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Auckland Cancer Society Research Centre, The University of Auckland, Private Bag 92019, Auckland, New Zealand
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAppendix I. PK/PD Model...Appendix II. PK Model...REFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAppendix I. PK/PD Model...Appendix II. PK Model...REFERENCES |
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A recent initiative in chemotherapy has been the development of drugs that exploit features of the tumor microenvironment (15)
, as shown by the benzotriazine-di-N-oxide TPZ, which is selectively cytotoxic to hypoxic cells (16
, 17)
and is currently in clinical trial (18)
. The PK (penetration) problem is especially critical for hypoxic cytotoxins; not only are their target cells distant from functional blood vessels, necessitating long extravascular diffusion distances, but these "bioreductive" drugs are activated by metabolic reduction via oxygen-inhibited pathways as shown for TPZ in Fig. 1
. This necessarily consumes the drug as it diffuses, potentially compromising its extravascular transport. Making multicellular spheroids more hypoxic has been shown to cause apparent resistance of the innermost cells to TPZ, consistent with such an extravascular transport problem (19)
. In addition, studies with the MCL model, in which tumor cells are grown on a permeable support to form multicellular layers, have demonstrated more directly that TPZ transport is impeded by rapid drug metabolism under hypoxic conditions (20, 21, 22)
. The latter studies included mathematical simulations that suggested the penetration problem to be severe enough to confer apparent resistance, although this conclusion depended on PD parameters (cytotoxicity) measured in other cell lines and was not tested experimentally. Thus, the two types of observations in the literature (resistance in spheroids, impeded transport in MCLs) have not been combined in a way that makes it possible to assess quantitatively whether the PK (penetration) problem is the main contributor to the observed PD (resistance) problem with TPZ. This is the main objective of the present study.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAppendix I. PK/PD Model...Appendix II. PK Model...REFERENCES |
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Growth of Monolayers, MCL, and Spheroids.
HT29 human colon carcinoma cells (obtained from Dr. David Ross, University of Colorado, Denver, CO) were passaged in 
MEM (Life Technologies, Inc., Grand Island, NY) with 5% fetal bovine serum (Life Technologies, Inc., Auckland, New Zealand) without antibiotics and were confirmed to be free of Mycoplasma using a PCR-ELISA assay (Roche Diagnositics GmbH, Mannheim, Germany). MCLs (Fig. 2)
were grown on collagen-coated Teflon supports (Millicell-CM cell culture inserts; Millipore Corp., Bedford, MA) as described previously (24
, 25)
by seeding 106 cells. After allowing cells to attach for 6 h, the inserts were submerged in stirred MEM containing 10% fetal bovine serum, 2 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 µg/ml), and grown for an additional 3 days. This medium was used for all subsequent experiments. Multicellular spheroids were grown in the same medium by seeding 105 cells into bacteriological P100 Petri dishes; 7 days later, they were transferred to Bellco spinner flasks (Invitrogen, CA) and grown for an additional 8 days with medium replacement every 3 days. Single cell suspensions were prepared by enzymatic dissociation of MCL or spheroids in 0.07% trypsin (Difco Laboratories, Detroit, MI) in saline containing trisodium citrate (14 mM, pH 7.6) for 10 min.
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Uptake, Metabolism, and Cytotoxicity in Single-Cell Suspensions.
Stirred suspensions (0.5–5 x 106 cells/ml) derived from HT29 spheroids were incubated with TPZ at a range of initial concentrations under 20% O2 or anoxia (<200 ppm O2, as confirmed with an Oxylite oxygen probe; Oxford Optronix, Oxford, United Kingdom). Suspensions were equilibrated with the appropriate gas phase for 1 h before the addition of drug. Cells and medium were separated by rapid centrifugation (16,000 x g, 1 min), followed by a brief second spin to remove medium from the walls of the tube. Concentrations of TPZ and its mono-N-oxide derivative SR 4317 were determined by HPLC of the extracellular medium and cell pellets (see below). Cell killing was determined by clonogenic assay of separate cell pellets in the same experiments; cells were washed by centrifugation in fresh medium, and serial dilutions were plated in Falcon P60 tissue culture dishes (Becton Dickinson Biosciences, Bedford, MA) at up to 105 cells/dish. Dishes were stained with methylene blue (2 g/liter in 50% ethanol) 14 days later, and colonies containing 
50 cells were counted. The surviving fraction was determined as the ratio of plating efficiency of treated cells to that of controls (exposed to an equal concentration of DMSO) at the same time. Metabolism and survival data were fitted simultaneously to the model described in "Appendix I."
