killing (Example 10) were
~ - ~ ~
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confirmed in clonogenic (Example 15, Fig. 31) and turnorigenic (Example 16, Table 34)
assays.
In addition, synergistic m~teht~s were effective in another cell line (HL60), also reported
to have homozygous abnormalities in expression of the p53 gene. See, Calabresse C et al.,
Biophys Biochem. Res. Com~n. 201:266-8 (1994). Protein kinase inhibitors are of interest for
human chemotherapy, since some are capable of d~m~in~ neoplastic cells that have evolved
mech~ni~m~ for drug r~si~t~n~e, Sarnpson, ~; Versantvoort, ~; Hiyarnoto, ~; Utz,~_, and mutagenic darnage to DNA is minimiz~-d in comparison to alkylating agents.
The present invention can help to circumvent the dilemma posed by m~liEn~nt cells that
have evolved drug resistance me~h~ni~m~ both by intensifying the cl~m~ginE 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. Tnt~ncification of TCI
effects can also be valuable in treatments of m~ n~ncy involving bone marrow transplantation,
where either the patient or the extracorporeal tissue can be subjected to a more rigorous
regimen of m~liEn~nt cell eradication.
Effects of dynarnic retardation were observed in a number of other tissue culture lines
developed from human m~liEn~nl~ies shown in Table 36. The effects of Aph as RA and STSP
as TCI on a line of m~lign~nt cells of epithelial origin is illustrated in Fig. 32. This cell line
(C33A) oriEin~ted from a human cervical carcinoma, Auersperg N et al., J Natl. Cancer
Inst 32:135-148 (1964) and is negative forp53. Shivastrava, suFra.
A useful result sho~,vn by the testing in various cell lines was that once the SMAX of an
RA for a particular synergistic match and cell population was established, the sarne range of
RA concentrations operated successfully with other TCI in classes complementary to that
particular RA. Thus, the conditions for SMAX must be established for each specified population
of cells, since differences in both the IC40 and the SMAX were observed with different
populations of m~ n~nt cells.
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. These
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examples and others are listed in Table 33 and the pertinent combined results ratios obtained
from MTT assays are provided in Tables 38-42.
The tests of RNR inhibitors shown in Table 8 provided the initial evidence that an
agent operated as an RA when used in a range less than the IC40 with respect to the growth of
human promonocytic lymphnm~ cells (U937). Table 37 shows a similar relationship between
the IC40 and the SMA~C for several different cell lines with synergistic m~tch~c involving either
dThd or Aph and STSP. These results also provided evidence that differences in the selectivity
of agents acting at operative concentrations of RA can be exploited to direct darnages to a
particular m~lign~nt cell population by an ap~u~opliate selection of RA and complementary TCI
for synergistic m~tch~. This applied approach must be based upon a practical knowledge of
differences in the chemosusceptibility of particular norrnal or m~lign~nt cell populations in the
treated host.
Cellular incorporation and metabolism to deoxythymidine triphosphate (dTTP) is
m~ ttod by thymidine kinase (Tk). Thus, the RA effect of dThd in a given cell population
depends upon a Tk+ phenotype. Certain neoplastic cell lines are deficient in Tk and, therefore,
relatively resistant to excess dThd. This was exemplified in tests of the Burkitt's lymphoma
cell line Raji: the IC40 for dThd was > 4 mM, compared to an IC40 of ~ 1 rnM in U937 cells.
The clinical treatment of a human m~lign~nt Iymphoma with characteristics of Raji cells using
dThd as the RA would be problematical because the relatively higher concentrations nrce~c~ry
for dThd to operate as an RA might produce intolerable side effects. A solution to this type
of transport enzyme deficiency is substitution of an alternative RNR inhibitor, such as HU,
which enters cells by diffusion. ~ Yarbo, ~. Fig. 35 shows that HU produced a
synergistic match with STSP in the Raji cells.
Dynamic retardation also proved successful in potenti~ting STSP damage to cells of
m~ n~nt epithelial origin. Examples included the cell line C33A, which originated from a
human cervical carcinoma and is negative forp~3, Shivastrava, supra, and the cell line ZR-75-1
which origin~ted from a human breast carcinoma. Engel, Cancer Res. 38:3352-3364,4327-4339 (1978). 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.
Thus, 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)).
In principle, cells susceptible to the effects of dynamic retardation may be of any type,
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including eukaryotic, prokaryotic and archae~cterizll; or~ni7~(1, free-living, and parasitic, or
growing in living hosts of the animal or plant or growing in m~nllf~rtllred environm~ nt~ This
invention can be expected to increase the ~l~m~gin~ 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 m~nuf~tllred environments. Thus, the invention may potentiate
damage inflicted upon an entire population of living cells or upon an entire plant, animal, or
other living org~Tli.cm, 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 delinP~te~ environment.
Clinical Strategy for Delivery of an RA and TCI
Application of a TCI for medical therapeutic purposes requires that its ~l~m~ging effect
be inflicted upon the ap~loy-iate 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 i~lentific~tion of synergistic m~t~h~c
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 ~plol,liate point for introduction of the TCI. See O'Dwyer, ~; Schilsky RL et al.,
Cancer Res 46:4184-88 (1986); Donehower RC, Hydroxyurea, In Chabner BA (ed.)
Pharmacologic Principles of Cancer Tre~tment, pp. 269-75, Philadelphia, PA, Saunders (1982);
Sessa, ~. 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 minim~lly cytotoxic and long act-
ing while the TCI is highly cytotoxic and short acting. In one such scenario, 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 ~plupliately scheduled
multiple dose regimen.
Confirm~tion of in vitro data by clinical trials, nevertheless, may require other types of
pre-clinical testing including animal studies to calibrate agent dosages more precisely and to
divulge unanticipated toxicities. See AF Gazdar et al., J. Natl. Cancer Inst. 82:117-24 (1990);
BA Chabner, J. Natl. Cancer Inst. 82:1083-85 (1990); MR Boyd, In Cancer, Principles and
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Practice of Oncology Update, pp. 1-12, ed. DeVita et al., vol. 3: Lipincott, Philadelphia (1989).
Dosages are calibrated in relation to a cell mass or number, the weight, surface area or blood
volume of a host.
A "ph~rm~rokinetic elimin~tion strategy" would work very effectively with either dThd,
Schilsky, ~, HU, Donehower, ~r~, or Aph, Sessa, ~, as the RA. Significant blood
and cerebrospinal fluid levels of dThd or HU have been achieved with continuous intravenous
infusions and levels can be m~int~int~1 for several days. Blumenreich MS et al., Cancer Res
44:2203-07 (1984); Schilsky, ~_. A major advantage of HU is that it is readily absorbed
after oral ingestion and blood levels peak in 2-4 hours. Donehower, ~. It also distributes
into the cerebrospinal fluid. When Aph is ~lminictered by continuous infusion, peak plasma
levels of 3 llg/ml can be achieved with minim~l toxicity. Sessa, supra.
The ultimate choice of timing and routes of ~-lmini~tration of an RA and TCI depends
upon specific ph~rrn~codynamic characteristics of absorption or metabolism of each agent in
a particular biologic system.
Direct methods for evaluating change in kinetics of the cell cycle in tissues removed
from a human host use DNA-specific labels and flow cytometry. Riccardi A. et al., Europ. J.
Cancer 27: 882-7 (1991); Mit~llh~hi et. al. Cancer 70:2540-6, 1992; Raza A. et al., Arch
Pathol. Lab. Med 115:873-9 (1991); Spyratos F et al., Cancer 69: 470-5 (1992); Kuo S-H and
Luh K-T, Acta Cytol. 37:355-7 (1993); Am J Surg Pathol 17:1003-10 (1993).
A major advantage of the present invention, however, is that such knowledge is not
cs~enti~l for some degree of synergy to be achieved, even with simultaneous application of the
RA and TCI. In the body of a living host, catabolism or elimination of any agent must be
gradual. Thus, the optimal blood concentration level of an agent might be reached at some
point during the ph~ okinetics of elimination.
Major advantages of the invented method are that the range of concentrations of agents
for effective synergistic actions can be relatively broad as indicated by SMA~ data shown herein,
and that the target interval during which the effect of a TCI will be maximum need not be
rigorously restricted.
A number of the agent concentrations found to be operative as RA in synergistic
m~tches, as demonstrated by MTT previously had been utilized as single chemotherapeutic
agents in humans or in animal trials. Several of these agents have been tested in dose ranges
that produced, or could be estimated to produce, plasma levels well above the in vitro ranges
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sufficient to achieve SMA~ shown by the MTT. Moreover, many of these agents would likely
be safe for therapeutic use, particulary at the lowered dosages made possible through practice
of this invention. Table 3~ lists examples of relevant data previously reported in the 5ei~ntific
or medical lil~,ldLLu~. References are Blumenreich, ~; Belt RJ et al., Cancer 46:455 (1980);
Allegra CJ et al., In Cancer Chemotherapy Principles and Practice, pp. 110-153, ed.
('h~hn~r BA and Collins JM, JB Lippincott Co. Philadelphia (1990); Sessa, ~; Calabressi,
supra; Buchholz, ~. The plasma levels shown in Table 35 1e~GSe11l values directly
measured in humans and published, or values estim~teA from reported dosages and blood
volume of dogs.
With this invention, 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. Although STSP and
homologues or analogues have been considered as anti-neoplastic agents, Schwartz, ~,
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 ~lecign~cl for the killing of a complex organism, extermination of agricultural or
domestic pests, selective poisoning of any number of living unicellular or multicellular
org~ni~m~ including any member of the animal and plant kingdoms, or cells infected by
mycoplasma, viruses, prions or other infectious agents may be possible.
In other specific embodiments of the invention, when the RA is ara-C, the TCI is not
dGuo. In other embo-liment~, when the RA is dThd or BrdU, the TCI is not ara-C. In still
other embo~liment~, 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. In further
embodiments of the invention, 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.
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The follo~,ving other uses are also contemplated:
(1) uses in chemotherapy or radiant energy therapies to e~ct~rmin~te neoplastic cells
in the human body or in tissues removed for autotransplantation or h~Lelv ,a~ nt~tio~;
(2) uses in immunotherapy or transplantation me~ .ine to control the excessive
proliferation of abnormally destructive immllnncytic clones, such as in graft vs. host reactions;
(3) uses in fertility control including destruction of germ line or conceptus tissues;
(4) medical anti-microbial therapies, systemic use with anti-viral, anti-bacterial or
anti-fungal agents;
(5) medical anti-malarial or other anti-parasitic chemotherapies;
(6) procedures for preventing in vitro c-~nt~rnin~tion of cell or organ cultures by
microbial infections;
(7) killing of neoplastic cells in vitro prior to autotransplantation of bone marrow;
(8) destruction of non-neoplastic but functionally abnormal cell clones, e.g.,
excessively proliferating immnn~ cells (autoimmunf disease) and psoriatic epidermal cells;
(9) to guide the synthesis or identification of new classes of agents which can be
applied as RA or TCI, and to lead to new utilizations of presently available; and
(10) to effect a biochemical organ ablation, e.g., thymectomy or prostatectomy.
The strategy designed to potentiate TCI actions during the somatic cell cycle may prove
to exert similar effects in germ line cells undergoing complete or reduction divisions, so that
the methods employed can in principle be applied to fertility control or sterilization.
The following examples set forth various aspects of the invention.
-
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Example 1
This example shows the relationship of progressively increased concentrations of dThd
to growth inhibition of a population of human m~ n~nt cells during the mean time of a single
cycle of cell-division.
Microcultures of human promonocytic lymphoma cells (U937) ("the U937 cells")
originally obtained from the American Type Culture Collection (CRL 1593) and later grown
by 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% N~ e~ and
antibiotics). The volurne of medium per well was 100 ~1 and the cell nurnber per well was
about l x 105. The plate was incubated at 37~ C for 24 hr.
A dye, 3-[4, 5-dimethylthiazol-2-yl]-2 ,S-diphenyl-tetrazolium bromide (MTT), was used
as a chromogenic indicator of the metabolically active cell mass. Viable cells metabolize the
dye and accurnulate a reduced form~7~n product (blue color) which is sohlbili7~ for
colorimetry. Mossman T, J. Im~nunol. 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, ~. The MTT method
is ideal for cells tested in suspension growth, and can also be used for some cells growing in
monolayers. It correlates well with in vivo biologic activities of agents used in clinical
chemotherapies and has been shown to be an apl,lu~l;ate indicator of cell sensitivity for clinical
applications of agents intended for use in chemotherapy. Alley, supra; Hofs, ~; Wilson JK
et al., Br. J. Cancer 62:189-94 (1990); Plowman, ~
Thirty ~Ll of 0.5% MTT in ph~sph~t~. buffered saline was added to each microculture
well at the appropriate time. After incubation at 37~C for 3 hr, 120 !11 of an aqueous solution
of 10 % SDS with 0.01 N HCI was added to release and dissolve formazan product. This was
accomplished by overnight incubation of the plate at 37~C. After careful mixing of the
microwell contents by placing the plates on an orbital shaker for 5 minutes the absorbencies
were quantitated by spectrophotometry at 570 nm with a Ceres UV 900 HDI microwell plate
reader (from Biûtek Instrurnents Inc, Winooski, VT 05404-0998). Absorbance data was
norrn~ d to a blank well cont~ining all reagents without cells and was collected semi-
~ntûm~tically.
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
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have been depleted or where cell growth is inhibited stain relatively weakly in comp~ri~on to
controls. In this method cells adherent to microwells are fixed by 5Id-1itio~l of 25~11 of 4%
form~ hyde 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. After
adding 100 111 of 50% ethanol to each well, the plates are ~git~tefl at slow speed on a Mini-
Orbital shaker for 20 minl-tes After an additional 40-60 minl-te~ adsorbance in each well is
measured on an ELISA reader at 570 nrn. Example 11. This procedure is valid when viable
cells adhere to plastic (see Grando SA et. al. Skin Pharmacol 6:137-47 (1993)).
Fig. 4 shows a plot of percent of growth inhibition for U937 cells as a function of
tre~tTn~nt with increasing concentrations of dThd.
Example 2
This Example shows flow cytometric analysis of the U937 cells exposed to a range of
concentrations of dThd below the IC40. Flow cytometric analyses were performed with a
Coulter Epics System (purchased from Coulter Corp, 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). ~ Shapiro NM, Practical Flow Cytometry, Man R Liss, NY
(1988); Nicoletti I et al., J Immunol Methods 139:271-9 (1991). Analyses of 10,000 nuclei
were performed with gr~ ted concentrations of dThd at serial time points. Histograrns of PI
fluorescent emissions at 675 ~ 10 nm were used to discriminate cell cycle fractions of the
target population. The results are shown in Fig. 5. The proportion of cells in each phase of
the cell cycle is le~l~sellled by stack bars above the X-axis as a fraction of those cells with
intact DNA structure. The fraction of cells with depleted DNA in each sample is le~l~senl~:d
below the X-axis as a percentage of the total cell number. Cells with depleted DNA are
presumed apoptotic. Crompton ST et. al. Biophys. Biochem. Res. Comm.183:532-537 (1992).
Each stack bar in Fig. 5 represents the cell cycle distribution of a population sample of
> 10,000 cells, analyzed for each indicated dThd concentration at indicated treatment times.
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Example 3
This Example 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 syncl~lu,li~Lion of human lymphoma
cells in the cell cycle hierarchy. Flow cytometric analyses were perforrned as in Example 2.
The results are shown in plot A of Fig. 5 from Example 2.
Example 4
This Example 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 Mrr assay was performed as in Example 1 except that the agent tested
was HU. The results, shown in Fig. 6, demonstrated that the IC40 for HU exceeded 2 mM.
Example ~
This Example 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
IC40 and sampled for flow cytometry at intervals of 8, 16, and 24 hours. Flow cytometric
analysis was performed as described in Example 2.
