Pharmacokinetic/Pharmacodynamic Modeling Identifies SN30000 and SN29751 as Tirapazamine Analogues with Improved Tissue Penetration and Hypoxic Cell Killing in Tumors

Hypoxia, frequently considered a hallmark of cancer, is a consequence of the inefficient vascularization of tumors (1). In a number of tumor types, hypoxia contributes to progression and is associated with poor prognosis and resistance to therapeutic agents through multiple mechanisms. For example, in the context of radiation therapy, hypoxic cells are less sensitive to radiation-induced DNA breakage and cell killing (2) and have increased invasive and metastatic potential (3), leading to failure because of metastatic disease outside the radiation field (4, 5). Additional hypoxia, induced through the antiangiogenic action of radiation (6), enhances tumor regrowth by stimulating vasculogenesis (7). These features, along with the relative absence of hypoxia in normal tissues (8), provide a strong rationale for targeting hypoxia, either by inhibiting pathways required for hypoxic cell survival (9, 10) or through the metabolic activation of bioreductive prodrugs under hypoxic conditions (11–13).

The most thoroughly investigated bioreductive prodrug, tirapazamine (TPZ), showed encouraging indications of activity in early clinical studies (14, 15) but failed to improve overall survival in a recent phase III trial with cisplatin/radiotherapy for advanced head and neck cancer (16). This disappointing outcome was shown to be at least partially due to failure in compliance with radiation therapy protocol (17) and is also likely to reflect lack of stratification for the most hypoxic tumors (18). Thus, there is a reasonable expectation that TPZ, or an improved analogue, could have a major impact on radiation therapy if developed appropriately.

Further exploration of TPZ analogues is merited because the benzotriazine di-N-oxide (BTO) class has two unique advantages over other bioreductive prodrugs for targeting hypoxia. First, the active cytotoxin is derived from the initial free radical reduction product (19, 20), which spontaneously transforms into DNA-damaging oxidizing radicals (21, 22), whereas its subsequent reduction products (the 1-oxide SR4317 and nor-oxide SR4330) are much less cytotoxic (19, 23). Hypoxic selectivity of TPZ derives from the rapid reoxidation of the initial radical to the parent prodrug by O₂ (20). In contrast, the active cytotoxins from other classes of bioreductive prodrugs (nitro compounds, quinones, and tertiary amine N-oxides) are 2-, 4-, or 6-electron reduction products, which, in many cases, can be generated by oxygen-insensitive two-electron reductases such as DT-diaphorase (24) or aldo-keto reductase 1C3 (25), compromising hypoxic selectivity. A second unique feature of TPZ is that it is activated under relatively mild hypoxia (26, 27); its oxygen sensitivity is approximately the inverse of that for radiotherapy, with half-maximal activation at ∼1 μmol/L O₂ in solution (27). One-electron reduction of other bioreductive prodrugs is more sensitive to inhibition by O₂ (28–30), which restricts activation to a smaller subset of essentially anoxic cells that are arguably of lesser importance for radiation therapy (31).

These features led us to ask whether the limited clinical activity of TPZ could be improved by rational drug design. Studies with three-dimensional cell culture models, including multicellular layers (MCL), have shown that TPZ is metabolized too rapidly to penetrate optimally into hypoxic tumor tissue (27, 32–35). We therefore hypothesized that improving the extravascular transport of TPZ would increase its therapeutic selectivity given that its penetration limitations will have a larger effect on activity in poorly perfused tumors than in well-perfused normal tissues. Measurement of tissue transport parameters in MCLs has made it possible to develop a spatially resolved pharmacokinetic/pharmacodynamic (SR-PKPD) model for TPZ, based on Green's function models of O₂ and TPZ transport, which describes its activity as a function of position within a microvascular network (36). This model has been validated as a tool for lead optimization by showing that it provides reliable prediction of activity against hypoxic cells in HT29 xenografts for a set of 16 BTOs (36). We have subsequently used the SR-PKPD model to guide lead optimization, focusing on lipophilic BTOs with potentially improved tissue diffusion coefficients (D; refs. 37, 38) but also holistically evaluating all other model parameters. The compounds include simple 3-amino-BTO derivatives (39, 40), 3-amino-BTOs with DNA-targeting functionality (41), 3-alkyl BTOs in which removal of the 3-amino H-bond donor usefully increases D (40, 42), and tricyclic triazine di-oxides (TTO) in which a lipophilic saturated ring fused to the 3-amino-BTO core also increases D (43). The corresponding 3-alkyl-TTO subclass (43) seeks to combine both features and builds on experience with the BTO series (39, 42) by adjusting other physicochemical properties to optimize the kinetics of bioreductive metabolism, solubility, and systemic PK.