Analytical Methods.
TPZ and SR 4317 in extracellular medium was assayed by HPLC after deproteinizing samples with 20 µl/ml of 70% (v/v) perchloric acid, centifugation, and adjustment of pH to 7 with 31.5 µl/ml of 50% (v/v) aqueous ammonia. In cell uptake experiments, the cell pellet was lyzed with 100 µl of ice-cold water, followed by deproteinization with 80% acetonitrile containing N,N-bis(2-chloroethyl)-N-methyl-N-(4-methyl-2-nitrobenzyl)ammonium chloride, as internal standard (27)
, evaporated to 50 µl and made up to 200 µl with mobile phase for HPLC injection. The HPLC system was a HP 1100 (Agilent, Palo Alto, CA) equipped with a diode-array detector and an Alltima C8 (150 x 2.1 mm, 5 µm; Alltech Associated Inc., Deerfield, IL) reverse phase column at a flow rate of 0.5 ml/min and an injection volume of 10–100 µl. The mobile phase comprised a gradient of acetronitrile in 0.45 M ammonium formate (pH 4.5). Absorbance was monitored at 462 nm for TPZ (to avoid a small coeluting peak at 13.3 min, which absorbs at the TPZ maximum of 266 nm) and 415 nm for SR4317 (retention time, 19.0 min). Standards at known concentrations were included in each experiment. The peak area was linearly related to the amount injected in the range 0.01–10 nmol (r2 > 0.999), and recovery from medium was >95% for both analytes.
14C and [3H] activity was determined by scintillation counting in 5 ml of Emulsifier-Safe scintillant using a Packard Tricarb 1500 Liquid Scintillation Analyzer (Canberra Packard, Meriden, CT).
HT29 Cell Volume and Intracellular Volume Fraction in MCLs.
Stirred suspensions of HT29 cells, dissociated from MCLs, were incubated at 5 x 106 cells/ml under the same gassing conditions as the metabolism and survival experiments. After the addition of [3H]2O and [14C]mannitol to preequilibrated vials, samples of 0.5 ml were taken every 15 min for 1 h, and the extracellular medium and cell pellet [solubilized with 1 ml of Soluene-350 (Packard) at 60°C for 1 h] were assayed for [3H] and 14C activity by scintillation counting as above but using Hionic-Fluor (Packard) organic scintillant for the cell pellet. The intracellular water volume in the pellet was determined from the difference between the total and extracellular water volumes as estimated from the activities of the [3H]2O and [14C]mannitol tracers, respectively. No time trend was observed, so all values were averaged. Intracellular volume fraction in HT29 MCLs was determined similarly, under the same conditions as flux experiments, after a 1-h equilibration after adding the tracers to both sides of the MCLs. Excess medium was aspirated, and the total [3H] and 14C activity in the MCL was determined after its removal and solubilization as above.
Diffusion of TPZ through MCLs (Flux Experiments).
MCL experiments were performed in custom-built diffusion chambers (25)
in which the MCL separates two well-stirred compartments, each containing 7 ml of medium under 5% CO2 in 95% O2 or 95% N2, which maintained pH at 7.4 ± 0.1 throughout the experiments. MCLs were equilibrated in the diffusion chambers for 1 h at 37°C before removal of 130 µl from the donor compartment and its replacement with an equal volume of medium containing TPZ (final concentration, 1–100 µM) and [14C]urea (internal standard; final concentration, 3 µM). Samples of 0.5 ml were taken from both compartments at intervals; 25 µl were used for scintillation counting, and the balance was stored at -80°C for HPLC. Similar experiments were performed using collagen-coated inserts without MCLs to check for chemical stability and to determine the effective diffusion coefficient of TPZ in the support membrane. Diffusion coefficients were determined from flux data by fitting the concentration-time profiles in both the donor and receiver compartment simultaneously, using the approach described in "Appendix II." The thickness of each MCL was determined from the flux of [14C]urea, using the diffusion coefficient of this internal standard as determined separately in HT29 MCLs, in which thickness was measured by frozen section immediately after the experiment (see "Results").