As shown in Fig. 7, in a range of 0.125-1 mM HU, treated cells continued to enter S
phase for up to 16 hr and no more than 20% of cells became DNA depleted. At the lowest
concentration of 0.13 mM HU there was evidence of a minim~l population movement into G~
and M phases from the exr~n-led S phase. This was consistent with a dynamic retardation of
S phase, as is also shown in Fig. 4 in cells treated with dThd.
At concentrations of HU > 1 mM, shown in Fig. 7 (see the 2 mM stack bar at 24 hr),
a major increase in cells with depleted DNA became evident. This reflected significant cell
darnage by high concentrations of HU as the m~lign~nt cells transited S phase.
Similar cell cycle changes to those obtained with low concentration of HU were
obtained with the RNR inhibitors dAde and dGuo when used in ranges below the IC40. At
a~ l;ate concentrations, each of these RNR inhibitory agents functioned as RA.
Example 6
This Example demonstrates that excess dThd produced apoptosis in m~lign~nt humancells.
Some ll~m~gin~ effect of > 1 mM dThd in human Iymphoma cell cultures was indicated
from earlier studies, e.g., Lockshin A et al., Cancer Res 44:2534-2349 (1984); Peterson AP et
=
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al., Basic Life Sci 31:313-34 (1985). Nevertheless, 3 mM dThd was used as a "mock control"
to emulate effects of the cytokine illt~r lOn on growth inhibition and static synchronization of
human m~ n;~nt lymphoma cells in the Gl to S phase transition, since it is known to cause cell
cycle arrest and is currently used for this purpose. See Grimley, ~. Krek W and DeCaprio
JA, Methods Enzymol 1995;254:114-24 (1995). During those tests, unexpectedly, the cell cycle
arrest caused by 3 mM dThd itself was accompanied by a pronounced DNA fr~gmP-nt~tion.
Samples of the U937 cells were exposed to a series of concentrations of dThd for 24
hr. Their DNA was isolated by lysis of cell samples in TE buffer cont~inin~ TRIS 10 mM,
EDTA 10 mM, SDS 0.5% and proteinase K 200 ~Lg/ml at 50OC for 2 h. The proteins were
precipitated in a lM solution of NaCl and centrifuged at 2,500 x g for 30 min at 400C.
DNAse free RNase (25 llg/ml) was added to the supernatant and incubated for 30 min at 370C.
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
slllrh~te and 0.05% (w/v) bromophenol blue. Samples were applied to se~ 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. ~ Tomei, ~; Obeid, supra; Gold, supra.
Example 7
This Example shows that low concentrations of dThd act as an RA to retard S phase.
Whole cell extracts (U937 cells) were prepared in an SDS 2X sample buffer (lOOmMTris.HCI (pH 6.8), 200mM DTT and 4% sodium dodecyl sulfate) and boiled for 5 minlltes
Protein concentrations were assayed, and samples of 100 ~g of protein per lane were applied
to 7.5% SDS-polyacrylamide gels (PAGE). Resolved proteins were transferred to Immobilon
P (Millipore), and pJ~b was localized with monoclonal anti-p~b (Santa Cruz, IF8) and a
chemilllmin~scence procedure (Amersham ECL). Samples for flow cytometry were prepared
and flow cytometry was performed as described in Example 2.
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Fig. 9 contrasts the effects of low and high concentrations of dThd. Whereas 0.2 mM
dThd permitted continuous cell transit, 3 mM dThd arrested cell cycle progression at G,/S.
For flow cytometry Fig. 8 a-c, samples were: (a) untreated controls; cells treated with (b) 0.2
mM dThd for 4 hrs showing that Fs was decreased, but that the cell distribution r~n~inPd
unform, or with (c) 3 rnM dThd, showing progressive depletion of the Fs with a trough in early
S phase (position of arrow), both indicative of G,/S phase arrest. These finflingc were
concictent with dynamic retardation of S phase transit by low dthd conl~entr~tions. The
irnmunoblot (d), shows protein mobility differences of pRb, specifically an zlc c.lm~ tion of
p~bP (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 sa~nple treated for 4 hours
or 1 1 hours.
Phosphorylation of pRb is f'CSenti~l to progression of the cell cycle from G~ into S
phase. Wiman KG FASEB J 7:841-5 (1993). In a hypophtl~phorylated state, pRb fails to
release the transcriptional activation factor E2F required for initiation of S phase (see Fig. 1).
Therefore, hypophosphorylated p~bP is associated with G, phase, while phosph~rylated pRbPPP
is associated with S phase transition. At the relatively low con~Pntration of 0.2 mM dThd,
shown by flow cytometry to be associated with S phase retardation, the phosphorylation of 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 Gl/S phase arrest,
pRb phosphorylation was inhibited.
Example 8
This Example shows how the biological interactions of dThd and STSP were quantitated
by an in vitro microculture indicator system.
Flat-bottom 96-well plates of tissue culture quality (Corning or Costar) were set up with
two-fold serial dilutions of each agent to be tested, e.g., the RA and TCI, using a multichannel
pipettor with adjustable volume, e.g., Costar or Rainin, to perform serial mixing. This set-up
is diagramrned in Fig. 12. Two rows and two columns in each plate were reserved as single
agent controls (RA or TCI), four wells were reserved in the lower right corner as untreated cell
controls and one well was reserved in the upper left corner as a blank with reagents only. In
each plate, a total of 59 wells then contained bivariate agent combinations, the bivariate serial
dilutions.
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Agents were serially diluted by serial mixing in two separate plates or series of wells,
and then were combined prior to addition of a fixed volume and density of the U937 cells in
a suspension calculated to deliver about 1 x 105 cells per well. In this F.x~mple, the dThd
concentrations and ratios to STSP were varied in two-fold steps along hori711nt~1 and vertical
rows. Thus, the diagonal axis from top left to bottom right showed constant ratios, while the
diagonal axis from bottom left to top right shows four fold increasing ratios of TCI / RA.
When present, a maximum of synergistic effects of RA and TCI usually have been found along
this diagonal.
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 104 cells per
microwell to provide optimal sensitivity of detection of cell damage in subsequent colorimetric
assays. The plate was incubated at 37OC for 4 h before the addition of serial dilutions of the
STSP. The final volume of medium was 100 ~ll/well. The plates were further in-uh~fed at
37OC 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 .ligiti7~d reflectance image of the actual plate used for this Example
of BYSD as captured by a Scanmaker 2 (Microtek) with Adobe photoshop software in a
M~elcintosh Quadra 800 and transferred to Aldus Persuasion 3.0 for labelling and printing at
300 dpi. In this MTT assay, norrnzlli7~-d absorbance data was collected semi-automatically from
each microculture well with a Ceres UV 900 HD1 plate reader, from ~iotek Instruments Inc.,
Winooski, VT 05404-0998, stored in an EIA file under the Ceres 900 program, transferred in
comma delimited format to a floppy diskette, and imported into a spread sheet program
(Borland Quattro Pro) for data manipulations. Table 4 shows the imported MTT data as
manipulated into a tabular format using an algorithm presented in Tables 3A-E. Table 4
shows data for dThd and STSP mathematically tr~n~l~ted 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).
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Example 9
This Example ~ ml~nc1Tates a correspondence of cell depletion, or growth inhibition, as
qll~ntit~tecl by MTT assays with cell darnage qll~ntit~ted by an assay for lactic dehydrogenase
(LDH) enzyme release. As a means to assure the significance of the MTT ~say in Example
8, results were co.l.~ucd in duplicate assays for Ml~ and release of LDH.
The MIS set up was identical to that described in Example 8. The relative activity of
LDH released into sup~ t~nt from each microculture well was ~letecterl by a coupled
enzvmatic assay, Decker et al., J. Tmm~lnt~l Methods 15:61 (1988), in which the chromogen
2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazoliurn chloride (INT) is converted to a red
INT-forrnazan product by NADH in the presence of diaphorase.
In this procedure, after incubation and at the time for assay, 50 111 of fresh medium was
added to each microculture well prior to assay and microcultures in the entire 96 well plate
were simultaneously sedimented at 400 x G for 10 min in a Becton Dickinson refrigerated table
top centrifuge using a plate carrier. A 50 111 sarnple from each microwell then was removed
carefully and transferred to a corresponding well in a clean 96 well plate. LDH activity was
qll~"~ cl by means ofthe CytoTox96 assay, Promega Corp., Madison, WI 53711, according
to the m~n~lf~rturers instructions. In tests to determine the total LDH in each microwell, 10
~11 of lysing reagent (from the CytoTox96 assay kit) was added to the fixed 100 ,ul volume with
cells and medium prior to the above step. UV 900 HDl plate reader. The LDH activity
proved stable in refrigerated samples for at least 5 days. Fig. 17, shows a digitized reflectance
image of the LDH plate (captured as in Example 8, Fig. 12).
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 104/cell in a test plate. Particularly for the latter purpose, all
of the cells in each microculture well were totally Iysed with Promega Iysing reagent prior to
the colorimetric measurement.
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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 (exactly as in
Example 8) were washed. Seventy-five percent of the well volurne was removed ~,vithout
disturbing the se~lim~ntPd cells, and medium was added to yield a 1/~ dilution. In three
additional washes and cell se~iim~nt~tions~ 50% of the mediurn 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 w~hings) including the untreated control cells, was then
assayed by the MTT method (total elapsed time of 70 hours). CRR are shown in Table 6.
As an adjunct to the delayed proliferative assay, we utilized an "imme~ te plate" to
qu~ntit~te the cell mass at the beginning of the experiment (No = original cell mass). The
"imm~ te plate" was prepared in parallel to the usual "test plate" shown in Fig. 12. It
required no more than a single column of microwells filled with 50 ~l of growth mediurn each,
and aliquots of cells in 50 ~ll identical to those transferred to the usual "test plate". The
imrnediate plate was incubated at 37~C to complete the MTT assay for the same time as used
for the test plate (i.e. 3 hr).
Fig. 20 is a plot of the U937 cell population growth in various concentrations of STSP
as a function of dThd concentration. Taking the mean final cell mass at 70 hours as N, each
data point represents a ratio of the mean cell mass of dThd-treated cells as fraction of N/No.
A ratio of N/No = l indicates no change in original cell mass (i.e. no growth of the targeted
population). A ratio of N/No > 1 indicates overall growth of the targeted population. A ratio
of N/No < l indicates overall population loss (cell killing).
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 (No) as ~leterrnined
in an "immediate plate". Taking the mean final cell mass at 70 hours as N, positive
percentages represent the % effective cytotoxicity (EC%) expressed as l-N/No and converted
to a percentage, in relation to concentration. Thus, EC obtained only for ratios of N/No < 1
(i.e. positive percentages). Fig. 21 shows that at 70 hours after treatment the m~ximllm
effective cytotoxicity for cells treated with 175 nM STSP was about 85%. The same result
could be achieved with 25 nM STSP in the presence of O.l mM dThd or 13 nM STSP in the
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presence of 0.5 mM dThd. Comparison to Fig. 20, shows that 0.1 mM dThd itself had
minim~l effect on cell growth as compared to controls.
More than 3mM dThd was required to achieve effective cytotoxicity of just 40% (EC40).
Example 11
This Exarnple 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 be~innin~ of tre~tm.ont
with dThd. The total duration of STSP tre~tm~nt in each plate was identical. Cnn~ictent with
a dynarnic 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. However, the consistent absence of a linear dose effect relationship and the greater ac-
tivity in the range of 0.05 mM-0.5 mM dThd as compared to effects of concentrations > 0.5
mM supported the concept of a potenti~ting effect by dynamic retardation rather than by a
stoichiometric metabolic competition. Table 7 shows the results of schedule dependent testing.
Example 12
This Example confirms that STSP induces apoptosis during S phase of the cell cycle
and that DNA fr~gm~nt~tion was l~:nh~nce~l by dThd.
The U937 cells treated with 50 nM STSP for 0-12 hours were analyzed by DNA gel
electrophoresis as described in Example 6. Fig. 23 is a DNA gel demonslldLillg 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 ~cc~ntll~t~A by dThd.
Using standard flow cytometry as described in Example 2, it was observed that U937
cells treated with 50 nM STSP gradually accumulated in the G2 and M phases (Fig. 24). This
effect was evident begirming at 4 hours after treatment and is shown at ~ hr and 24 hours after
lllcnt. In Fig. 24, the proportion of cells in each phase of the cell cycle is represented by
stack bars above the X-axis as a fraction of those cells with intact DNA structure, while the
fraction of cells with depleted DNA in each sample is represented below the X-axis as a
percentage of the total cell number. This fraction of presumed apoptotic cells was 4~ ~d
by the relatively decreased PI fluorescence of cells during flow cytometry (see Cromptons,
~). An accolll~3a.lying reduction of cells in S phase indicated that the accumulation of cells
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observed in G2 and M phases might le~les~llt survivors of a more numerous population that
became depleted during S phase. Thus, 50 nM STSP behaved as a TCI with a target interval
in S phase.
In a more sophisticated use of flow cytometry, DNA strand breaks were c~etected in
nuclei by means of termin~l transferase and homopolymer tailing as described by Gorczyca W.
et al., Cancer Res 53:1945-1951 (1993). Samples of 1 X 106 cells were fixed in 1% buffered
formaldehyde at OoC for 5 min~ltec, washed with PBS, resuspended in 70% ethanol and stored
at -200C for ~8 hours. Cells were rehydrated in PBS, then suspended in 100 ~Ll of cacodylate
buffer and 2.~ mM CoCI2 to prepare them for the termin~l transferase reaction. Biotin-labelled
16-dUTP and terrnin~l transferase (Boehringer Mannheim, GmbH) were added to provide the
final conce~ dLion of 0.5 nM and 500 units/ml, respectively, and reacted with the cells at 370C
for 30 minutes. Cells were rinsed in PBS, resuspended in a 4X saline-citrate buffer with 0.1%
triton X100, 5 % (w/v) non-fat powdered milk and 2.5 ,ug/ml FITC-labelled avidin, Boehringer
M~nnhf im, ~, and incubated at room temperature for 30 minutes. Excess avidin-FITC was
removed by washing the cells with PBS cont~ining 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 fr~gment~tion to establish that S phase is a target interval of STSP. Each histogram
shows a 3-(1im~n~ional 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
propidiurn 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 incorporation at sites of DNA fr~gm~nt~tion (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
fr~gm~nt~tion, 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 minim~l apoptosis.
The large increase in cells positioned in G2/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
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labelled cells after 4 hours of tre~tntent c~f 0.2 M dThd followed by addition of 25 nM STSP
lasting an additional 7 hours.
Similar results are shown for cel]s treated similarly with two other indole carb~oles
(KT5926, Figs. 25(f and g) and K252a, Figs. 25(h and i). Thus, the presence of dThd was
associated with a dramatic concomitant reduction in the accumulation of cells in G2 and M
phases compared with that produced by STSP or related indole carbazole compounds alone.
Flow cytometry, with or without a DNA probe is almost es~er~ti~l for defining the target
interval even when a DNA ladder pattern is shown, since DNA damage might be initi~ted in
one phase and expressed in another. Zc~eri ZF et al., FASEB J 7:470-478 (1993); Fisher,
supra; Cotter TG et al., Anticancer Res 12:773-9 (1992); T.in(lenhoi ML et al., Cancer Res
55:1242-7 (1995). If DNA fragmentation is not identified by DNA gel electrophoresis, other
techniques such as employing Hoechst dye may be employed to analy~e the cell cycle position
of DNA darnage. Sun XM et al., Analytic Biochem. 204:351-356 (1992); Chen U,
Immunology 85:366-379 (1992).
Example 13
This Exarnple shows that detention of cells in G, or Go phase of the cell cycle reduced
potentiation by dThd of STSP-in-lllced apoptosis.
Tre~tment of human promonocytic Iymphoma 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, ~. In this example, U937 cells were
differçnti~t~ d by treatment in suspension with 10 nM TPA (Sigma Chemicals) for 48 hours.
After 48 hours in floatation, followed by 24 h of substrate attachment, the fraction of cells in
S phase (Fs) was analyzed by flow cytometry as in Example 2, and results showed that TPA
forced these cells into Gl or Go phases of the cell cycle.