Here, we identify a 3-alkyl BTO (SN29751) and a 3-alkyl TTO (SN30000) as the preferred compounds from this SR-PKPD–guided lead optimization program. We characterize their physicochemical properties, hypoxia-selective cytotoxicity, cellular pharmacology, and PK in plasma and (by modeling) in hypoxic regions of tumors. Further, we show that these novel TPZ analogues provide improved therapeutic activity against human tumor xenografts, making them potential development candidates for human therapy.

TPZ (44) and SN29751 (45) were synthesized as reported, and SN30000 by modification³

Cell lines, obtained from the American Type Culture Collection, were cultured as monolayers from Mycoplasma-free frozen stocks at <3-month intervals in αMEM with 5% heat-inactivated fetal bovine serum without antibiotics by weekly passage. Multicellular spheroids were grown in spinner flasks with 10% FCS and dissociated to give single-cell suspensions for drug metabolism and clonogenicity assays or for inoculation into mice. MCLs were grown as described (36).

As described previously (36, 39), log-phase cells were exposed to drugs for 4 hours under 20% O₂ or anoxia (Bactron anaerobic chamber), and growth was assessed by sulforhodamine B staining 4 to 5 days later. The IC₅₀ was determined by nonlinear regression to the standard Hill equation, and the intra-experiment hypoxic cytotoxicity ratio (HCR) calculated as aerobic IC₅₀/anoxic IC₅₀.

Metabolic consumption of drugs and clonogenic cell killing were measured simultaneously using magnetically stirred single-cell suspensions (1-2 × 10⁶ cells/mL) in medium without serum, equilibrated with 5% CO₂/N₂ (<100 ppm O₂) or 5% CO₂/20% O₂ as described previously (35). Samples were removed at intervals and centrifuged; supernatants were stored at −80°C for HPLC. The apparent first-order rate constant for drug metabolism (k_met) was determined from the concentration-time data by linear regression. Cell pellets were resuspended in fresh medium; cell number was determined by Coulter counter, and viability by 0.4% trypan blue exclusion. Cells were plated to determine clonogenic survival. The data were fitted using the previous (36) cellular PKPD model in which the rate of log cell kill [LCK; defined as the negative log of the surviving fraction (SF)] at time t is proportional to both the rate of drug metabolism and the parent drug concentration C:

where dM/dt = k_met × C/φ is the metabolized drug per unit cell volume, φ is the HT29 cell volume fraction determined from the cell count as previously described (35), γ is the proportionality constant, and C₀ is the initial measured drug concentration. To allow for decreasing drug concentrations during exposure, Eq. A was integrated to give

where the exposure integral E, at each sampling time, t, is given by:

E was plotted against log SF to obtain the potency coefficient γ by regression as previously validated for TPZ (35) and TPZ analogues (36).

Clonogenic cell killing was measured over a range of oxygen concentrations in stirred cell suspensions as previously described (27), using lower cell densities (3 × 10⁵ cells/mL) to minimize the effect of cellular respiration on solution oxygen concentrations (C_s), which were monitored using an OxyLite 2000 O₂ luminescent probe (Oxford Optronix Ltd). Drug concentrations were also monitored, at 30- to 60-minute intervals, by HPLC, and the cell survival data were fitted as above. The potency as a function of C_s was fitted to a Hill equation to estimate K_O2 (C_s to halve anoxic potency) by regression.

Diffusion through HT29 and SiHa MCLs was determined as described previously (36, 37) using a two-chamber diffusion apparatus (46). The medium was equilibrated with 95% O₂ to suppress bioreductive metabolism. Compounds were added to the donor compartments with ¹⁴C-urea and samples taken at intervals to assay drug (by HPLC) and ¹⁴C-urea (liquid scintillation counting) in both compartments. MCL thicknesses and drug diffusion coefficients (D_MCL) were fitted to the concentration-time profiles of urea and drug, respectively, using a Fick's second law mathematical model (36).