Cytotoxicity of TPZ in Anoxic MCLs.
MCL cytotoxicity studies were performed in the same apparatus as the transport studies. MCLs were equilibrated under anoxia for 2 h, during which time [14C]urea flux was measured to determine the thickness of each MCL as above. TPZ was then introduced into both donor and receiver compartments (0, 50, 75, or 100 µM), and MCLs were incubated for an additional 1 or 2 h. Initial and final TPZ concentrations were measured by HPLC. After exposure, the MCLs were trypsinized and plated for clonogenic assay.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAppendix I. PK/PD Model...Appendix II. PK Model...REFERENCES |
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5 x 106 cells/ml). Given that the mass balance was dominated by the extracellular compartment, it was appropriate to monitor overall metabolism by assaying the extracellular medium only.
The kinetics of TPZ metabolism under anoxia was determined by following TPZ loss and SR 4317 formation at a range of cell densities and initial TPZ concentrations (10–150 µM), as shown for a typical experiment in Fig. 3
. TPZ loss was first order at high concentrations, but faster relative metabolism was evident at <10 µM. No loss of TPZ was seen in anoxic culture medium without cells, even at low concentrations (data not shown). For each experiment, the TPZ and SR 4317 concentrations were simultaneously fitted to a metabolism model (see "Appendix I," Eq. A2
) with first-order and saturable (Michaelis-Menten) terms as shown in Fig. 3
. The estimated metabolism parameters were then averaged across experiments and used as initial parameter estimates to fit all data across all experiments simultaneously (28 concentration-time profiles), to give the values shown in Table 1
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. Anoxic cytotoxicity was greater for high than low TPZ concentrations at equivalent concentration (C) x time (T), as shown by the greater killing with 100 µM TPZ for 0.5 h than 50 µM for 1 h in Fig. 4
. The C-T dependence was fitted well (Fig. 4
, lines) by a PD model in which the rate of log cell killing is proportional to the product of the TPZ concentration and the rate of its metabolism (see "Appendix I"). At low cell density, when the TPZ concentration can be approximated as a constant, this model gives a log cell kill approximately proportional to exposure time and concentration squared (see "Appendix I").
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. At this time, MCL thicknesses were approximately 140 µm. Previous studies have shown that transport of TPZ into hypoxic MCLs is significantly impeded by its bioreductive metabolism (20, 21, 22)
. Thus, prediction of PD response in MCLs requires information about the TPZ concentration-time profile as a function of distance into the MCL.
Diffusion coefficients were determined following the approach described previously for EMT6 and MGH-U1 MCLs (20)
, but using an apparatus that allowed measurement of TPZ concentrations on both sides of the MCL (22
, 25)
; this more tightly constrains fitting of the data to the transport model (see "Appendix II"). Typical raw data are shown for TPZ and the internal standard, urea, in HT29 MCLs in Fig. 5
. The key parameter values are reported in Table 2
. First, the apparent diffusion coefficients of urea and TPZ were determined in the collagen-coated Teflon support membrane (Ds). Second, flux of urea through Teflon supports bearing MCLs of known thickness [197 ± 22 µm (mean ± SE) for six MCLs as determined by histology] was fitted to determine the diffusion coefficient for urea in HT29 MCLs (DMCL). All these values were independent of O2 concentration (data not shown). TPZ flux through MCLs was then investigated using 95% O2 in the gas phase, which effectively suppressed bioreductive metabolism as shown by the TPZ mass balance and lack of formation of SR 4317 in Fig. 5
. The data were well fitted as simple Fickian diffusion, with DMCL as the sole fitted parameter, using the flux of the urea internal standard to estimate the thickness of each MCL. DMCL for TPZ was independent of the initial TPZ concentration in the donor compartment over the range 1–90 µM (Fig. 6A)
, with a best estimate of (0.40 ± 0.01) x 10-6 cm2s-1 (Table 2)
. This was approximately 2-fold and 3-fold lower that the TPZ diffusion coefficients measured previously in V79 and MGH-U1 MCL (0.74 ± 0.03 and 1.3 ± 0.2 cm2s-1), respectively (20)
. Similar trends were seen in urea and sucrose diffusion coefficients determined in previous studies (20
, 24)
and probably reflects differences in extracellular space and tortuosity between MCL grown from different cell lines.