After washing, the cells were placed in fresh growth medium, and treated + dThd (0.19
mM) or I STSP (25 nM) for 7 more hours. DNA extraction and gel electrophoresis was
pel~~ ed as in Example 6. Fig. 27 shows that TPA differentiation significantly reduced dThd
potentiation of DNA fragmentation during STSP treatment.
Example 14
This example shows that potentiation b~,y dThd of STSP damage was due to dThd inhibition
of ribonucleotide reductase (RNR)
-
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In this Example, dCyt was added into cultures of U937 cells treated with dThd and I
STSP. MTT assays were performed as described in Example 8 and DNA extraction and gel
electrophoresis were performed as described in Example 6.
Fig. 28 shows decreased DNA fragmentation in samples subjected to both dThd and
STSP when they also were treated with dCyt.
Example 15
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 m~lign~nt lymphoma
cells (IJ937).
F.limin~tion of a targeted cell population by a cytotoxic agent or agent combinations can
not be assured if cells with generative capacity survive. The clonogenic assay in soft agar or
methylcellulose is a practical and useful means of determining long-terrn effects of cytotoxic
agents on a m~lign~nt cell population, since m~lign~nt cells are often capable of growing and
forming colonies in these media. The colony forming efficiency of any line or population of
m~lign~nt cells must be established from the colony count in relationship to the inoculum
number and varies over a wide range. With U937 cells, control studies showed an efficiency
of at least 50% after 3-5 days of culture in methylcellulose. In this method, stock cell
suspension was prepared in fresh growth medium (8 x 105 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 50~1 of selected concentrations of dThd
and/or STSP were transferred to d~ign~ted 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. and made up in a series of sterile tubes: (a) 0.4 ml MC stock
solution, (b) 50 ,ul of NuSerum, (c) 2 x 104 cells from the treated cell suspension from the 24-
well plate (about 250 1ll) and (d) complete growth medium to make a final volume of 1 ml
(final cell density = about 2 x 104 cells). A syringe with 18 g. needle was used to transfer
load lO0 ~11 of each sample the final cell preparation to each of triplicate wells in a 96-well
plate. Thus the final cell number was about 2,000 cells/well. Plates were ex~min~cl daily for
colony formation. At days 3-5, colonies were counted. and colonies were overlaid with MTT:
25111, incubated overnight at 37~C and lO0 !11 of 4% forrnalin was added to aid visualization
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and photography. Fig. 31 shows results of a elonogenic assay at 96 hours. The effeetive
eytotoxieity was ealeulated as 1-c/co where C = the number of colonies formed by a sample
of treated eells and C0= the number of eolonies formed by an equal sarnple of ullL~edlt:d eells.
~7hereas the m~k;1~ l effeetive eytotoxieity of either 0.1 mM dThd or 20 nM STSP alone was
40-60% at 96 hours, almost total eytotoxieity was observed when eells were treated with an
optimal synergistic match of both agents.
Example 16
This Exarnple shows, by means of a turnorigenie assay, that exposure of eells to a
synergistie mateh of an RA (dThd) and a TCI (STSP) for less than a mean generation time
produced long term biologie damage to a targeted population of human m~lign~nt lymphoma
eells (U937).
The biologic behavior of xenografts of human malignant eells in immuno~ ,plessedmiee ean approximate natural growth conditions in a patient, since the cells receive a loeal
supply of plasma nutrients and ean survive for several weeks. Thus, inoeulated eells may
survive in a latent state prior to growth, a condition not readily duplieated in vitro. Inoeulum
size is eritieal for observation of tumor growth within a praetieal time frame. Although
immunocolnl,lolllised mice do not mount an effective cell-mediated immllne response to
xenografted tissue, small inoeula may be damaged by maerophages or natural killer eells before
developing an effective vascular support. Preliminary experiments showed that subeutaneous
inoeulation of 1 x 107 human m~lign~nt Iymphoma cells (U937) was tumorigenie in athymic
nude mice. For test of tumorigenicity, U937 cells cultivated in plastic flasks with growth
medium as described in Example 1 and growing logarithmically according to repeated cell
counts at intervals of 24 hours, were treated: (a) dThd 0.2 mM for 12 hr; (b) STSP 25 nM for
8 hours; (c) a combination of dThd 0.2 mM for 4 hr followed by STSP 25 nM for 7 hr; or (d)
no tre~tm~nt (controls). After tre~tment, cells were sedimented and resuspended in serum-free
RPMI 1640. In each group four athymic Nu/Nu weanling mice of mean weight 17 gm were
inoculated subeutaneously into both subseapular regions with 0.1 ml of RPMI 1640 eont~ining
10' eells (viability > 90%). Formation of tumors in these subcutaneous sites was observed
during the next 14 days. One control mouse died prematurely. At 14 days, all animals were
saerificed, tumors were photographed, tumors were carefully dissected, and tumor weights were
reeorded. Tumors were verified histologieally. The animals receiving both dThd and STSP
developed no tumors.
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Table 34 shows the mean weights of tumors for ~nim~l~ receiving dThd only, STSP
only, and no agent st~tictical evaluation. Dirre~ ces in the fate of the dThd/STSP treated cells
and single agent treated or control cells all were statistically significant.
Comparison of the results of Examples 10, 15 and 16 to the results of Examples 7 and
8 shows that an MIS for synergistic ln~trhin~ of RA and TCI may underestim~te the effective
cytotoxicity as reflected in delayed cell killing, clonogenic assay or tumorigenic assay.
The feasibility of therapeutic applications in man or in animals within the latter
respective ranges of agent combinations is obvious from published data showing toleration of
plasma levels of up to 6 mM dThd in human subjects, Blumenreich, ~, and animal tests
of STSP as an anti-hypertensive with up to 700 ~lg/kg IV in rats and 130 ~lg/kg in dogs. See
Buchholz RA et al. In Cellular and Molecular Mech~ni.cm.c in Hypertension, p. 199-204,
Plenum Press, NY (1991) and Hypertension 17:91-100 (1991). And see Table 35.
Example 17
This Example demonstrates a use of this invention in tre~tm~nt of a yeast or fungal
infections:
In this example, the IC40 for an HU or other RA and the ICso for STSP or other TCI are
deterrnined with respect to growth inhibition of a yeast or fungal population during a
predeterrnined generation time. A series of 96-well plates is filled with ~plol,liate liquid or
semi-solid agar-based growth medium, see McGinnis MR and Rinaldi MG in Lorian, ~,
cont~ining bivariate concentrations of the RA (maximum concentration = IC40) and the TCI
(maximum concentration = IC50) as described in Example 8. Each microwell is inoculated with
an equal number of the yeast or fungal org~nicm~ (100-2000 colony forming units/well). At
16-96 h after inoculation, and incubation at 30-350C, parallel tests are performed to measure
effects of the bivariate RI and TCI combinations on fungal growth using: (l) visible turbidity
is noted in liquid medium and a digitized tr~n~mi~cion or reflectance image is obtained for
computer analysis, or (2) the MTT assay or other colorimetric assay is performed; and collected
data are analyzed by combined results ratios and O/S differential plots; or (3) colony formation.
Colony formation is described as follows. Plates with liquid media are agitated on an
orbital shaker and 5-10 ,ul of medium is aspirated from each well and diluted up to 1:100.
Samples from plates with semi-solid medium are obtained by mixing each well contents in 100
111 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
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in~.t-~t~rl at 370C overnight and the yeast or fungal colonies are eounted on eaeh plate
aeeording to standard mierobiologie proeedures. Lorian, supra, pp. 53-197. The pelet;~ ge
inhibition of proliferation is ealculated by taking the ratio of number of colonies formed by
treated org~ni~mc to the number of eolonies formed by ~~ at~d eontrol org~ni~m~ Results
of the data analysis by data algorithms show a pot.-nti~ting action of the RA on colony growth
inhibition by a TCI in a range of eoneentrations below the RA IC40.
Example 18
This Example demonstrates a use of the invention in pot.onti~ting damage to cells inf~et~d by
a virus and selectivity due to changes in thymidine kinase (Tk) or pRb
In eells infeeted by DNA viruses, the DNA biosynthesis is susceptible to dynamicretardation. Such viruses inelude members of the herpesvirus family. Many human m~lign~nt
cells of Burkitt's Iymphoma origin carry incomplete genomes of the Epstein-Barr herpesvirus
group virus and may express a thymidine kinase (Tk) of viral origin in addition to any
endogenous Tk aetivity. Infection of eells with herpesvirus thus provides potential avenues for
selective potentiation of TCI damage to the virus-infeeted cells using dThd aeting as the RA.
In this example, cells are infected with a strain of cytomegalovirus aceording to
procedure deseribed by Berezesky IK et al., Exp and Mol Pathol. 14:337-49 (1971) and seeded
into 96-well plates. The IC40 of dThd is determined for uninfected cells. The virus infection
proeeeds over a period of several days with visible ehanges in infeeted foei: eells beeome
enlarged and rounded with increases in both nuelear and cytoplasmic volume. Beginning at
times from 0-24 h after infection, the cells in multiwell plates are exposed to a series of
seheduled and bivariate combinations with RA including dThd, HU, or Aph (maximumcol1ce~ dLions = IC40 ) for 2-4 hrs followed by STSP (maximum concentration - 50 nM) for
up to 24 h total duration. Cell damage is assessed using either the the LDH, MTT, or crystal
violet assays. Results are compared to uninfected control cells treated id~ntie~lly with the
bivariate eombinations of dThd and STSP and data are analyzed using the appl~ liate set of
formulas for CRR or 0/S plots as deseribed in Example 8.
As compared to the uninfected eontrols, eells in the infeeted eulture show inereased
rates of cell damage as cletermin~d by increased LDH release at serial time points for each
combination of RA and STSP.
In a related proeedure, human m~lign~nt Iymphoma cells are infeeted by herpes simplex
virus as deseribed by Bedoya V et al., J. Natl. Cancer Inst. 41:635-52 (1968). The IC40 of
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dThd is clet~rmine~l for uninfected cells. Beginning at times from O to 2 h after infection the
potentiation of cell damage by bivariate combinations of RA (IC40 m~imum) and STSP (50
nM m~imum) 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
darnage at serial time points during the combined agent tre~tmerlt.~. Analysis of the data and
calculations of Sm"" show action of dThd as an RA and potentiation of STSP in a range of dThd
concentrations below the IC40.
Cells infected by oncogenic DNA viruses, such as hurnan papillomaviruses implicated
in human carcinogenesis are dynamically retarded by the action of a virus that synth~ci7~s a
protein able to inhibit the function of pRb. Winman KG, FASEB J. 7:841-5, 1993. Virus
transformed cells, such as cells that ~nt~cede 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. Moreover, transfection of cells with
oncogenic virus proteins serves as an RA to synergize with effects of STSP or other TCI.
Example 19
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.
Drug interactions are an issue of major medical and pharmacologic concern. In
chemotherapy, agent antagonisms can ~limini~h specificity of agent actions and increase side
effects. In tests with STSP as a TCI, which was shown in Example to exert biochemical
effects during G2 phase of the cell cycle, the inventors attempted to learn whether other agents
acting in late S phase or early G2 phase might enhance its effect by acting as RA. In fact, the
inventors discovered that caffeine which is reported to influence the cell cycle at G2 phase
(Schlegel R; Harris MO; Belinsky GS J Cell Biochem 57:351-61(1995) strongly antagonized
the action of STSP. This is shown in a differential O/S plot where the concentrations of
caffeine tested were in a range of 0.1 - 2 mM and the concentrations of caffeine were in a
range of 3.1 -50 nM.
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Example 20
This Example shows that the MIS and auxiliary data algorithms may be used to test for
synergistic interactions of drugs, toxic substances or other environment~l hazards for
genotoxicty.
Envir-)~ment~l of medical exposure of hllm~nc, animals or plant life to potentially
hazardous agents which are genotoxic such as some of the compounds producing DNA lesions
shown in Table 2 or Fig. 11 is a matter of considerable societal concern. By means of the
MIS, cell cultures can be employed as a surrogate target population to analyze potential
genotoxic effects to the extent that such effects may be cell cycle related. In this approach
effects of a compound with unknown properties is tested according to the algorithms ~ c~l~sed
herein. Agents which have effects related to the cell cycle are potentially genotoxic, but may
be reversible. On the other hand, an agent which is not primarily genotoxic may become
genotoxic when synergistically m~trhrrl with a low and overtly non-hazardous concentration
of another agent. This type of occurrence has been a major concern in the ~rrn~th of
episodes of major environmental stress such as oil or forest fires, or in military operations
where toxic s-~bst~nce~ may be released with malicious intent.
In this example, the agent tested was DEET (N,N-Diethyl-m-toluarnide) which is
commonly applied in minim~l dilutions as a topical insect repellant and has been a subject of
safety investigations Osimitz TG and Grothaus RH J Arn Mosq Control Assoc, 11:274-8
(1995). At concentrations above 0.5 mM, applicants identified induction of apoptosis in the
U937 cells as well as in Jurkat T cells and Daudi B cells. In MTT tests, preliminary results
with dThd or HU as prospective RA in ranges of about 0.05 to 3 mM showed no significant
~nh~nrement of the DEET-induced apoptosis. Additional tests of this and other agents using
the MIS and auxiliary data analysis are thus possible.
Example 21
This Example shows that the MIS and au~iliary data algorithms can be used to measure
effects of a radiation source as a TCI or an RA for use in synergistic matches.
The role of p53 expression, and the cell cycle in determining cell sensitivity to radiation
exposure has been a subject of many investigations (See, Kuerbitz SJ et al. Proc. Natl. Acad.
Sci. USA 89:749]-5 1992)
Using 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
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Ml~l assay. Serial t~,vo-fold changes in calibrated radioactive dosages are comhin~d with two-
fold serial dilutions of an agent being tested ~ 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 m~ m The IC40 and
EC50 are cleterrnined by MTT ~say as shown in Example 1 and in Example 10 at
approxim~t~.ly 24 hr. The specific range of activity required is b~ed upon dosimetry
calculations. Using 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 G2 ph~es. This
is anticipated from extensive published literature (See for e~arnple Kuerbitz, ~a, Giocanti
N et al. Cancer Res 53:2105-11(1993)
The effect of dThd, HU, aphidicolin or other RA becomes evident if it is ~f~mini~t~red
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 ~rlmini~tration 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 discemed an enhancing effect upon damage produced by a single exposure of the U937
cells to 2.5 to 10 Gy of garnma radiation. Thus, STSP is a candidate for testing either as RA
or TCI.
The use of tritiated water in these e~pelilllents 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 incorporated 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. 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
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increm~ntSll 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
This Example demonstrates a use of the invention in the tr.o~trnent of a fal~;i~u,--
malaria infection.
The malaria plasmodium falciparum (p. falciparum) is a eukaryotic organism that grows
by division within human erythrocytes of m~rnm~ n 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 hllm~nc, 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 ribonucleotide re~ rt~e have been characterized for the m~l~ri~l
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).
In a series of experiments, human A positive erythrocytes (RBCs) infected with p.
falciparum W2 or D6 strains (Oduola AMJ et al. Exper. Parasitol. 66: 86-95 (1988)) by an
in vitro method (Milhous WK et al. Antimicrob. Agents Chemother. 27:525-530 (1985) were
exposed to serial concentrations of staurosporine, hydroxyurea or aphidicolin during the stage
of rapid multiplication at 24 hr after the RBC infection. Parasite growth was monitored by
uptake of 3H-hypo~nthine which becomes incorporated into the parasitic DNA. Based upon
radioactivity measurements in 96-well plates with serial agent dilutions, the inhibitory
concentrations (IC50) in strain W2/D6 were staurosporine, 0.15 ~1M/0.19 ~M, hydroxyurea, 219
M/175.2 !lM and aphidicolin 0.123 ~lM/0,40 !lM respectively.
These results show that use of HU or APH as RA with STSP as a TCI is practicable.
" In f~lrther work, the MIS method of agent combinations in serial dilutions is applied, using the
parasite infected RBC system. Absolute radioactivity counts in each well are used as an
indication of the extent of parasitic infection (i.e. higher counts indicate greater infection).