Drug concentrations were determined with a 150 mm × 2.1 mm Alltima C8 reverse phase column and Agilent 1100 HPLC using photodiode array detection. For in vitro studies, culture supernatants were analyzed by direct injection (35). Plasma samples (below) were analyzed following previous methods (36) after precipitation of proteins with acetonitrile, evaporation to dryness in a Speed-Vac concentrator, and reconstitution in 125 μL of 45 mmol/L ammonium formate buffer (pH 4.5), of which 100 μL were injected.

All animal experiments followed institutional protocols as previously described (36). Specific pathogen-free CD-1 nu/nu mice (∼25 g) or Sprague-Dawley rats (∼200 g) were ear-tagged and randomized to treatment groups. Freshly prepared solutions of compounds were given i.p. to mice at 20 mL/kg body weight or to rats at 10 mL/kg (25 mL/kg for TPZ), using 1.33-fold dose increments. The maximum tolerated dose (MTD) was defined as the highest dose that caused no drug-related deaths, body weight loss of more than 15%, or severe morbidity in a group of three to six animals, with an observation time of 28 days.

Mice were injected i.p. or i.v. at 75% of the MTD for each compound, with sampling by cardiac puncture under terminal CO₂ anesthesia. Rats were injected i.p. at 178 and 316 μmol/kg, and blood samples of 20 to 40 μL were obtained serially from the saphenous vein without anesthesia. Blood was immediately centrifuged (3,000 × g, 3 minutes), and plasma stored at −80°C for HPLC. Noncompartmental PK parameters were determined by WinNonLin v 5 (Pharsight Corp.).

Tumor xenografts were grown s.c. in the dorsal flank of CD-1 nude mice, 1.5 cm from the base of the tail, by inoculation of 10⁷ cells. Treatment was initiated when tumors reached a mean diameter of 9 to 11 mm, using i.p. dosing with either a single dose or multidose [bidaily at 9 a.m. and 3 p.m. for 4 consecutive days (BID1-4)] schedule, at various times before or after irradiation. Mice were irradiated without anesthesia using a cobalt-60 gamma source, either whole-body (15 or 20 Gy) for single dose studies or locally using a custom-built restraining jig for multidose studies (2 or 2.5 Gy × 8). Tumor response was assessed by clonogenic assay of cell suspensions from xenografts removed 18 hours after the end of treatment (excision assay) as previously described (36). LCK was calculated from the difference in log (clonogens/g) for treated and control groups. Statistical significance of drug effects was tested using one-way ANOVA with Dunnett's test for multiple comparisons. Alternatively, tumors were measured three times weekly using calipers until tumors reached three times the pretreatment volume (regrowth assay).

Oxygen and drug concentration gradients were calculated using Green's function methods in a mapped microvascular network (500 × 500 × 230 μm) from a rat R3230Ac tumor as previously described (36), based on the tissue diffusion coefficients and rate constants for drug metabolism measured in vitro above with inflow drug concentration defined by the measured plasma PK. Drug-induced cell kill at each point in the tumor microregion was then calculated using the above cellular PKPD model. Radiation-induced cell kill at each point was also calculated using reported linear-quadratic model parameters (36). The predicted surviving fraction for drug and radiation was averaged over the whole tumor microregion to calculate the overall LCK. The difference between the surviving fraction for drug with radiation and the surviving fraction for radiation alone gave the predicted drug-induced LCK in the radiation-resistant hypoxic cell population.

An overview of the algorithm we used to identify improved TPZ analogues is shown in Fig. 1A. Of 281 BTO and TTO compounds synthesized from five different structural classes, 225 had sufficient solubility to determine hypoxia-selective cytotoxicity in IC₅₀ assays. One hundred eighty-two passed decision point “A” in Fig. 1A (HCR >20 against both HT29 and SiHa cells), and 173 of these were evaluated in additional tissue culture models to determine the parameters required for SR-PKPD modeling. This included measurement of clonogenic cell killing and metabolic drug consumption under anoxia (first-order rate constant k_met,0) in stirred HT29 cell suspensions. We also calculated tumor tissue diffusion coefficients (D) from physicochemical parameters (MW, log P at pH 7, hydrogen bond donors and acceptors) using relationships previously established with a training set of BTOs in three-dimensional MCL cultures (38). The ranges of values for k_met,0 and D are shown in Fig. 1B, which illustrates the diversity of extravascular transport properties; compounds lying below the diagonal line are predicted to have superior penetration distances into hypoxic HT29 tissue relative to TPZ (40).