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, and flux into the receiver compartment was suppressed, with accompanying formation of SR 4317 (Fig. 5D)
. These features, coupled with the lack of effect of anoxia on urea flux (Fig. 5, A and B)
, pointed to bioreductive metabolism in the MCL as the reason for impeded TPZ transport. We tested whether the decrease in TPZ flux could be quantitatively accounted for by the rate of TPZ metabolism observed in single cells above. This required scaling the single cell metabolism parameters to correct for the higher cell density in MCLs. We used [3H]2O and [14C]mannitol as markers for total and extracellular water volumes, respectively, to determine the average intracellular water volume in cell pellets of HT29 cells dissociated enzymatically from MCLs (1.23 pl4
). Using this measured cell volume, we then estimated the intracellular volume fraction in MCLs (
c) needed to account for the transport impediment in anoxic MCLs by fitting the anoxic MCL flux data using the oxic DMCL and the single cell metabolism model. The fitted value of c showed no significant trend with concentration (Fig. 6B)
, provided that both the Michaelis Menten and first order terms were included. We then asked whether the average value of c corresponds to the actual cell volume fraction in intact MCLs by measuring the latter directly, again using the [3H]2O/[14C]mannitol method. The measured MCL intracellular volume fraction, e, was indeed in good agreement with c (Table 1)
. The concentration independence of c, and its agreement with e, demonstrates that the metabolism model in single cells provides an excellent description of TPZ metabolism at the tissue-like cell densities in MCLs.
TPZ Cytotoxicity in HT29 MCLs.
Cell killing was quantified by exposing anoxic MCLs to TPZ at up to 100 µM for 1 or 2 h, with the drug added to both sides of the MCLs, then dissociating with trypsin to determine clonogenic survival. Cytotoxicity was greatly reduced relative to exposure of single cell suspensions under equivalent conditions, as shown in Fig. 7A
. This ignores any difference in actual TPZ concentrations in the cells as a result of compromised transport into anoxic MCLs. To test whether the transport problem quantitatively accounts for the apparent resistance in MCLs, we calculated the expected killing using the PK (transport) and PD (cytotoxicity) parameters determined above. For each MCL, the effective thickness was determined from the urea flux (before the addition of TPZ), and TPZ concentration-time profile and cell killing was calculated as a function of distance from the MCL surface as shown for a representative MCL in Fig. 7B
. Spatially averaging across this MCL gave a predicted mean surviving fraction of 0.20, whereas a value of 0.002 would be expected in single cell culture under the same exposure conditions. The measured surviving fraction for this MCL was 0.23. Across the 23 MCLs investigated, the predicted surviving fractions were in good agreement with measured cell killing (Fig. 7C)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAppendix I. PK/PD Model...Appendix II. PK Model...REFERENCES |
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. To assess the extent to which this multicellular resistance is caused by failure of TPZ to penetrate anoxic MCLs, we have measured the transport of TPZ through HT29 MCLs. This provided a mathematical model of TPZ transport that has allowed us to calculate the concentration-time profile as a function of distance into MCLs (Fig. 7B)
. We have also developed a PK/PD model that relates cell killing to TPZ exposure, and to its metabolic activation, in isolated HT29 cells. Combining the PK/PD model for cell suspensions with the MCL transport model has enabled us to predict cell killing as a function of position in MCLs (Fig. 7B)
. Averaging this calculated surviving fraction over the whole MCL provided predicted values in good agreement with the measured (average) surviving fraction (Fig. 7C)
. The success of this spatially resolved PK/PD model indicates that there is no need to invoke any mechanism other than drug penetration limitations to account for the apparent resistance of cells in multicellular structures to TPZ. The corollary of this is that the intrinsic sensitivity of HT29 cells to TPZ is the same whether these cells are exposed in intact MCLs or as single cells, at least when the cells are exposed to the drug promptly after their isolation from MCLs. This has implications for the development of new analogues of TPZ, suggesting that critical elements of the overall PK/PD model such as rates of anoxic metabolism and cytotoxic potency can be determined meaningfully using single cell suspensions. We are currently using this approach, in conjunction with measurement of penetration through MCLs and the oxygen dependence of metabolism and cytotoxicity, to model response in HT29 tumors, and have recently demonstrated that this has strong predictive value (unpublished data).