Thus in synergistic action of an RA and TCI, the number of counts is lower than expected by
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sllmm~tion of the results using either agent alone. The algorithm applied for data analysis
therefore is the same as that used analysis of LDH release in Example 9. Using Table 3A-C,
both CRR and O/S plots may be obtained.
For tests in vivo, 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 tr~n~mitted
by sacrificing an infected animal when the percentage parasitemia is a~.v~ lately 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 intraperitoneally into a fresh
animal (0.5 ml of the diluted sample).
In one type of treatment of the ~nim~ HU is ~Aminictered orally, be~ lhlg on dayzero or at 24 hours after an infection, to achieve a plasma concentration in a range up to 150
M. After 2-24 hr, in different experiments, STSP at 700 llg/kg (Buchhol~, ~), is~ mini~tl-red IV in a single dose. 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 treatrnents may be
repeated as indicated by initial results. Thin blood smears are prepared with drops of blood
from tail veins. Smears are fixed in methanol, stained with Giemsa and numbers of infected
red blood cells per 50 oil irnmersion fields (100 X) are counted. Reduction of parasitemia is
m~imllm in the HU/STSP treated mice and the mean survival time of these mice is increased
relative to the other groups of mice.
Example 23
This Example demonstrates that the success of STSP as a TCI in inducing apoptosis
with multiple RA in cells lacking functional pS3 may be explained by some key molecular and
cell biologic changes associated with the actions of STSP.
In the present Example human m~lign~nt Iymphoma cells (U937) were treated with
STSP or a synergistic match of dThd and STSP. For irnmunoblot analyses of CDC2 and MAP
kinases samples of S x 106 cells were extracted in SDS sample buffer and subjected to 12%
SDS-PAGE and immunoblotting as described in Example 4. The cyclin-dependent kinase
p34Cdc2 was detected with antibody clone #1 from Signal Transduction Laboratories. For
detection of histone phosphorylation activity p34Cdcz was immunoprecipitated with monoclonal
antibody (Santa Cruz, #17) using protein A sepharose and reacted with H1 substrate
(Boehringer Mannheim) in the presence of ~32P-ATP (Amersham). Phosphorylated product was
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separated and resolved by 12% SDS-PAGE and visualized by autoradiography with X-Omat
film (Kodak). MAP kinases were det~ct~cl by im~nunoblotting with monoclonal antibodies
from ph~ningen (San Diego, CA). For assay of enzymatic activities, MAP kinases were
immllnoprecipitated 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
(Ph~rmin~en). The protooncogene product c-myc was detected by imrnunoblotting with an
antibody from Oncogene Sciences.
All of the above proteins were of interest in relation to the action of STSP in causing
apoptosis and acting in synergistic match with dThd. Fig. 36 shows that dephosphorylation of
p34Cdc2 on p-Y-15 and p-T-14 is a critical factor in regulating cell movement through G2 phase
into M phase. The ratio of MAP kinases JunK (JNK) and Erk-2 were of interest in relation
to the molecular met~h~ni.~m 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. (Xia et al,
supra) and increased ~xlules~ion of the protein c-myc is a critical factor in the initiation and
m~int-on~nre of S phase (F.isçnm~n RN and Cooper JA, Nature 378:438-439, 1995). The agent
aurintricarboxylic acid (ATA) has been of interest as an inhibitor of endonucleases and
apoptosis.
Fig. 37~ is an immunoblot showing the effect of STSP and + ATA on the
phosphorylation Of p34CDC2,
Fig. 37B is an irnmunoblot demonstrating that STSP induced a functional activation of
cdc2 as shown both by the ability to phosphorylate histone protein (H1).
Fig. 37C is an immunoblot showing the effect of STSP and 1 ATA on c-myc
expression.
Fig. 38 is an immunoblot showing the effect of STSP and + dThd on MAP kinases.
Results demonstrated that phosphorylation of CDC2 was inhibited by STSP + dThd, but
not by dThd alone, and that levels of c-myc normally elevated in S phase were reduced. In
addition, the synergistic combination of dThd and STSP was associated with strikingly
increased JunK and decreased Erk-2 M~P kinase activities. This may be significant in
, relationship to cell cycle control mechz~ni~m~
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Description of Tables
Table lA&B: Functional categories and examples of proven or potential RA .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-MTTcombined results ratios from tabular format.
Table 4: Example 8 Combined results ratios from MTT data for example of dThd andSTSP (% cytotoxicity of three plates was averaged for this example.
Table 5: Example 9 Combined results ratios from LDH data for example of dThd andSTSP (% cytotoxicity of three plates was averaged for this example.
Table 6: Example 10 Combined results ratios from MTT data showing dThd potentiation
of STSP cell damage at 48 hr after washing.
Table 7: Example l l Combined results ratios from MTT data showing schedule testing
of dThd potentiation of STSP cell damage.
Table 9: Combined results ratios from MTT data for BrdU and STSP
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 STSPTable 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 for dThd and K252a
Table 23: Combined results ratios from MTT data for dThd and KT5926
Table 24: Combined results ratios from MTT data for dThd and KT5720
Table 25: Combined results ratios from MTT data forThd and Aph
Table 26: Combined results ratios from MTT data for dThd and ara-C
Table 27: Combined results ratios from MTT data forHU and K252A
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Table 28: Combined results ratios from MTT data for HU and Aph
Table 29; Combined results ratios from Ml~ data for dThd and cisplatin
Table 30: Combined results ratios from MTT data for HU and cisplatin
Table 31: Combined results ratios from MTT data for Aph and K252a
Table 32: Combined results ratios from MTT data for etoposide and daunorubicin.
Table 33: Summary of data for synergistic m~t(~.ht~ in U937 cells.
Table 34: Tumor formation in athymic nude mice innoculated with U937 cells.
Table 35: Examples relating SMAX for specified RA to blood levels previously reported in
chemotherapy.
Table 36: Characteristics of human m~ n~nt cells lines successfully treated by dThd or
other RA to potentiate the action of STSP or other TCI.
Table 37: Evidence for Cell dep~n~i~nce of IC40 and SMAX in synergistic matches with
STSP.
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.
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Table 1. Functional categories and examples of potential restraining agents (RA)
F.ctim~terl position of Potential Restr~inin~ Agent (RA)
Reference Point
Specific target of action ¦ Re~ sc~ e examples
class I
nucleoside transport dipyridamole, Tk
antagonist(~
transition purine or pyrimidine 6-thioguanine, pentost~tin,
metabolism 5'-~7~.ytidine,
ribonucleotide reductase (RNR) dThd, ~r~!ll, dAde, dGuo,
H~J,
trimidox, desferoxarnine
N-hydroxy-N'-aminoguanid
ine (HAG) Schiff bases
expression of
early S phase genes dihydrofolate rednct~ce Ml~, aminopterin
thymidylate synthase fluorin~t~d uracilc
pRb phosphorylation, E6 oncoprotein~
gene e~ ion transferred dominant
negative gene(~, ~nti.ce.n.ce
molecules(~
growth factor inhibitor
receptor antibody
class IIA
S phase DNA polymerases ~phidicolin, .ara-C
nascent DNA
synthesis
class IIB
DNA intercalation daunorubicin. isoquinolines
S phase DNA breakage/repair bleomycins~ ~amma rays
DNA lesions etoposide. camptothecin
DNA alkylation nitro~en mustards
DNA cross-linkage mitomycin C~ cisplatin
-
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~,stim~teA position of Potential Restr~ining Agent (RA)
Reference Point
Specific target of action I Representative examples
class III
S to G2 protein kin~ces, STSP~, ~?~
phase (including cyclin-dependent KT5926(~, quercetin
kinases)
DNA supramolecular
organization, histone phophorylation, ~(~, growth factor
chromosome nucleoprotein associations inhibitor:
con~en.c~tion competitive ligand or
antibody
* Actions of agents underlined are demonstrated in working examples, potential
actions of other agents are inferred from previously published data. Some RA such as
STSP and K~2a 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
- been clinically tested in hllm~nc,
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Table 2. Functional classes of targeted cytotoxic insults (TCI)
Fetim~t~d Targeted Cytotoxic Insults (TCI)
position of
Target Interval Targets of ~l~m~inE~ action Rc~ ;uLa~ e examples
class A
G, to S initis~tion of S phase u.. col,.ul~u.
phase
nucleotide metabolism metabolic ~~n7~yme inhihitors
early S phase
gene expression gene ~ iVII growth factor antagonists,
.ee molecules~
class Bl
S phsse DNA polymerases ~hidicolin Ara-C
class BZ
DNA intercalation ~rtin(lmycin' daunorubicin-
S to Gl isoquinolines
phase DNA cross-linkage cisplatin. carboplatin. multiple
DNA lesions lkyl~tino ;~oents. niLlusoul~a~
mitomYcin C
DNA methylation 5'-~7~rytidine
DNA strand integrity bleomycin. ~o~mm~l rays. etoposide
class C
G2 phase nucleoproteins, protein kinases, STSP, K~2A~. KT5926~.
cyclin-rh~p~n~l~nt kinases other protein kinase inhibitors.
olomoucine~. caffeine,
ilmofosine
DNA supramolecular organi_ation etoposide
: 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 .i.op~,nriin,o upon the cell type or strength
and duration of application. Agents marked with ~ have not yet been tested clinically.
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TABLE 3
ALGORITHM WITH SPREADSHEET FORMULAS FOR ANALYSIS OF MIS DATA
(formula locatlons mdlcated by cell letter and number e.g. Cl9)
Gencral Headings and ~, . c ' cells Numbers in C18 to 018 indic:lte positions
in which . vanables are entered of columns in the 96-well plate
Al: (T) W8] ~DATE C18: (T) W31 1
Cl: (T) W31] ~EXP # D18: (T) W31 2
El: (T) W32] ~CELLTYPE E18: (T) W32 3
Gl: (T) W3Z] ~ASSAY F18: (T) W32 4
G18: (T) [W32~5
A2: (T) rw8] ~enterda~e H18: (T) IW31]6