A first iteration of the SR-PKPD model was then run to predict the hypoxic cytotoxicity differential [HCD = LCK in the radiobiologically hypoxic region (<4 μmol/L O₂)/aerobic region (>30 μmol/L O₂)] of each compound in HT29 xenografts and the plasma area under the curve (AUC) required for a hypoxic LCK of >0.5. Compounds passed this decision point (“B” in Fig. 1A) if HCD >1 and the required AUC was <333 μmol/L h, which is twice that of TPZ at its MTD in initial PK studies in this mouse strain (36). For 115 compounds, we determined the MTD in CD-1 nude mice and the plasma AUC at 75% of MTD. The SR-PKPD model was then rerun using the measured AUC to identify compounds with LCK >0.3 at 75% MTD. Compounds passing this third decision point (“C” in Fig. 1A) were then evaluated in vivo by testing their ability to kill radiation-resistant hypoxic cells in HT29 xenografts following a single drug dose at 75% of MTD. Sixteen of the 18 compounds predicted to be active showed statistically significant activity, whereas a further 12 compounds predicted to be inactive were also tested to evaluate the algorithm and were all found to be inactive. SN29751 was identified as the most promising of the BTO series (42) and SN30000 as the preferred analogue in the new TTO series.⁴

A more detailed investigation of each component of the pharmacology of these new lead compounds was undertaken. The metabolism of the prodrugs in HT29 cultures, illustrated in Fig. 1D, showed hypoxia-dependent loss of the parent compounds with formation of the 1-oxides (and for SN30000 a trace of the nor-oxide 3) identified by comparison of retention time and absorbance spectra with authentic standards (Supplementary Fig. S1). For all experiments, the rate constant for bioreductive consumption (k_met,0) was highest for SN30000 (1.53 ± 0.21 min⁻¹; errors are SEM throughout), intermediate for TPZ (1.30 ± 0.04 min⁻¹), and lowest for SN29751 (0.87 ± 0.03 min⁻¹).

SN29751 and SN30000, like TPZ, showed hypoxia-selective cytotoxicity in HT29 and SiHa IC₅₀ (antiproliferative) assays, whereas the reduced metabolites were substantially less cytotoxic and lacked hypoxic selectivity (Fig. 2A). When this assay was extended to eight cell lines, SN30000 was consistently more potent than TPZ under anoxia, whereas SN29751 was less potent (Fig. 2B). Notably, the anoxic potency of both compounds was highly correlated with that of TPZ across the cell lines (Fig. 2B; R² = 0.97 and 0.93 for SN29751 and SN30000, respectively), suggesting a common mechanism of action. Consistent with its higher potency under anoxia, the HCR was consistently higher for SN30000 than for TPZ and SN29751 across all cell lines (Fig. 2C). These findings were confirmed for HT29 cells by clonogenic assay (Fig. 2D), which showed the highest anoxic potency (coefficient γ in Table 1) and HCR values for SN30000.

The diffusion coefficients of SN30000 and SN29751 in HT29 MCL, calculated from their physicochemical properties, were higher than that of TPZ (Fig. 1B). These predictions were confirmed by measuring penetration through aerobic HT29 MCL (Fig. 3A). At these high oxygen concentrations, no reduced metabolites were observed and diffusion coefficients (D_MCL) could be fitted as simple diffusion without metabolism (Fig. 3B); the values for SN30000 and SN29751 were respectively 3- and 2.5-fold higher than that for TPZ (Table 1). This same trend (SN30000 > SN29751 > TPZ) was seen in SiHa MCL (Supplementary Fig. S2).