The finding that extravascular transport limitations account for resistance of MCL cultures to TPZ does not necessarily preclude the existence of other forms of multicellular resistance if these persist for several hours after enzymatic dissociation of the three-dimensional structures. The "contact effect," first demonstrated by the resistance of cells in small spheroids to ionizing radiation (1)
, is known to show such persistence (4
, 6) . Similarly, resistance because of cytokinetic changes in MCLs, or to other relatively slow changes in gene expression, cannot be expected to reverse quickly after trypsinization of MCLs or spheroids. Whether there are subsequent changes in sensitivity as cells adapt to growth as log-phase monolayers is not addressed by this study, but we see it is an advantage that the PK/PD model approach described here does not require use of monolayers that potentially introduce artifacts in chemosensitivity testing (5
, 6
, 13)
. Given that TPZ has been shown to act as a hypoxia-selective TopoII poison (28)
, and that cells in three-dimensional contact can be resistant to TopoII poisons relative to monolayers because of changes in TopoII phosphorylation and nuclear accumulation (29)
, differences in TPZ sensitivity might well be expected between monolayers and cells in MCLs or tumors. Changes in sensitivity to apoptosis via cell contact (5)
could also contribute and might become important in modeling tumor response if cell contact after TPZ exposure suppressed apoptotic cell killing.
Determination of the parameters of the PK/PD model for HT29 cells, as required for this study, has also strengthened understanding of key steps in the mechanism of action of TPZ. The anoxic cytotoxicity of TPZ against HT29 single cell suspensions is in good agreement with an independent study showing similar cell kill after 1-h exposure of HT29 cell suspensions (30)
. The approximate quadratic dependence on concentration seen with HT29 cells is a general finding across cell lines (30
, 31)
. We show (see "Appendix I") that the observed dependence of killing on C2 x T at low cell density is consistent with the proposed "dual action" of TPZ cytotoxicity (32
, 33)
. In this model, the rate of killing is proportional to the rate of metabolism of TPZ to a DNA-oxidizing radical (shown as TPZ* in Fig. 1
) and to the TPZ concentration. The latter is consistent with the proposed second step in the action of TPZ in which it oxidizes the initial DNA radicals to generate DNA strand breaks, shown as step 2 in Fig. 1
. The importance of the first step is consistent with other studies that have demonstrated a strong correlation between the rate of TPZ metabolism and cytotoxicity in several cell lines (30
, 31) .
The detailed investigation of TPZ metabolism in HT29 cells extends previous studies with cell lysates and isolated enzymes (34
, 35) and cell suspensions (30
, 31
, 36
, 37)
, the latter study reporting first order metabolism in HT29 cells at rates comparable with the present study. The previous studies did not detect saturable metabolism of TPZ at low concentrations, but the presence of two kinetic components was clearly evident over the wide concentration range investigated in this study. The kinetic data do not define the Km of this component with precision (Table 1)
, but the saturable reductase(s) are important at concentrations <10 µM, which makes them significant in anoxic MCLs when the input TPZ concentration is 25 µM. We also predict that the Michaelis-Menten kinetic component will be important in vivo, because PK/PD modeling indicates that much of the exposure in hypoxic regions is at concentrations below 10 µM.5
The nonsaturable first order component may be largely attributable to reduction by cytochrome P450 reductase, which is a promiscuous electron donor in the endoplasmic reticulum (38)
and is probably the quantitatively major TPZ reductase (39
, 40)
. It is tempting to speculate that the saturable component is caused by the TPZ reductase(s) in the nuclear matrix, which is considered to make a disproportionate contribution to TPZ cytotoxicity (41)
. However, there is no indication from our PK/PD modeling that the saturable component makes a larger contribution to cell killing.