C2: (T) IW31] ~enterexperiment # 118: (1) lW32]7
E2: (T) [W32] ~enter cell ~ype J18: (T) [W32]8
G2: ( 1) rW32] ~enter as5a,v K18: (T) tW32]9
L18: (T) IW32]10
D4: (T) [W31] ~MAXIMUM CONCENTRATION M18: (1) [W29]11
E4: (T) tW32] ~Moles or '~.d N18: (1) tW16]12
F4: (T) rW32] ~HOURS OF TREATMENT 018: (T) IW28] ~MEANS
A5:(T)[W8] ~TCI(vertical) BeginningofTablewithll r ~cd~ ; ~ data
C5: (T) [W31] ~entername of TCI B19: (T) W10 ~A
D5:(T)[W31]~enter value Cl9:(T) W31 +A15=Blank b.. "~L.. ' well
E5: (T) [W32] ~enter units Dl9: (T) W31 +A16
F5: (T) [W32] ~enterhours E19: (T) W32 +A17
Fl9: (1) W32 +A18
A7: (T) W8] ~RA (horizontal) Gl9: (T) [W32] +AI9
C7:(T) W31 ~entername of R~ Hl9:(T)lW31] +A20
D7:(T) W31 ~entervalue 119:(T)[W32] +A21
E7: (T) W32 ~enter units J19: (T) tW32] +A22
F7:(T) W32 ~enter hours Kl9:(T)[W32] +A23
Ll9: (1) IW32] +A24
F9: (T) [W32] "enter hours M19: (T) tW29] +A25
G9: (T) [W32] 'HOURS TOTAL Nl9: (1) IW16] +A26
019: (1) IW28] @~AVG(M19NI9)
Al l: (T) W8] ~PURPOSE:
Bll:(T) .W10 "Detectagent B20:(1) W10 ~n
Dll:(T) W31 'andsynergistic C20:(T) W31 +A27
Ell: (T) W32 'match forgrowth D20: (T) W31 +A28
Fll:(T) W32 'inhibition E20:(T) W32 +A29
F20: (T) W32 +A30
A13: (T) W8] ~ABSORBANCE DATA G20: (T) iw321 +A31
C13: (T) W31] 'enterubO- ' nm H20: (T) [W31] +A32
G13:(T) W32] ~Immedia~eassavvalue 120:(1)[W32] +A33
113: (T) tW32] +G2 J20: (T) IW32] +A34
A14: (T) [W8] ~/File Import/Comma~Clear~a:leial~ ~~ K20: (T) [W32] +A35
G14: (T) [W32] ~enterpla~e numoer L20: (T) IW32] +A36
M20: (T) [W29] +A37
A15: (T) lW8] 'ABSORBANCE OF BACKGROUND N20: (T) [W16] +A38
BLANK 020: (T) [W28] (~AVG(M20.. N20)
Absorbance data from a diskette is entered into
cells A15 through Al 10 by the macro ~1 ~- h I cell A14 B21: (T) tW10 ~C
This data (not shown) is transferred to a tabular format in cells C2 1: (T) [W3 1 +A39
B19 through N26 as indicated below (in B19 through N26) D21: (T) [W31 +A40
B16: (T) tW10] 'CONTROLS FE221 ~ tWW32 +A41
C16: (T) [W31] ~ REM: CHECKPOSITIONOF G21:(T)[W32] +A43
BACKGROUND BLANK! H21: (T) [W31] +A44
G16: (T) [W32] ~REM:CHECK LOCATION FORN0 121: (T) [W32] +A45
J21: (T) [W32] +A46
B17: (T) W10 'MEAN K21: (T) W32 +A47
C17: (T) W31 ~AVG(M25.. N26) L21: (T) W32 +A48
D 17: (T) W3 1 "COEFFICIENT OF VARIATION = M2 1: (T) W29 +A49
E17: (T) W32 +$N$29/$NS28 N21: (T) W16 +A50
TABLE 3A
RUIE 26~
CA 0222~682 l997-l2-23
W O 97/01344 PCTrUS96/10921
-74-
021:(T)[W28] ~AVG(M21.. N21)
of Tabl~with li r ~cd ~1 i dnta
B22:(T) WIO ~D B25:(T) W10 ~G
C22:(T) W31 +ASI C25:(T) W31 +A87
D22:(T) W31 +A52 D25:(T) W31 +A88
E22.(T) W32 +A53 E25:(T) W32 +A89
F22 (T) W32 +A54 F25:(T) W32 +A90
G22:(T)[W32] +ASS G25:(n tW32~ +A91
H22:(T)[W31] +A56 H25:(T)[W31] +A92
122:(T)[W32] +A57 125:(T)[W32] +A93
J22:(T)[W32] +A58 J25:(T)rW32] +A94
~22:(T)[W32] +AS9 K25:(T)[W32] +A9S
L22:(T)[W32] +A60 L25:(T)[W32] +A96
M22:(T)[W29] +A61 M25:(T)[W29] +A97
N22:(T)[W16] +A62 N25:(T)[W16] +A98
022:(T)[W28]6~AVG(M22.. N22) 025:(T)[W32](~AVG(M25.N25)
B23:(T) WIO ~E B26:(7-) WIO ~H
C23:(1~ W31 +A63 C26:(T) W31 +A99
D23:(T) W31 +A64 D26:(T) W31 +AI00
E23:(T) W32 +A65 E26:(T) W32- +AIOI
F23:(T) W32 +A66 F26:(T) W32 +A102
G23:(T)[W32] +A67 G26:(T)[W32] +A103
H23:(T)[W31] +A68 H26:(T)[W31] +A104
123:(1)[W32] +A69 l26:(T)tW32] +AIOS
J23:(T)[W32] +A70 J26:(-1-)[W32] +A106
1;23:(T)[W32] +A71 K26:(-1-)[W32] +A107
L23:(T)[W32] +A72 L26:(T)tW32] +A108
M23:(T)tW29] +A73 M26:(T)[W29] +A109
N23:(T)[W16] + A74 N26:(1)[W16] +AII0
023:(T)[W28] ~AVGlM23N23)
End of Table with 1. ~ ~ cd ' i dnta
B24:(T) WIO ~F
C24:(T) W31 +A75
D24:(T) W31 +A76
E24:(1) W32 +A77
F24:(T) W32 +A78
G24:(-1-)[W32] +A79
H24:(T)[W31] +A80
124:(T)[W32] +A81
J24:(T)[W32] +A82
K24:(T)[W32] +A83
L24:(T)[W32] +A84
M24:(T)[W29] +A85
N24:(T)[W16] +A86
024:(T)[W28](~AVG(M24..N24)
TABLE 3B
SU~ 3~ 8~
CA 0222~682 I997-I2-23
WO 97/01344 PCT/US96/10921
- 75 -
Beginning of nnnlyses of ~ ~ datn
Delta = absolutc :''~ c from means of ~' ; for
control RA and TCI dilutions intended to re~eal nny outlier
values
B27: (T) W10 ~DELTA B30: (T) WIO ' INHIBITION
C27: (T) W31 ~ABS( +C29-C25) C30: (T) W31 (1-C29/$NS28)
D27:(T) W31, (~ABS(+D29-D25) D30:(T) W31 (1-D29/$N$28)
E27: ~r) W32 (-~ABS( +E29-E25) E30: (T) W32 (1-E29/$N$28)
~F27: (T) W32 ~ABS( +F29-F25) F30: (T~ W32 (1-F29/$N$28)
G27: (T) tW32] (~ABS( +G29-G25) G30: (T) [W32] (1-G29/$N$28)
H27: (T) [W31] ( ~ABS(+H29-H25) H30: (T) [W31] (1-H29/$N$28)
127: (T) tW32] ~alABS( +129-125) 130: (T) [W32I (1-129/$NS28)
J27: (T) [W32] @ABS( +J29-J25) J30: (T) [W32] (1-J29/SNS28)
K27: ('1') rW32] (~,ABS( +K29-K25) K30: (T) [W32] (1-K29/$N$28)
L27: ('1') [W32] ~ABS( +L29-L25) L30: (T) [W32] (1-L29/SNS28)
N27: ~ [W16] ~CONTROL M30: (T) tW29] "COEFFICIENTOFVARIATION
N30: (T) tW16] +SN$29/$N$28)
B28: (T) [W10' ~VALUES
C28: ('I ) tW31 ~ABS( +C29-C26) Henders for the tnble to nnalyze BVSD
D28:(T)rW31 (~ABS(+D29-D26~ B31:(T) W10 +C7
E28: (T) rw32' ( ~ABS( +E29-E26) C31: (T) W31 +F7
F28:(T)[W32 C~ABS(+F29-F26) D31:(T) W31 ~Hrs
G28: ('1~ tW32] (~ABS( +G29~26) D33: (T) W31 ~BIVARIATE SERIAL
H28: (T) rW311 ( ~ABS( +H29-H26) E33: (T) W32 ' DILUTION ANALYSIS (BVSD)
128: (T) [W32] ~.ABS( +129-126)
l28: (T) [W32] ~),ABS( +l29-J26) D35: (T) [W31] ~TABULATION OF
K28: (T) [W32I ~ABS( +K29-K26) E35: ( r) [W32I ~COMBINED RESULTS RATIOS (CRR)
L28: (T) [W32] ~ABS( +L29-L26)
M28: (T) ~ ,W,29] ''MEAN B36: (T) [W10] +G2
N28: (T) rW~6] ~AVG(M25.. N26) C36: (T) tW31] +A2
D38:(T) W31] +F9
Menn ~nlues orRA ~ ' used to cnlculate % growth E38: (T) W32] ~H
inbibition in tbe, ~ ' ' cells B30.J30 G38: (1') W32I +C5
B29: (T) W10 ~MEAN 138: ('1') [W32] ~for
C29: (T) W31 (~AVG(C25... C26) J38: (T) [W32] +F5
D29:(r) W31 (clAVG(D25.. D26) K38:(T)tW32]~H
E29: (T) W32 (-~IAVG(E25,,E26)
F29: (T) ,W32 ~AVG(F25,,F26) B39: (T) IWIO] +$C$5
G29: ('1) IW32] (--,AVG(G25,,G26) G39: (T) lW32] +C7
H29: (T) ,rW31] ( ~AVG(H25,.H26) 139: (T) [W32] ~for
129: (T) [W32] ~lAVG(125.. 126) J39: (T) [W32] +F7
l29: (T) [W32] ~.AVG(J25.. J26) K39: (T) [W32] ~H
K29: (T) [W32] (--,AVG(K25.. K26)
L29: (T) [W32] ~AVG(L25... L26) B40: (T) [W10I +$E$5
M29: (T) LrW29] STANDARD DEVIATION
N29: (T) [W16] ~STD(M25.... N26) B41: (T) [W10] ~Growth Inhibition [%]
TABLE 3C
SUBSTITUTE SH EET (RULE 26)
CA 0222~682 1997-12-23
W O 97101344 PCTrUS96/10921
76
Beginning of rormul~ls for the CRR
B42~ W10 +D5 B44:(T)[W10] +B43/2
C42: (T) W31 (1-~AVG(M19.. N19)/$C$17) C44: (T) W31 (1-~AVG(M21.. N21)/$C$17)
D42:(T) W31 +C42/($C42+D$50) D44:(1) W31 +C44/($C44+D$50) ~,
E42: (T) W32 ~BACKGROUND BLANK E44: (T) W32 1-(C21/SC$17)
G42: (T) W32J l-(DI9/SCS17) F44: (T) W32 +E44/($C44 +E$50)
H42: (T) W31] +G4V($C42 +G$50) G44: (T) [W32] 1-(D21/$C$17)
142: (T) [W321 1-(EI9/$C$17) H44: (T) [W311 +G44/($C44 +G$50) "
J42: (T) [W321 +142/($C42 +1$50) 144: (T) [W32] 1-(E21/$C$17)
K42: (T) tW32] 1-(F19/$C$17) J44: (T) [W32] +144/(SC44 +1$50)
L42: (T) tW321 +K4V($C42 +K$50) K44: (1) tW32] 1~1:21/SCS17)
M42: (T) tW29] 1-(GI9/SCS17) L44: (T) tW32] +K44/(SC44 +KSS0)
N42: (1) tW 16] +M4V($C42 +M$50) M44: (T) tW29] 1-(G21/$CS17)
042: ( I ) tW28] 1-(HI9/SCS17) N44: (T) tW161 +M44(SC44 +MSS0)
P42: (T) [W17] +042/(SC42 +O$50) 044: (T) [W28] 1~H21/$CS17)
Q42: (T) W27 1-(119/$CS17) P44: (T) jW17] +044/($C44 +OSS0)
R42:(1) W18 +Q4V($C42+Q$50) Q44:(T) W27 1~121/SC$17)
S42: (1) W25 1-(119/$CS17) R44: (T) W18 +0~44/($C44 +QSS0)
T42: (T) Wl9. +S42/($C42 +S$50) S44: (T) W25 1-(J21/$CS17)
U42: (T) W28 1-(KI9/SCS17) T44: (T) Wl9 +S44/($C44 +SSS0)
V42: (T) Wl9 +U4V($C42 +U$50) U44: (T) W28 1-(K21/SCS17)
W42:(T)[W2'] 1-(LI9/SCS17) V44:(1) WI9 +U44/($C44+U$50)
X42: (T) [W18] +W42/($C42 +W$50) W44: (T) [W2.-] 1-(L21/$C$17)
X44: (T) [W18] +W44/($C44 +WSS0)
B43: (T) W10 +B4V2
C43: (T) W31 (1-~AVG(M20.. N20)/$C$17) B45: (T) W10 +B44/2
D43: (T) W31 +C43/($C43 +D$50) C45: (T) W31 (l~AVG(M22.. N22)/$CS17)
E43: (1) W32 1-(C20/SCS17) D45: (T) W31 +C45/($C45 +D$50)
F43: (T) W32 +E43/($C43 +E$50) E45: (T) W32 1-(C22/SC$17)
G43: (1) [W32] 1-(D20/SCS17) F45: (~1') W32 +E45/(SC45 +E$50)
H43: (T) [W31] +G43/(SC43 +GSS0) G45: (1) tW32~ 1-(D2VSCS17)
143: (T) tW32] 1-(E20/SCS17) H45: (T) tW31] +G45/(SC45 +GS50)
J43: (T) [W32] +143/($C43 +IS50) 145: (T) tW32] 1-(E2V5CS17)
K43: (1) tW32] 1~F20/SCSl7) J45: ('1) tW32] +145/(SC45 +IS50)
L43: (T) [W32] +K43/(SC43 +KS50) K45: (1) [W32] 1-(F2VSCS17)
M43: (1) tW29] 1-(G20/SCS17) L45: (T) [W32] +K45/(SC45 +KSS0)
N43: (T) [W16] +M43/(SC43 +MSS0) M45: (1) tW29] 1~G22/SC$17)
043: (T) tW28] 1-(H20/SCS17) N45: (T) tW16] +M45/($C45 +M$50)
P43: (1) [W17] +043/($C43 +OSS0) 045: (T) [W28] 1-(H22/$C$17)
Q43: (T) -W27 1-(120/$CS17) P45: (T) rW17] +045/(SC45 +O$50)
R43: (T) W18 +Q43/($C43 +QS50) Q45: (1) W27 1-(122/$CS17)
S43: (T) W25 1-(J20/SCS17) R45: (T) W18 +Q45/($C45 +QSS0)
T43: (1) W19 +S43/(SC43 +SSS0) S45: (T) W25 1-(J2V$CS17)
U43: (T) W28 1-(K20/SC$17) T45: (T) Wl9 +S45/($C45 +S$50)
V43: (T) Wl9 +U43/($C43 +USS0) U45: (T) W28 1-(K2V$CS17)
W43: (T) tW2 ] 1-(L20/SC$17) V45: (T) WI9 +U45/(SC45 +USS0)
X43: (T) [W18] +W43/($C43 +W$50) W45: (T) tW2: ] 1-(L2V$C$17)
X45: (T) [W181 +W45/($C45 +WSS0)
TABLE 3D
SUB5TITUTE SHEET (RULE 26)
CA 0222~682 1997-12-23
W O 97/01344 PCT~US96/10921
-77-
C~ ' of formul~ls for CRR
B46: (T) W10] +B45n F48: (T) [W32] +E50/(SDS0 +ES50)
C46: tT) W31] (1-t~AVG(M23.. N23)/$C$17) H48: tT) rW31] +GS0/($D50 +G$50)
D46:(T) W31] +C46/($C46+D$50) J48:(T)[W32] +IS0/($D50+1$50)
E46:tT) W32] 1-(C23/$C$17) L48:(T)fW32] +K50/(SD50+KS50)
F46: (T) W32] +E46/($C46 +E$50) N48: tT) rW16] +MS0/(SC48 +MSS0)
G46: tT) [W32] I-(D23/$C$17) P48: tT) W17] +O50/($C48 +0$50)
H46: (T) [W311 +G46/($C46 +G$50) R48: tT) 'W 18 +Q50/($C48 +Q$50)
146: (T) [W32] 1-(E23/$C$17) T48: (T) Wl9 +S50/($C48 +S$50)
~46: (T) rW32] +146/($C46 +1$50) V48: tT) Wl9 +US0/($C48 +USS0)
K46: (T) IW32] 1-(F23/$C$17) X48: (T) W18 +Wso/(SC48 +W550)
L46: (T) [W32] +K46/($C46 +K$50)
M46: (T) [W29~ 1-(G23/$CS17) B50: t,T) rW10 ~Growth Inhibition
N46: (T) rw 16] +M46/($C46 +M$50) DSo: tT) rW31 1 -t~AVGtB25... B26)/$CS 17
046: t,T) [W28] 1-(H23/$CS17) E50: tT) lW32 1-~AVG(C25..... C26)/$CS17
P46: tT) rW17~ +046/($C46 +O$50) G50: tT) rw32 1~AVG(D25...... D26)/$C$17
Q46: tT) W27] 1-(123/$C$17) IS0: tT) rW32] 1-t;i),AVGtE25.. E26)/SC$17
R46: tT) W181 +Q46/($C46 +Q$50)K50: tT) rW32] 1~AVG(F25....... F26)/SC$17
S46: (T) .W25 1-tJ23/$C$17) M50: tT) [W29] 1~,AVG(G25... G26)/SC$17