We defined the oxygen dependence of SN29751 and SN30000 activation using the same approach as previously described (27), thus testing the assumption in the initial SR-PKPD screen (Fig. 1A) that this was the same as for TPZ. As illustrated for SN30000 (Fig. 3C), the slope of the survival versus exposure integral plots decreased with increasing oxygen concentration in solution. These slopes were used to determine cytotoxic potency as a function of oxygen (Fig. 3D). The oxygen concentration required to halve the anoxic potency of SN30000 and SN29751 (K_O2 ∼1 μmol/L; Table 1) was not significantly different (one-way ANOVA, P = 0.26) from that for TPZ. Metabolic consumption of the prodrugs, in the same experiments, showed similar oxygen dependence (Supplementary Fig. S3) with 50% inhibition at approximately 0.6 to 1 μmol/L O₂.

Following single i.p. administration, the MTD in male CD-1 nude mice was 1,000 μmol/kg (370 mg/kg) for SN29751 and 750 μmol/kg (275 mg/kg) for SN30000, that is, 5.6- and 4.2-fold higher molar doses than for TPZ (178 μmol/kg, 31.7 mg/kg), respectively. As summarized in Supplementary Table S1, these differences in host toxicity (SN29751 < SN30000 < TPZ) were also seen in the three other mouse strains investigated (C3H/HeN, C57/Bl6, and Rag-1^−/−) and with other dosing schedules in CD-1 nude mice. For example, the MTD for twice-daily dosing (9 a.m. and 3 p.m.) for 4 consecutive days was 421 μmol/kg for SN29751, 237 μmol/kg for SN30000, and 75 μmol/kg for TPZ.

Histopathology of CD-1 nude mice following single drug doses or the BID1-4 schedule at 1-1.3 × MTD showed a qualitatively similar organ toxicity profile for SN29751, SN30000, and TPZ (Supplementary Table S2). For single doses, bone marrow hypoplasia, gastrointestinal toxicity, and airway epithelium vacuolation were the most pronounced acute findings. Following the BID1-4 schedule, histopathologic changes were similar to the single-dose study for SN29751, but gastrointestinal toxicity was the only acute finding for SN30000 and TPZ. As reported previously for TPZ in mice (47), significant retinal toxicity was observed 28 days after treatment with all three agents (Supplementary Fig. S4).

Initial screening of PK following single i.p. doses at 75% MTD in CD-1 nude mice (Fig. 4A) indicated similar plasma half-lives of all three compounds (28, 35, and 32 minutes for TPZ, SN29751, and SN30000, respectively; Table 1). However, increased plasma concentrations were achieved for the analogues, resulting in AUC values of 49, 162, and 79 μmol-h/L for TPZ, SN29751, and SN30000, respectively, at these equitoxic doses (Table 1). Comparison with i.v. dosing showed an i.p. bioavailability of 77% for SN30000 (Supplementary Table S3).

Comparison of toxicity and plasma PK in Sprague-Dawley rats also showed higher MTD values for the analogues (178 μmol/kg for TPZ and 316 μmol/kg for SN30000 in both sexes; 316 μmol/kg for SN29751 in females and 421 μmol/kg in males) after single i.p. doses. Representative plasma PK is shown in Fig. 4B; C_max, terminal half lives, and AUC (Supplementary Table S4) were all higher for the analogues than for TPZ.

The measured parameters of the SR-PKPD model, summarized in Table 1, allow a complete description of PK and PD (cell killing) at each point within HT29 xenografts; the model output is shown in Fig. 4C for all three compounds at 75% of MTD in CD-1 nude mice. The model predicts a large increase in hypoxic cell killing by SN30000 and SN29751 relative to TPZ at equivalent host toxicity. In both cases, killing shows excellent complementarity to that calculated for a large single dose of radiation (20 Gy), which spares the hypoxic cells (dashed line in Fig. 4C). To model overall LCK by each drug when used in combination with radiation, we assumed independent action of both agents and summed all tissue regions for the combination, subtracting LCK for radiation alone. This additional LCK and the model-predicted hypoxic selectivity (HCD) both showed a clear increase for SN30000 and SN29751 relative to TPZ at equivalent host toxicity (Fig. 4D). We also simu`lated the activity of the compounds against an HT29 tumor notionally grown in Sprague-Dawley rats, based on the measured plasma PK at MTD. The spatially resolved PD predictions (Supplementary Fig. S5) again show a large improvement in hypoxic cell killing for the analogues relative to TPZ at equivalent host toxicity, and the combined activity with radiation for this “virtual therapeutic trial” (Fig. 4D) is again predicted to be markedly superior to TPZ.