This study further extends the use of the MCL model for cytotoxicity (PD) studies, in which the effective thickness of the tissue can be measured accurately by the flux of a tracer (e.g., urea) during the experiment. To achieve the level of accuracy required for this study, precise measurement of MCL thickness and medium TPZ concentrations was essential because small differences in the penetration distance or initial drug concentration can lead to large differences in the average cell kill. The present validation of MCLs as an experimental model for quantifying TPZ transport, and the spatially resolved PK/PD model for cytotoxicity in MCLs, supports the use of these tools for developing an analogue of TPZ in which extravascular transport limitations are minimized. More generally, the present study points to a methodology with considerable potential for assisting lead optimization in anticancer drug development by providing information on PK as a function of distance from blood vessels in tumors. Thus, in vitro studies with multicellular layers have the potential to not only elucidate the relative importance of transport limitations in multicellular resistance but also to introduce transport considerations in the early stages of drug design. This PK/PD model is currently being extended by incorporating the O2 dependence of TPZ metabolism and cytotoxicity, and by applying it to three-dimensional diffusion in tumor microvascular networks.
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Appendix I. PK/PD Model for TPZ Metabolism and Cytotoxicity in Single Cell Suspensions. |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAppendix I. PK/PD Model...Appendix II. PK Model...REFERENCES |
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 | (A1) |
is the intracellular volume fraction determined from the cell density (Coulter count) and cell volume, [TPZ] is the concentration of TPZ averaged over the whole culture volume at time t, and [TPZ]0 is the corresponding initial TPZ concentration. Given the low cellular uptake factor for TPZ (see "Results"), [TPZ] is well approximated by the extracellular concentration at low values of . Anoxic TPZ metabolism in stirred single cell suspensions had both a first order and saturable (Michaelis-Menten) component over the concentration range examined; thus
 | (A2) |
 | (A3) |
by an empirical model in which the rate of fall in the log-surviving fraction (SF) was proportional to the rate of its bioreductive metabolism and to the TPZ concentration:
 | (A4) |
is a proportionality constant. The data fit was improved by including a lag period, Tlag, in which there was no cell killing:
 | (A5) |
gave more consistent values (coefficient of variation, 2.0%). All equations were solved numerically using a fourth order Runge-Kutta method in Modelmaker version 4.0 (Cherwell Scientific Ltd., Oxford, UK), fitting all [TPZ], [SR4317], and SF data simultaneously by nonlinear least squares regression. Note that in the limiting case of low cell density, [TPZ] 
[TPZ]0, the predicted amount of metabolism is approximately
 | (A6) |
 | (A7) |
 | (A8) |
by linear regression for each experiment. |
Appendix II. PK Model for TPZ Transport in MCLs. |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAppendix I. PK/PD Model...Appendix II. PK Model...REFERENCES |
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 | (A9) |
 | (A10) |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH National Cancer Institute Grant PO1-CA82566 and by the Health Research Council of New Zealand. 
2 To whom requests for reprints should be addressed, at Auckland Cancer Society Research Centre, The University of Auckland, Private Bag 92019, Auckland, New Zealand. Phone: 64-9-3737-599, ext. 86939; Fax: 64-9-3737-571; E-mail: k.hicks{at}auckland.ac.nz
3 The abbreviations used are: PD, pharmacodynamic; HPLC, high-performance liquid chromatography; MCL, multicellular layer; PK, pharmacokinetic; TPZ, tirapazamine.
4 There was a significant (P < 0.01) difference in cell volume between anoxic (0% O2) cell suspensions (1.40 ± 0.02 pl; n = 12) and hyperoxic (95% O2) cell suspensions (1.09 ± 0.06 pl; n = 16). For purposes of scaling the intracellular volume fraction from cell suspensions to MCLs, the pooled average of these values has been used.