T46: (T) W19 +S46/($C46 +S$50) 050: tT) rW28] 1-~a)AVG(H25.. H26)/$CS17
U46: (T) W28 1-(K23/$C$17) Q50: tT) IW27] 1~,AVG(125.... 126)/$C$17
V46: (T) Wlg +U46/($C46 +US50)Sso: tT) [W25] 1-~AVG(J25...... J26)/SC$17
W46: (T) [W25] 1-(L23/$C$17) U50: (T) rW28] 1~AVG(K25.... K26)/$CS17
X46: (T) [W18] +W46/($C46 +W$50) W50: (T) rW25] 1-t~,AVG(L25.. L26)/$C$17
B47: tT) WI0 +B46n B51: tT) W10' +E7
C47: tT) W31 (1~AVGIM24.. N24)/$C$17) C51: tT) -W31' +C7
D47:tT) W31 +C47/($C47+D$50) ESl:tT) W32 +D7
E47: (T) W32 1-(C24/SCS17) G51: tT) W32 +E51/2
F47: (T) W32 +E47/($C47 +E$50) 151: tT) r~32] +GSl/2
G47: t,T) [W321 1-(D24/$CS17) KSI: tT) rW32] +ISl/2
H47: tT) tW31] +G47/($C47 +G$50) M51: tT) [W29] +KSl/2
147: tT) [W32] I{E24/$CS17) O51: tT) [W28] +MSl/2
J47: t~T) [W32] +147/(SC47 +1$50) Q51: tT) lW27] +O51/2
K47: tT) rW32~ 1-(F24/$C$17) SSI: tT) rW25] +Q5112
L47: tT) [W32] +K47/($C47 +KSS0) U51: tT) rW28] +S51n
M47: (T) [W29] 1-(G24/SC$17) W51: tT) lW25] +USI/2
N47: tT) [W16] +M47/($C47 +MS50)
047: (T) [W28] 1-(H24/$CS17) End of formul:ls for the CRR
P47: tT) [W17] +047/($C47 +O$50)
Q47: tT) W27 1{124/$CS17)
R47: (T) W18 +Q47/($C47 +Q$50)
S47: tT) W25 1~J24/$C$17)
T47: (T) WI9 +S47/($C47 +SS50)
U47: tT) W28 1-(K24/$C$17)
V47: tT) W19 +U47/($C47 +U$50)
W47: (T) rW2. ] 1-(L24/SC$17)
X47: tT) IW18] +W47/($C47 +W$50)
TABLE 3E
N~E~ (RUlE ~6)
CA 0222~682 1997-12-23
W O97/01344 PCTrUS96/10921
-7~-
8eginning of formulas for graphic I . B63: (TOIW101 +B61/2
HeadcrsforO/Sptots C63:(T) W31 100-(CZI/$C$17)~100
D63: (T) 'W31 100~D21/SC$17)~100
E54: (T) [W32] ~TABULATION OF OBSERVED RESULTS E63: (1) W32 100-(E21/$C$17)~100
F63: (T) W32 100-(F21/$C$17)~100
E55: (T) [W32] ~AND HYPOTHETICAL SUMMATION ("S") G63: lT) [W32] 100~G21/$CS17)~100
H63: (T) IW31] 100~H21/$C$17)~100
B57: (T) [W10] +$C$5 163: ('1') [W32] 100-(121/SC$17)~100
E57: (T) rW32]' REM: CORRECTANY SUM > 100 ~ J63: ('1') lW32] 100~J21/SC$17)~100
TO BE = 100 K63: (T) IW32] 100-(K21/$C$17)~100
L63: (T) LW32] 100-(L21/$C$17)~100
M63: ('1) [W29] 100~AVG(M21..N21)/$C$17*100
Formulas for O/S plots
B58: (T) [W10] +$E$5
B64: (T) 'W10' ~SUM
B59: (T) W10' +DS C64: ('1') W31 +C$71+$M63
D59: (T) W31 100-(D19/$C$17)~100 D64: (T) W31 +DS71+SM63
E59: (T) W32' 100-(E19/$C$17)~100 E64: (T) W32 +E$71+SM63
F59: (T) W32 100~FI9/SC$17)~100 F64: (T) W32 +F$71+5M63
G59 (T) [W32] 100-(GI9/$C$17)~100 G64: (T) [W32] +G$71+$M63
H59: ('1') [W31] 100-(H19/$C$17)~100 H64: (T) IW31] +H$71+$M63
159: (T) [W32] 100-(119/$C$17)~100 164: ('1') lW32] +1$71+$M63
159: (T) [W32] 100-(119/$C$17)~100 J64: (T) [W32] +J$71+SM63
KS9: (T) [W32] 100-(KI9/$C$17)#100 K64: (T) [W32] +K$71+$M63
L59: (T) [W32~ 100-(L19/$C$17)~100 L64: (T) IW32] +L$71+$M63
M59: (T) [W29] (1-~AVG(M19.. N19)/$C$17)~ 100 M64: (T) [W29] +M$71+$M63
B60: (T) 'W10' ~SUM B65: (T) W10' +B63/2
C60: (T) W31 +C$71+$M59 C65: (T) W31 100-(C22/$C$17)~100
D60: (T) W31 +D$71+$M59 D65: (T) W31 100-(D22/SC$17)''100
E60: (T) W32' +E$71+SMS9 E65: ('1') W32' 100-(E22/sCSl7)~loo
F60: (T) W32 +F$71+$M59 F65: (T) W32' 100~F22/$C$17)~100
G60: (T) ~W32] +G$71+SM59 G65: (1) lW32J 100-(G22/$C$17)~100
H60: ('1') ~W31] +H$71+$M59 H65: (T) [W31] 100-(H22/$C$17)~100
160: (T) [W32] +1$71+$M59 165: ('1~ [W32] 100-(122/$C$17)~100
160: (T) tW32] +J$71+$M59 J65: (T) [W32] 100-(J22/SCS17)~100
K60: (T) [W32] +K$71+$M59 K65: (T) IW32] 100~K22/SC$17)~100
L60: (T) [W32] +L$71+$M59 L65: (T) IW32] 100-(L22/SCS17)''100
M60: (T) [W29] +M$71+$M59 M65: ('1') [W29] 100-~AVG(M22.. N22)/$C$17~100
B61: (T) 'W10' +B59n
C61: (T) 'W31 100-(C20/$C$ 17)~ 100 B66: (1') 'W 10' ~SUM
D61: (T) W31 100-(D20/$C$17)#100 C66: ('1') 'W31 +C$71+$M65
E61: (T) W32' 100-(E20/$C$17)~100 D66: (T) W31 +DS71+$M65
F61: (T) W32' 100-(F20/SC$17)~100 E66: (T) W32' +E$71+$M65
G61: (T) tW32] 100-(G20/$C$17)~100 F66: (T) W32' +F$71+$M65
H61: (T) [W31] 100-(H20/$C$17)~100 G66: (T) IW32] +G$71+SM65
161: (T) [W32] 100-(120/$C$17)~100 H66: (T) [W31] +H$71+$M65
J61: (T) [W32] 100~J20/$C$17)~100 166: (T) LW32] +1$71+SM65
K61: (T) [W32] 100-(K20/$C$17)~100 J66: ('1') tW32] +J$71+$M65
L61: (T) [W32] 100-(L20/$C$17)~100 K66: (T) [W32] +K$71+SM65
M61: (T) LW29] 100-~AVG(M20.. N20)/$C$17)~100 L66: (T) [W32] +LS71+SM65
M66: (T) tW29] +M$71+$M65
B62: (T) W10' ~SUM
C62: (T) W31 +C$71+$M61 B67: (T) W10' +B65n
D62: (T) W31 +D$71+SM61 C67: (T) 'W31 100-(C23/$C$17)~100
E62: (T) W32' +E$71+$M61 D67: (T) W31 100-(D23/$C$17)'100
F62: (T) W32' +F$71+$M61 E67: (T) W32' 100-(E23/$C$17)~100
G62:(T)[W32]+G$71+$M61 F67:(T) W32' 100-(F23/$C$17)~100
H62: (T) [W31] +H$71+$M61 G67: (T) [W32] 100-(G23/$C$17)~100
162: (T) [W32] +1$71+$M61 H67: (1) [W31] 100-(H23/$C$17)~100
J62: (T) [W32] +J$71+SM61 167: (T) LW32] 100-(123/$C$17)~100
K62: (T) [W32] +K$71+$M61 J67: (T) LW32] 100-(J23/$C$17)''100
L62: (T) [W32] +L$71+SM61 K67: (T) [W32] 100-(K23/$C$17)~100
M62: (T) [W29] +M$71+SM61 L67: (T) [W32] 100-(L23/$C$17)~100
M67: (T) LW29] 100~AVG(M23..N23)/$C$17~100
TABLE 3F
SU8S 1 l l UTE SHEET (RULE 26~
CA 0222~682 1997-12-23
W O97/01344 PCTrUS96110921
-79-
formuldsforO/Splotscontinued
B68: (T) WI0] ~SUM C72: (T) [W31] +C$71+$M71
C68: (T) W31 +C$71+$M67 D72: (T) [W31] +D$71+$M71
D68: (T) W31 +D$71+$M67 E72: (T) [W32] +E$71+$M71
E68: (T) W32 +E$71+$M67 F72: (T) [W32] +F$71+$M71
F68: (T) W32 +F$71+$M67 G72: (T) lW32] +G$71+$M71
G68: (T) [W32] +G$71+$M67 H72: (T) [W31] +H$71+$M71
H68: (T) [W31] +H$71+$M67 172: (T) [W32] +1$71+SM71
168: ~ iW32] +1$71+$M67 J72: (T) [W32] +J$71+$M71
J68: (T) [W32] +J$71+$M67 K72: (T) [W32] +K$71+$M71
K68: (T) [W32] +K$71+$M67 L72: (T) [W32] +L$71+$M71
L68: (T) [W32] +L$71+$M67 M72: (T) [W29] +M$71+$M71
M68: ~ IW29] +M$71+SM67
B73: (T) [W10] +$E$7
B69: (T) [W10] +B67/2 C73: (T) [W31] +D7
C69: ~ [W31] I 00-(C24/$C$ 17)~ 100 D73: (T) ~W31] +C73/2
D69: ~ IW31] 100-(D24/$C$17)~100 E73: (T) lW32] +D73/2
E69: (T) [W32] 100-(E24/$C$17)~100 F73: (T) [W32] +E73/2
F69: (T) [W32] IOO~F24/$C$17)~100 G73: (1) lW32] +F7312
G69: (T) [W32] 100-(G24/$C$17)~100 H73: (T) [W31] +G73/2
H69: (T) [W31] 100-(H24/$C$17)~100 173: (T) [W321 +H73/2
169: (T) [W32] 100-(124/$C$17)~100 J73: (T) [W32] +173/2
J69: (T) [W32] 100-(J24/$C$17)~100 K73: (T) lW321 +J73/2
K69: (T) [W32] 100-(K24/$C$17)~100 L73: (T) [W32] +IC73/2
L69: (T) [W32] 100-(L24/$C$17)~100 M73: (1-) [W29] 0
M69: (T) [W29] 100-~AVG(M24..N24)/$C$17-100
B74: (1~ rW10] +C7
B70: (T) W10 ~SUM
C70: (T) W31 +CS71+SM69 B75: (T) W10 ~to raph
D70: (T) W31 +DS71+$M69 C75: (T) W31 1-(~ AVG(C25..... C26)/$CS17)
E70: ~1~ W32 +E$71+$M69 D75: (T) W31 1-((c~AV~(n7C n26)/$CS17)
F70: ('1-) W32 +F$71+SM69 E75: ~) W32 1-((~),AVG(E25... E26)/$CS17)
G70: (T) [W321 +G$71+SM69 F75: (T) W32 1-(~AVG(F25..... F26)/SCS17)
H70: (T) [W31] +H$71+SM69 G75: (T) lW32J l~(--,AVG(G25.... G26)/$CS17)
170: (1') [W32] +1$71+SM69 H75: (T) [W31] 1-((L~)AVG(H25.. H26)/$C$17)
J70: (T) [W32] +J$71+$M69 175: (T) [W32] 1-(~AVG(125.. 126)/$C$17)
K70: (T) rW32] +K$71+$M69 J75: (T) [W32~ AVG(J25...... J26)/$C$17)
L70: (T) [W32] +L$71+$M69 K75: (T) [W32] 1-~(3,AVG(K25... K26)/$CS17)
M70: (T) [W29] +M$71+$M69 L75: (T) [W32] 1-(~'AVG(L25.... L26)/$C$17)
M75: (T) [W29] 1-((~AVG(M25... M26)/$C$17)
B71: (T) WI0 0
C71: (T) W31 I OO~@~AVG(C25C26)/$C$ 17) ~ 100
D71: (T) W31 100-(~AVG(D25D26)/$CS17) ~100
E71: (T) W32 100-(@~AVG(E25E26)/$C$17) *100
F71: (T) W32 100-(~AVG(F25F26)/$C$17) ~'100
G71: (T) [W32~ 100-(~AVG(G25G26)/SC$1i) ~100
H71: (T) [W31] I 00-(( ~AVG(H25H26)/$C$ 17) ~t I 00
171: (T) [W32] 100-(~AVG(125..126)/$CS 17) ~ I 00
J71: ~r) [W32] 100-(CAVG(J25..J26)/SCS17) ~100
K71: (T) [W32] 100-((3AVG(K~ )/$C$17) ~100
L71: (T) [W32] 100-(CAVG(L25..L26)/$CS17) ~100
M71: (T) IW29] 100-((~AVG(M25..M26)/$CS17) ~'100
TABLE 3G
SU8SmUTE ~tEET (RULE 26)
CA 0222~682 1997-12-23
W O 97/01344 PCT~US96/10921
-80-
Formuhs for :' ~ c ' O/S plots
D78: (T) [W31] ~TABULATIONS OF
H78: (T) IW31] ~TABULATIONS OF
D79: (T) [W31] ~O/S DIFFERENTIALS
H79: (T) [W31] ~O/S DIFFERENTIALS
B80: (T) tW10] +$C$5
B81: (T) [W10] +$E$5
Numbers in C81 to Lll indic~te positions
of columns in the 96-well phte
C81: (T) W31 I B84: (T) W 10] +B 83n
D81: (1) W31 2 C84: (T) W31] +C63-C64
E81:(T) W32 3 D84:(T) W31]+D63-D64
F81: (T) W32 4 E84: (T) W32] +E63-E64
G81: (T) [W32] 5 F84: (T) W32] +F63-F64
H81: (T) [W31] 6 G84: (T) lw32] +G63-G64
181: ('1) [W32] 7 H84: (T) [W31] +H63-H64
l81: (T) [W32] 8 184: tT) [W32] +163-164
K81: (T) [W32] 9 J84: (T) [W32] +J63-J64
L81: (T) [W32] 10 K84: (T) ~W32] +K63-K64
L84: (T) [W32] +L63-L64
B82: (T) W10 +D5
C82:(T) W31 +C59-C60 B85:(I) W10 +B84/2
D82: (T) W31 +D59-D60 C85: (T) W31 +C65-C66
E82: (T) W32 +ES9-E60 D85: (T) W31 +D65-D66
F82: (T) W32 +F59-F60 E85: (1) W32 +E65-E66
G82: (T) IW32~ +G59-G60 F85: (T) W32 +F65-F66
H82: (T) [W31] +H59-H60 G85: (T) lW32J +Ci65-G66
182: (T) [W32] +159-160 H85: ('1~ [W31] +H65-H66
J82: (1) [W32] +J59-J60 185: (T) [W32] +165-166
K82: (1) [W32] +K59-K60 J85: (1) [W32] +J65-l66
L82: (T) [W32] +L59-L60 K85: (T) ~W32] +K65-K66
L85: (T) [W32] +L65-L66
B83: (T) W10' +B82n
C83: (T) W31 +C61-C62 B86: (T) W10~ +B85/2
D83:(T) W31 +D61-D62 C86:(T) W31 +C67-C68
E83: (T) W32 +E61-E62 D86: ~T) W31 +D67-D68
F83: (T) W32 +F61-F62 E86: (T) W32 +E67-E68
G83: (T) [W321 +G61-Ci62 F86: (T) W32 +F67-F68
H83: (T) IW31] +H61-H62 G86: (T) iW321 +G67-G68
183: (T) [W32] +161-162 H86: (1) IW31] +H67-H68
J83: (T) [W32] +J61-J62 186: (1) [W32] +167-168
K83: (T) [W32] +K61-K62 J86: (T) [W32] +J67-J68
L83: (1) tW32] +L61-L62 K86: (T) [W32] +K67-K68
L86: (T) IW32] +L67-L68
B87: (T) W10 +B8612
C87:(1) W31 +C69~70
D87: (T) W31 +D69-D70
E87: (T) W32 +E69-E70
F87: (I) W32 +F69-F70
G87: (T) [W32] +G69-G70
H87: (T) [W31] +H69-H70
187: (T) [W32] +169-170
J87: (T) [W32] +J69-J70
K87: (T) [W32] +K69-K70
L87: (T) [W32] +L69-L70
TABLE 3H
SUBSmUTE SHE~ (RULE 26)
CA 02225682 1997-12-23
W O 97/01344 PCT~US96/10921
- 81 -
An X-sxis series used for grsph of % growth inhibition
(with RA and TCI data)
C90: (T) [W31] ''~ ' series
C91: (T) W31
D91: (T) W31 +C91/2
E91: (T) W32 +D91/2
F91: (T) W32 +E91/2
G91: (T) [W32] +F91/2
H9 1: (T) [W3 1 ] +G9 In
191: (T) [W32~ +H91/2
J91: (T) [W32] +191/2
K91: (T) [W32] +J91/2
L91: (T) [W32] +K91/2
TABLE 31
SUBSTITUTE StlE~ tRULE 263
CA 0222~682 1997-12-23
W O 97/01344 PCT~US96/10921
- 82 -
Formulss for analysis of cell growth