5 F. B. Pruijn, J. R. Sturman, H. D. S. Liyanage, K. O. Hicks, M. P. Hay, and W. R. Wilson, Extravascular transport of drugs in tumor tissue: effect of lipophilicity on diffusion of tirapazamine analogs in multicellular layer cultures, submitted for publication.
Received 11/18/02. Revised 5/15/03. Accepted 7/ 8/03.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAppendix I. PK/PD Model...Appendix II. PK Model...REFERENCES |
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correlate with etoposide resistance in multicell spheroids and xenograft tumors.. Cancer Res., 60: 5747-5753, 2000.This article has been cited by other articles:
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  W. R. Wilson, S. M. Stribbling, F. B. Pruijn, S. P. Syddall, A. V. Patterson, H.D. S. Liyanage, E. Smith, K. J. Botting, and M. Tercel Nitro-chloromethylbenzindolines: hypoxia-activated prodrugs of potent adenine N3 DNA minor groove alkylators Mol. Cancer Ther., October 1, 2009; 8(10): 2903 - 2913. [Abstract] [Full Text] [PDF]
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 O. Tredan, A. B. Garbens, A. S. Lalani, and I. F. Tannock The Hypoxia-Activated ProDrug AQ4N Penetrates Deeply in Tumor Tissues and Complements the Limited Distribution of Mitoxantrone Cancer Res., February 1, 2009; 69(3): 940 - 947. [Abstract] [Full Text] [PDF]
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 O. Tredan, C. M. Galmarini, K. Patel, and I. F. Tannock Drug Resistance and the Solid Tumor Microenvironment J Natl Cancer Inst, October 3, 2007; 99(19): 1441 - 1454. [Abstract] [Full Text] [PDF]
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 A. V. Patterson, D. M. Ferry, S. J. Edmunds, Y. Gu, R. S. Singleton, K. Patel, S. M. Pullen, K. O. Hicks, S. P. Syddall, G. J. Atwell, et al. Mechanism of Action and Preclinical Antitumor Activity of the Novel Hypoxia-Activated DNA Cross-Linking Agent PR-104 Clin. Cancer Res., July 1, 2007; 13(13): 3922 - 3932. [Abstract] [Full Text] [PDF]
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K. O. Hicks, F. B. Pruijn, T. W. Secomb, M. P. Hay, R. Hsu, J. M. Brown, W. A. Denny, M. W. Dewhirst, and W. R. Wilson Use of three-dimensional tissue cultures to model extravascular transport and predict in vivo activity of hypoxia-targeted anticancer drugs. J Natl Cancer Inst, August 16, 2006; 98(16): 1118 - 1128. [Abstract] [Full Text] [PDF]
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U. Emmenegger, G. C. Morton, G. Francia, Y. Shaked, M. Franco, A. Weinerman, S. Man, and R. S. Kerbel Low-Dose Metronomic Daily Cyclophosphamide and Weekly Tirapazamine: A Well-Tolerated Combination Regimen with Enhanced Efficacy That Exploits Tumor Hypoxia Cancer Res., February 1, 2006; 66(3): 1664 - 1674. [Abstract] [Full Text] [PDF]
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 L. A. Kunz-Schughart, J. P. Freyer, F. Hofstaedter, and R. Ebner The Use of 3-D Cultures for High-Throughput Screening: The Multicellular Spheroid Model J Biomol Screen, June 1, 2004; 9(4): 273 - 285. [Abstract] [PDF]
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B. G. Siim, F. B. Pruijn, J. R. Sturman, A. Hogg, M. P. Hay, J. M. Brown, and W. R. Wilson Selective Potentiation of the Hypoxic Cytotoxicity of Tirapazamine by Its 1-N-Oxide Metabolite SR 4317 Cancer Res., January 15, 2004; 64(2): 736 - 742. [Abstract] [Full Text] [PDF]
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), which decays to form a DNA-oxidizing radical (TPZ*) such as the hydroxyl radical 
, 
, 
: 50 µM; 
, 
: 14 µM). A, TPZ loss. B, SR4317 production. The data were fitted to the metabolism model outlined in "Appendix I" 