based upon ratios of -' ; st the end of a test B102: (T) W10 +B101/2to aLau. ' - at the onset of a test (as applied for MTT assay) C102: (T) W31 +C21/$H$95
D102: (T) W31 +D21/$HS9S
Headers for section of formulas E102: (T) W32 +EZI/$H$95
D94: (T) [W31] ~TABULATIONS OF GROWTH = N/N0 F102: (T) W32 +F21/$H$95
F94: (T) [W32] 'FROM IMMEDIATE PLATE: G102: (T) [W32] +G21/SH$95
J94: (T) [W32] ~Ratio H102: (T) [W31] +H21/$H$95
K94: (T) [W32] ~Fraction 1102: (T) [W32~ +121/$H$95
1102: (T) [W32] +J21/$H$95
E9S: (T) [W32] ~N0 = irnmediate assay = K102: (T) [W32] +K21/$H$95
F9S: (T) i-W32] 'enter mean absorbance vaiue L102: (T) [W32] +L21/$H$95
l9S: (T) [W32] +C17/+H9S N102: (T) iW16] +021/$H$95
K9S: (T) i~W32] +H9S/C17
B103: (1) W10 +B102/2
B98: (T) [W101 +$C$5 C103: (T) W31 +C22/$H$95
N98:(T)[W16] 11 D103:(T) W31 +D22/$H$95
E103: (T) W32 +E22/$HS9S
B99: (T) [W10] +$E$5 F103: (T) W32 +F22/$H$95
G103: (T) [W321 +G22/$H$95
Numbers in C99 to N99 indicate positions H103: (T) rW31] +H22/$H$95
of cotumns in the 96-well plate 1103: (T) [W32] +122/SH$95
C99: (T) W31 1 J103: (T) [W32] +J22/$HS9S
D99: (T) W31 2 K103: (T) [W32] +K22/$H$95
E99: (T) W32 3 L103: (T) [W32] +L22/$H$95
F99: (T) W32 4 N103: (T) [W16] + 022/$H$95
G99: (T) [W32] ~
H99: (T) [W31] G B104: (T) W10 +B103/2
199: (T) [W32] 7 C104: (T) W31 +C23/$H$95
J99: (T) [W32] 8 D104: (T) W31 +D23/SH$95
K99: (T) [W32] 9 E104: (T) W32 +E23/SHS9S
L99: (T) [W32] 10 F104: (T) W32 +F23/SH$95
M99: (T) [W16] 11 G104: (T) i-W32] +G23/$H$95
N99: (T) [W16] 12 H104: (T) [W31] +H23/$H$95
1104: (T) i-W32] +123/SH$95
Formulas J104: f~T) [W32] +J23/SH$95
B100: (T) W10 +$D$5 K104: (T) [W32] +K23/$HS95
D100: (T) W31 +DI9/$H$95 L104: (T) iW32] +L23/SHS9S
E100: (T) W32 +EI9/$H$95 N104: (T) [W16] +023/SHS9S
F100: (T) W32 +FI9/$H$95
G100: (T) [W32~ +GI9/$H$95 B105: (T) W10 +B104/2
H100: (T) [W31] +HI9/$H$95 C105: (T) W31 +C24/$H$95
1100: (T) [W32] +119/$H$95 D105: (T) W31 +D24/$H$95
J100: (T) [W32] +JI9/$H$95 E105: (T) W32 +E24/$H$95
K100: (T) [W32] +KI9/$H$95 F105: (T) W32 +F24/$H$95
L100: (T) ~W32] +LI9/$H$95 G105: (T) [W32] +G24/SH$95
N100: (T) [W16] +OI9/$H$95 H105: (T) iW31] +H24/$H$95
1105: (T) [W32] +124/$H$95
B101: (T) W10 +B100/2 J105: (T) [W32] +J24/SH$95
C101: (T) W31 +C20/$HS95 K105: (T) [W32] +K24/$H$95
D101: (T) W31 +D20/$H$95 L105: (T) iW32] +L24/$HS9S
E101: (1~ W32 +E20/$H$95 N105: (T) [W16] +024/$H$95
F101: (T) W31 +F20/$HS9S
G101: (T) [W32~ +G20/$H$95 B107: (T) W10 'MEANS
H101: (T) [W31] +H20/$H$95 C107: (T) W31 +C29/$H$95
1101:(T)~W32]+120/$H$95 . D107:(T) W31 +D29/$H$95
J101: (T) [W32] +J20/$H$95 E107: (T) W32 +E29/$H$95
~ K101: (T) [W32] +K20/$H$95 F107: (T) W32 +F29/$H$95
L101: (T) IW32] +L20/$H$95 G107: (T) [W32~ +G29/$H$95
N101: (T) [W16] +020/$H$95 H107: (T) [W31] +H29/$H$95
1107: (T) [W32] +129/$H$95
J107: (T) [W32] +J29/$H$95
K107: (T) [W32] +K29/$H$95
L107: (T) [W32] +L29/$H$95
N107: (T) [W16] +C17/H95
TABLE 3J
SUB~ E~ (~U~E 26)
CA 02225682 l997-l2-23
W O 97/01344 PCTrUS96/10921
-83-
Formulas continued
B109: (T) [W10] +$E$7
C109: (T) rW31] +D7
D109: (T) [W31] +C109/2
E109: (T) [W321 +D109/2
F109: ~T~ rW32] +E109/2
G109: ~Tl lW32] +F109/2
H109: (T) rW31] +G109/2
1109: (T) rW32] +H109/2
J109: (T) rVV32] +1109/2
K109: ~T~ rUV32] +J109/2
L109: ~T~ rVV32] +K109/2
N109: (T) rW16] 0
TABLE3K
SUBSmUTE SHE~ (RUI E 26)
CA 0222~682 l997-l2-23
W O 97/01344 PCT/US96/10921
-84-
Section of ~ c ' ' for - - of immediate data
C120: (T) W31 +A139
Hesders forsection D120: (T) W31 +A140
Bl lO: (T) [W10] +D7 E120: (T) W32 +A141
F120: (T) W32 +A142
D113: (T) [W31] "IMMEDIATE DATA G120: (T) ~W32~ +A143
E113: (T) [W32] 'EXP # H120: (T) LW31] +A144
F113: (T) [W32] +G14 1120: (T) [W32~ +A145
J120: (T) [W32] +A146
A114 (1~ [W8] '~/File, Import/CommaJ~Clear/a:leial*.~~ K120: (T) [W32] +A147
Al ; ~ data from n diskette is entered into L120: (T) IW32] +A148
cells A115 through A210 by the macro in ~ ' ' - cell A114 M120: (T) [W29] +A 149
This dnta (not shown) is trnnsfered to n tnbular formnt in cells N120: (T) LW16] +AI50
C118 through N125 ns indicnted below (in C118 through N125)
C 121 : (T) W31 +A 151
Headers for section D121: (T) W31 +A152
C 116: (T) [W31] ~IMMEDIATE MTT DATA E 121: (T) W32 +AI 53
D116:(1')[W31]'PLATE# F121:(T) W32 +A154
E116: (T) [W321 'enter plate # G121: (T) [W32] +AI55
H121: (T) [W31] +A156
Numbers in C117 to N117 indicnte positions 1121: (T) IW32] +A157
of columns in the 96-well pblte J121: (T) [W32] +A158
Cl 17: (T) W31 I K121: (T) IW32] +AI 59
D117: (T) W31 2 L121: (T) [W32] +A160
E117: (T) W32 3 M121: (T) lW29] +A161
F117:(T) W32 4 N121:(T)[W16]+A162
G117: (T) [W32] 5
H117: (1') lW31] 6 C122: (T) W31 +A163
1117: (T) lW32] 7 D122: (T) W31 +A164
J117: (T) [W32] 8 E122: (T) W32 +A165
K117: (T) IW32] 9 F122: (T) W32 +A166
L117: (T) [W32] 10 G122: (T) [W32] +A167
M117: (1) lW29] 11 H122: (T) [W31] +A168
N117: (~1) [W16] 12 1122: (T) [W32] +A169
1122: (T) tW32] +A170
Beginning of Tnble ' ~ cells) K122: (T) lW32] +A171
with t r ~cl ~ dnta L122: (T) [W32] +A172
C118: (T) W31 +AI I5 M122: (r) [W29] +A173
D118: (T) W31 +A 116 N 122: (T) lW16] +A174
E118: (T) W32 +A117
F118:(T) W32 +A118 C123:(T) W31 +A175
G118: (T) [W32] +AI I9 D123: (T) W31 +A176
H118: (T) IW31] +A120 E123: (T) W32 +A177
1118: (T) [W32] +A121 F123: (T) W32 +A178
J118: (T) IW32] +A122 G123: (T) [W32~ +A 179
K118: (T) IW32] +A123 H123: (T) lW31] +A180
L118: (T) [W32] +A124 1123: (T) [W32] +A181
M 118: (T~ [W29] +A 125 l 123: (T) [W32] +A 182
N118: (T) [W16] +A126 K123: (T) lW32] +A183
L123: (T) lW32] +A184
Cl 19: (T) W31 +A127 M123: (T) lW29] +AI 85
Dll9:(T) W31 +A128 N123:(T)[W16]+A186
E119: (T) W32 +A129
Fl19: (T) W32 +A130 C124: (T) W31 +AI87
Gl l 9: (T) [W321 +A 131 D 124: (T) W31 +A 188
H119:(T)rW31]+A132 E124:(T) W32 +A189
1119: (T) [W32] +A133 F124: (T) W32 +A190
1119: (T) [W32] +A134 G124: (T) [W32] +A191
K119: (T) [W32] +A135 H124: (T) [W31] +A192
Ll l 9: (T) lW32] +A 136 1124: (Tj [W32] +A 193
Ml l9: (T) [W29] +A137 J 124: (T) lW32] +A194
Nl l9: (T) [W16] +A138 K124: (T) [W32] +A195
L124: (T) [W32] +A196
M124: (T) lW29] +A197
N124: (T) [W16] +A198
TABLE3L
SUElSTlTUTE 5H EE~ tRULE 2~)
CA 02225682 l997-l2-23
W O 97/01344 PCT/US96/10921
-85-
Mean data from columns of wells in immediate ~ssay plate
for use as N,(MlTasssyinExsmples)
C125: (T) W31 +AI99 D126: (T) LW31] 1
D125: (T) W31 +A200 E126: (T) [W32] 2
E125: (T) W32 +A201 F126: (T) [W32] 3
F125: (T) W32 +A202
G125: ('1') lW32~ +A203 B127: ('1) W10 ~MEAN
H125: (T) [W31] +A204 C127: (T) W31 ~n),AVG(C118... C125)
1125:(T)lW32]+A205 D127:(T) W31 ~ ~AVG(D118.... D125)
1125:(T)1W32]+A206 E127:(T) W32 (-~AVG(E118.... E125)
K125: (T) rW32] +A207 F127: (T) W32~ ~a)AVG(F118..... F125)
L125~ W32] +A208 G127: (T) lW321 (~AVG(G118..... G125)
M 125: (T) [W29] +A209 H127: (1) [W31] ~ IAVG(HI I 8.. H125)
N125: (T) lW16] +A210 1127: (T) [W32] (-'AVG(1118.. 1125)
J127: (T) rW32] Ca,)AVG~J118..J125)
End of ~ - cells K127:(T)IW32]~O,AVG(K118........ K125)
witht. - .cd ~ 1 data L127:(T)[W32](~'AVG(L118........ L125)
M127: (T) IW29] (--,AVG(M118..... M125)
N 127: (T) [W 16] (nlAVG (N118.. N125)
TABLE 3M
SUBSTITUTE SHEET ~RUI E 2~i)
CA 0222~682 l997-l2-23
W O 97/01344 PCT~US96/10921
-86- =
HeadersforTabul:ltionofCellLossin% B136:(F2) WlO +B135/2
C136: (F2) W31 1-C103
C 130: IW31] 'TABULATIONS OF CELL LOSS = I -(N/N0) D 136: (F2) W31 1 -D 103
1130: [W32] "N0 = E136: (F2) W32 1-E103
J130: [W32] +H96 F136: (F2) W32 1-F103
G136: (F2) tW32] 1-G103
C131: ~F2) IW31] 'REM: DELETENEGATIVE VALUES H136: (F2) IW31] 1-H103
1136: (F2) [W32] 1-1103
Numbers in C132 to L132 indicate positions J136: (F2) lW32] 1-J103
of the columns in the 96-well phlte K136: (F2) lW32] 1-K103
C 132: U W31 I L136: (F2) LW32] I-LI 03
D132: U W31 2
E132: U W32 3
F132: U W32 4 B137: (F2) W10 +B136/2
G132: U [W321 5 C137: (F2) W31 1-C104
H132: U [W31] 6 D137: (F2) W31 1-D104
1132: U [W32] 7 E137: (F2) W32 1-E104
J132: U [W32] 8 F137: (F2) W32 1-F104
K132: U IW32] 9 G137: (F2) lw32] 1-G104
L132: U IW32] 10 H137: (F2) [W31] 1-H104
1137: (F2) [W32] 1-1104
Formul:ls for % Cell Loss J137: (F2) [W32] 1-J104
K137: (F2) [W32] 1-K104
B133: (F2) W10] +$D$5 L137: (F2) lW32] 1-L104
C133: (F2) W31] 1-C100
D133: (F2) W31] 1-D100 B138:(F2) W10 +B137/2
E133: (F2) W32] 1-E100 C138: (F2) W31 1-C105
F133: (F2) W32] 1-F100 D138: (F2) W31 1-D105
G133: (F2) iw32] 1 -G100 E 138: (F2) W32 1 -E105
H133: (F2) lW31] I-H 100 F138: (F2) W32 1 -F105
1133: (F2) [W32] 1-1100 G138: (F2) [W32J l-G105
J133: (F2) lW32] 1-J100 H138: (F2) lW31] 1-H105
K133: (F2) lW32] 1-K100 1138: (F2) lW32] 1-1105
L133: (F2) lW32] 1-L100 1138: (F2) [W32] 1-J105
K138: (F2) [W32] 1-K105
B134: (F2) W10' +B133/2 L138: (F2) [W32] 1-L105
C134: (F2) W31 1-C101
D134:(F2) W31 1-D101 B140:(F2)'W10 "Means
E134: (F2) W32 1-E101 C140: (F2) W31 1-C107
F134: (F2) W32 1-F101 D140: (F2) W31 1-D107
G134: (F2) iw32] 1-G101 E140: (F2) W32 1-E107
H134: (F2) [W31] 1-H101 F140: (F2) W32 1-F107
1134: (F2) [W32] 1-1101 G140: (F2) ~W321 1-G107
J134: (F2) [W32] 1-J101 H140: (F2) [W31] 1-H107
K134: (F2) [W32] 1-K101 1140: (F2) [W32] 1-1107
L134: (F2) [W32] 1-L101 J140: (F2) [W32] 1-J107
K140: (F2) [W32] 1-K107
B135: (F2) W10 +B134/2 L140: (F2) [W32] 1-L107
C135: (F2) W31 1-C102
D135: (F2) W31 1-D102 D142: (F2) IW31] +C142/2
E135: (F2) W32 1-E102 E142: (F2) [W32] +D142/2
F135: (F2) W32 1-F102 F142: (F2) [W32] +E142/2
G135: (F2) lW32] 1-G102 G142: (F2) [W32] +F142/2
H135: (F2) [W31] 1-H102 H142: (F2) [W31] +G142/2
1135: (F2) IW32] 1-1102 1142: (F2) [W32] +H142/2
J135: (F2) [W32] 1-J102 J142: (F2) [W32] +1142/2
K135: (F2) [W32] 1-K102 K142: (F2) [W32] +J142/2
L135: (F2) [W32] 1-L102 L142: (F2) [W32] +K142/2
TABLE3N
SUBS 111 UTE SHE~ tRULE 26)
CA 0222~682 1997-12-23
W O 97/01344 PCT~US96/10921
-87-
Formulas for the CRR to be used in an LDH
or; , ~ '- assay
B42: (F0) W9 +E5 B44: (F0) W9 +B43/2
C42: (F2) W7 ~IAVG(M19.. N19)/$C$17 C44: (F2) W7 ~AVG(M21.. N21)/$C$17
D42: (Fl) W7 +C42/($C42+D$50) D44: (Fl) W7 +C44/($C44+D$50)
E42: (F0) W6' 'blank well E44: (F2) W6 +C21/$CS17
G42: (F2) W6 +DI9/$C$17 F44: (Fl) W6' +E44/($C44+E$50)
H42: (Fl) W6 +G42/($C42+G$50) G44: (F2) IW61 +D21/SCS17
142: fi2) IW6] (El9/SC$17) H44: (Fl) [W6] +G44/($C44+GS50)
142: (Fl) [W6~ +142/($C42+1$50) 144: (F2) tW6] (E21/SCS17)
K42: (F2) [W6] (F19/SC517) J44: (Fl) [W6] +144/(SC44+1S50)
L42: (Fl) [W6] +K42/($C42+K$50) K44: (F2) [W6] (F21/SCS17)
M42: (F2) [W7] (Gl9/$CS17) L44: (Fl) [W6] +K44/($C44+KSS0)
N42: (Fl) [W7] +M42/($C42+M$50) M44: (F2) [W7] (G21/SCS17)
042: (FZ) [W7] (H19/$CS17) N44: (Fl) W7] +M44/($C44+MSS0)
P42: (Fl) rW6] +042/($C42+0$50) 044: (F2) W7] (H21/$CS17)
Q42: (F2) W6 (119/SCS17) P44: (Fl) ,W6] +044/(SC44+0SS0)
R42: (Fl) w6 +Q42/($C42+Q$50) Q44: (F2) W6](121/$CS17)
S42: (F2) W6 (J19/$CS17) R44: (Fl) W61 +Q44/($C44+QSS0)
T42: (Fl) WS +S42/($C42+SSS0) S44: (F2) W6 (J21/$CS17)
U42: (F2) W6 (Kl9/$CS17) T44: (Fl) WS +S44/($C44+S$50)
V42: (Fl) W5 +U42/($C42+U$50) U44: (F2) W6 (K21/$CS17)
W42: (F2) [W~] (L19/$C$17) V44: (Fl) WS +U44/(SC44+US50)
X42: (Fl) [W5] +W42/($C42+W$50) W44: (F2) [W~] (L21/$CS17)
X44: (Fl) [WS] +W44/($C44+WSS0)
B43: (F0) W9 +B42/2
C43: (F2) W7 ~AVG(M20.. N20)/$C$17 B45: (F0) W9 +B44/2
D43: (Fl) W7 +C43/($C43+D$50) C45: (F2) W7 ~AVG(M22.. N22)/SCS17
1~43: (F2) w6 +C20/$CS17 D45: (Fl) W7 +C45/(SC45+DSS0)
F43: (Fl) W6 +E43/(SC43+ESS0) E45: (F2) W6 +C22/SCS17
G43: (F2) [W6] +D20/$CS17 F45: (Fl) W6 +E45/(SC45+ESS0)
H43: (Fl) tW6] +G43/($C43+G$50) G45: (F2) [W6~ +D22/$CS17
143: (F2) [W6] (E20/SCS17) H45: (Fl) [W6] +G45/(SC45+GSS0)
J43: (Fl) [W6] +143/($C43+1$50) 145: (F2) [w6] (E22/SCS17)
K43: (F2) [w6] (F20/SCS17) J45: (Fl) [W6] +145/(SC45+1$50)
L43: (Fl) [W6] +K43/($C43+K$50) K45: (F2) [W6] (F22/SCS17)
M43: (F2) [W7] (G20/$CS17) L45: (Fl) [W6] +K45/($C45+KS50)
N43: (Fl) [W7] +M43/($C43+M$50) M45: (F2) [W7] (G22/SCS17)
043: (F2) [W7] (H20/SCS17) N45: (Fl) [W7] +M45/($C45+M$50)
P43: (Fl) [W6] +043/($C43+0$50) 045: (F2) [W7] (H22/$C$17)
Q43: (F2) -W6 (120/SCS17) P45: (Fl) [W6] +045/($C45+0$50)
R43: (Fl) W6' +Q43/(SC43+Q$50) Q45: (F2) w6 (122/SCS17)
S43: (F2) w6 (J20/$CS17) R45: (Fl) w6 +Q45/($C45+Q$50)
T43: (Fl) WS +S43/($C43+S$50) S45: (F2) ,w6 (J22/SCS17)
U43: (F2) W6 (K20/$C$17) T45: (Fl) W5 +S45/($C45+SS50)
V43: (Fl) WS +U43/($C43+U$50) U45: (F2) W6 (K22/$CS17)
W43: (F2) [W6] (L20/SC$17) V45: (Fl) WS +U45/(SC45+USS0)
X43: (Fl) [WS] +W43/($C43+W$50) W45: (F2) [W6] (L22/$CS17)
X45: (Fl) [W5] +W45/($C45+W$50)
TABLE 30
Sl~ $~1EE~ LE 26~
CA 0222~682 1997-12-23
W O 97/01344 PCT~US96/10921
-88
-
B46: (F0) [W9 +B45/2 BS0: [W9] 'Release Multiple
C46: (F2) [W7 ~AVG(M23.. N23)/$C$17 ES0: (F2) [W6] ~AVG(C25.... C26)/$CS17D46: (Fl) [W7 +C46/($C46+D$50) GS0: (F2) [W61 ~AVG(D25.... D26)/$C$17
E46: (F2) [W6 +C23/$C$17 150: (F2) [W6] ~AVG(E25.... E26)/$C$17F46: (Fl) [W6 +E46/(SC46+E$50) ICS0: (F2) [W6] ~!AVG(F25.... F26)i$C$17G46: (F2) [W61 +D23/$CS17 MS0: (F2) [W7] QAVG(G25...... G26)/$C$17H46: (Fl) [W6] +G46/($C46+G$50) OS0: (F2) [W7] C!AVG(H25..... H26)/SC$17146: (F2) [W6] (E23/$C$17) QS0: (F2) [W6] ( ;),AVG(125.. 126)/$CS17J46: (Fl) [W6] +146/($C46+1SS0) SS0: (F2) [W6] ~.AVG(125.. J26)/SCS17
K46: (F2) [W6] (F23/$C$17) US0: (F2) [W6] C~AVG(K25... K26)/$C$17
L46: (Fl) [W6] +K46/($C46+K$50) W50: (F2) [W6] ~~Av~(l 7~ 1 ~6)/$C$17
M46: (F2) [W7] (G23/$CS17)
N46: (Fl) [W7] +M46/($C46+M$50) BSI: U [W9] +F7
046: (F2) IW7] (H23/SC$17) CSI: U [W7] +C7
P46: (F I ) ~W6] +046/($C46+0$50) ES 1: (F I ) [W6] +E7
Q46: (F2) W6' (123/$CS17) GSI: (Fl) [W6] +ESI/2
R46: (Fl) W6 +Q46/($C46+Q$50) ISI: (Fl) [W6] +GSI/2
S46: (F2) ,W6 (J23/$CS17) KSI: (Fl) [W6] +ISI/2
T46: (Fl) WS. +S46/($C46+S$50) M51: (Fl) [W7] +KSI/2
U46: (F2) W6, (K23/$C$17) O51: (Fl) [W7] +M51/2
V46: (Fl) W5 +U46/($C46+U$50) Q51: (Fl) [W6] +OSI/2
W46: (F2) [W~] (L23/$C$17) SSI: (Fl) [W61 +Q51/2
X46: (Fl) [W5] +W46/($C46+W$50) U51: (Fl) [W6] +SSl/2
WSI: (Fl) [W6] +USI/2
B47: (F0) W9 +B46/2 - -
C47: (F2) W7 ~AVG(M24N24)/$CS17
D47: (FI) W7 +C47/($C47+D$50)
E47: (F2) W6 +C24/SCS17
F47: (F l ) W6 +E47/(SC47+E$50)
G47: (F2) [W6] +D24/$C$17
H47: (Fl) [W6] +G47/($C47+G$50)
147: (F2) [W6] (E24/SCS17)
J47: (Fl) [W6] +147/(SC47+1SS0)
K47: (F2) [W6] (F24/SC$17)
L47: (Fl) [W6] +K47/(SC47+KS50)
M47: (F2) [W7] (G24/$CS17)
N47: (Fl) [W7] +M47/($C47+M$50)
047: (F2) [W7] (H24/$CS17)
P47: (Fl) [W6] +047/($C47+0$50)
Q47: (F2) 'W6' (124/$C$17)
R47: (F l ) W6 +Q47/($C47+Q$50)
S47: (F2) W6 (J24/SCS17)
T47: (Fl) W5 +S47/(SC47+S$50)
U47: (F2) W6 (K24/$CS17)
V47: (Fl) W5 +U47/(SC47+U$50)
W47: (F2) [W~] (L24/$CS17)
X47: (Fl) [WS] +W47/($C47+W$50)
TABLE3P
$~ a~E~ (~UIE2G)
CA 02225682 l997-l2-23
W O 97/01344 PCT~US96/10921
-89-
O O .., co ~ O
~ O --
.. ~~e 0~ t ~ ~
al
~ C~o
_ _ _ ~ C~ ~ --
~ 0~ a~ ~ ~ O
~ ~ . O
~> ~ r_ o c~
ae ~ ;;e
I_ <D C" ~ _ _ O
~r ~ ~ ~ CD G~ O
_ _ _ ~ _ _ _
cn ~ ~ c~ 9 o
<.~ o ~ o
O C~
~ 0~ ~ ~ 0
~ ~ ~ ~ R ~ ~ ~ O
cn ~ ~ ~ q ~ O:
~ _ _ _ ~
O _
z L O 0~ ~ ~ ~ ~ 0~ O.
. 0~ , ~ ;~ O
C~ ~ ~ ~ o~
~1 Z
I--m5 ~ , o
9 ~ 4, ~ C .
T ~
~ 01 ~ ~Q ~ ~ O
~ . ~ O
O --~ ~r, ~ _ _ =
~ ~ 9~'-
~ -o 0~ q ~ ~ ~
''~ O o .~ _ _ _ _
~ ~ . 0 0 00
Q ~ 1~
I~ _ _ ~ ~- _ ~
~ ~ ~ ~o ~
C~l ~ ~ ~O ~~ ~ * m ~n O ~ . ==
a~ ~ c~
-- ~) ~
~ ~ CO
SUI~STITVTE SHEE~ (RULE 26)
CA 02225682 l997-l2-23
W O 97/01344 PCTrUS96/10921
-90-
O O . O O O O ~n --~ ~=-- -- ~' ~ - ~ '
,0 ,~, g ~ ~ ~ O g ~) ~ ~ O ~ a2 ~
d o o-- O r~ 0 0 r- ~ 0 " ' ~ o
o o o o o o -- . -- . -- o
-- _ _ o o o _ o ~ ~ ~ ~ ~ ~ ~ ~ ~ o
O o o ~ _ - o _ ~ o
~_ ~ <~ ~ _ o o _ o o ~ 0 ~ i~ ~ ~ o
C~ ~ ,
~ 0 m O O O C~ ~. ~ O ~'' In , ~ 0
~i
_ _ O _ O c, ~ ~D 0 0 ~> <~ . O
_~ ,0 ~ ~ O <oD O O O _ ~' ~
o ~ o ~~ ~ - 0 8 ~ ~ ~ ~ ~ ~ ~ o
~ ' ~ O 0 0 0 0 ~ ~D ~ O
~ cn
~ ' '~ o ' o o- - o o. o ~
o : O ~ ~:
0 ~ 0 -- ~ I . .I 0 0 0 0 0 0 ~r~ o
I I ~ . ~ 0, ~ _ 0, m ~ -::~ o:::3 o o - _ o
z ~ ~ ~, ~ ~ _ O C~ 0 0 0 0 0 0 o O
Q Z
O ~ O ~ O~ 3 O O ~ ~ o
,0, .0 ' ~ ~ o . O
C~ , O, ~_~0Ocn g O 0 0 0 0 0 0 ~~ o
~ ~ ~ ~ ~ _ = ~ 3 ~ ~ 0 ~
_ _ __ _
~_ ~ ~ 0 'J ~ ~0D ~ ~~ ~ :~ 0 ~ 0 ~ 0 0 ~ --
~ > _ O 3 O O
2 ~ .D 0~, ~ 0 0 0 0 0 ~e oO,
I _ ~ o o o . o o, . o o o
S ~ ' o O O O ~ ~ ~ 0 0 ~ U~ C Y
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SUBSTlTUTE SHE~ ~RULE 26~
CA 02225682 l997-l2-23
W O 97/01344 PCTrUS96/10921
-91 -
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W O 97/01344 PCT~US96/10921
-92-
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W O 97/01344 PCTrUS96/10921
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W O 97/01344 PCT~US96/10921
-94-
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CA 02225682 1997-12-23
W O 97/01344 PCT~US96/10921
_95_
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W O 97/01344 PCTAUS96/10921
-96-
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CA 02225682 l997-l2-23
WO 97/01344 PCT/US96/10921
-97-
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CA 02225682 l997-l2-23
W O 97/01344 PCTrUS96/10921
-98-
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W O 97/01344 PCTrUS96/10921
_99_
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CA 02225682 l997-l2-23
W O 97/01344 PCTrUS96/10921
-100-
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CA 02225682 l997-l2-23
W O 97/01344 PCTrUS96/10921
-101-
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CA 02225682 1997-12-23
W O 97/01344 PCTrUS96/10921
-102-
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CA 02225682 l997-l2-23
W O 97/01344 PCTrUS96/10921
-103-
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CA 02225682 1997-12-23
W O 97/01344 PCTrUS96110921
-104-
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CA 02225682 l997-l2-23
W O 97/01344 PCT~US96/10921
-105-
Table 33. Sumrnar~ of Data for Synergistic Matches in U937 cells
RA TCI data for
combined results ratio
BrdU STSP Table 9
dAde STSP Table 10
dGuo STSP Table 11
HU STSP Table 12
MTX STSP Table 13
Floxuridine STSP Table 14
Aph STSP Table 15
ara-C STSP Table 16
STSP bleomycin Table 17
STSP mitomycin C Table 18
STSP ci.~pl~tin Table 19
STSP daunorubicin Table 20
STSP etoposide Table 21
dThd K252A Table 22
dThd KT5926 Table 23
dThd KT5720 Table 24
dThd Aph Table 25
dThd Ara-C Table 26
HU K252A Table27
HU Aph Table 28
dThd cisplatin Table 29
HU cisplatin Table 30
Aph K252a Table 31
etoposidedaunorubicin Table 32
SU8STTTUTE SHEE~ (RULE 26)
CA 02225682 l997-l2-23
W O 97/01344 PCTrUS96/10921
-106- ~
Table 34. Tumor formation in athymic nude mice innoc~ te~l with human m~ t
Iymphoma cells (U937)
Treatment group # Mean tumor weight Standard Error p Value
(grams) compared to
control
Control 3 2.22 0.36
dThd only 4 1.69 - 0.65 > 0.1
STSP only 4 0.78 0.52 >0.05
dThd/STSP 4 0.0 > 0.01
Su~9Ulk~~~ (RUlE26)
CA 02225682 1997-12-23
W O 97/01344 PCTrUS96/10921
-107-
Table 35 Examples relating SMAX for specified RA to blood levels previously reported in chemotherapy
- RA S~rA~C~ Examples plasma levels# references
dThd0.05-0.5 mM Table4 upto 6 mM Blumenreich
- MS et al.
HU 0.06-0.5 mM Table 12 2.5 mM Belt RJ et al.
MTX 20-80 nM Table 13 up to 1 mM Allegra CJ et al.
Aph .02-1 ,ug/ml Table 15 up to 3 ,ug/ml Sessa C et al.
ara-C0.05-0.3 ~bM Table 16 up to 50 ~M Calabresi P &
Chabner BA
STSP 20-30 nM Table 17 # UP to 500 nM Buchholz RA
~ The range of SMAX was ascertained in relevant examples in~ tf~rl
# Conservative estirnate of ~ x i, -1- plasma level based upon total blood volume
in dog and human
SUBST1TUTE S~ (RULE 26~
CA 02225682 1997-12-23
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-108-
Table 36 Characteristics of human m~lign~nt cell lines which were succPs~fully treated by dThd
or other RA to potentiate the action of STSP or other TCI
Human ATCC Cell type p 53 Clinical Source
Cell line ID #(~ Expression
U937 CRL promonocyte absent l\/i~lign~nt Iymphoma/large cell
1 593 histiocytic
HL-60 CCL promyelocyte absent Chronic myelogenous leukemia
240
Daudi CCL B-cell mutant l~ lign~nt lymphoma/Burkitt's
213 type
Raji CCL 86 B-cell mutant ~align~nt lymphoma/Burkitt's
type
Jurkat TIB 152 T-cell mutant Acute T-cell leukemia
C33A HTB 31 epithelial mutant cervical cancer, negative for
human papillomavirus
entifir~tion number from catalog of American Type Culture Collection, Rockville, MD 20852
SUg~ u~t$WEE~(~UIE2'6)
CA 02225682 1997-12-23
W O 97/01344 PCT~US96/10921
-109-
Table 37. Evidence for cell dependence of IC40 and SMAX in synergistic matches with STSP.
RA Cellline IC40 sblAx
dThd Hl-60 promyelocytes >0.4 mM 0.04-O.lmM
JurkatTCellleukemia >3mM 0.1-lmM
Aph Daudi lymphoma cells >8 ,ug/ml 0.5-3 ,ug/ml
C33 A cervical carcinoma >8 ~g/ml 0.8-8 ,ug/ml
SW~a~ S~EE~ (~IJIE26)
CA 02225682 1997-12-23
W O 97101344 PCT/US96/10921
-1 10-
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