The SR-PKPD model predictions were in good agreement with the measured killing of HT29 cells in xenografts by excision assay (Fig. 5A), which showed greater LCK for SN30000 and SN29751 than TPZ when administered at 75% of MTD 5 minutes after a single dose of radiation (to sterilize well-oxygenated cells). A similar large increase (∼3-fold) in hypoxic tumor cell killing was seen with SiHa and H460 xenografts (Fig. 5A). No significant activity was seen with the drugs alone (without radiation) against any of the xenografts, consistent with selective killing of the hypoxic subpopulation (data not shown). The analogues were also generally superior to TPZ when administered 30 minutes before each fraction of a BID1-4 radiotherapy schedule (Fig. 5A). The time course of interaction between the drugs and radiation against SiHa tumors (Fig. 5B) also showed higher activity of SN29751 and SN30000 and showed that the compounds were active both before and after irradiation, as for TPZ, showing that the mechanism is hypoxic cell killing rather than direct radiosensitization.

The activity of SN30000 with fractionated radiation was also evaluated in SiHa tumors using tumor regrowth as the endpoint (Fig. 5C), showing significant activity additional to radiation at the two highest drug doses tested. Comparison with TPZ in the same experiment is shown in Fig. 5D, where the time for tumor regrowth is compared with body weight loss (as a measure of toxicity). Both TPZ and SN30000 showed dose-dependent body weight loss and inhibition of tumor regrowth, but with greater antitumor activity for SN30000 relative to toxicity.

This study identifies novel TPZ analogues with improved formulation properties (aqueous solubility), cytotoxic potency against hypoxic cells in culture, tissue penetration characteristics, and therapeutic activity against hypoxic cells in multiple tumor models. Perhaps most notably, the approach we have taken represents a departure from the usual strategy for lead optimization during anticancer drug development in which potency and selectivity in monolayer cell cultures are used to select compounds for evaluation in in vivo models. In contrast, we use SR-PKPD modeling to make a holistic assessment of the molecular features that contribute to antitumor activity in vivo. The SR-PKPD model for TPZ analogues incorporates the relationships between prodrug metabolism, cytotoxicity, and oxygen concentration as determined in vitro; the systemic (plasma) pharmacokinetics of the compounds at tolerated doses; and their extravascular transport (tissue penetration) in tumors as assessed with the MCL model. The latter aspect enables calculation of drug concentration gradients in a representative microvascular network and, thus, prediction of cell killing probability at each position in the tumor tissue.

The utility of this SR-PKPD approach is shown by confirmed hypoxia-selective cell killing in HT29 xenografts for 16 of 18 compounds predicted by the model to be active (one of the two false positives showed a hypoxic LCK of 0.60 ± 0.36, which did not reach statistical significance) and the quantitative agreement between model prediction and measured hypoxic cell killing in HT29 xenografts for TPZ, SN29751, and SN30000 [compare Figs. 4A and 5A (left)]. In contrast, 12 of 12 compounds predicted by the SR-PKPD model to be inactive (despite good hypoxic selectivity in vitro) were confirmed as true negatives. This suggests that the assumptions adopted in the modeling are acceptable for this drug series, including that the microvascular geometry of the rat R3230Ac tumor (used as the basis for our Green's function oxygen and drug transport models) is representative of human tumor xenografts and that the intrinsic sensitivity of HT29 cells is the same in single-cell culture and tumors.

Notably, the inactive compounds in vivo showed hypoxic potency and selectivity for HT29 cells in culture in the same range as for the active compounds, and linear regression showed that neither hypoxic potency (IC₅₀; r² = 0.07, P = 0.16) nor selectivity (log₁₀ HCR; r² = 0.02, P = 0.47) in vitro correlated with hypoxic LCK in tumors (Supplementary Fig. S6). Thus, the key determinants of therapeutic activity are systemic PK relative to toxicity and intratumor PK [consistent with the evidence that this is a limiting feature for TPZ itself (32); refs. 27, 33–36]; these features are explicitly represented in the model. In the case of SN30000, the improvement over TPZ is mainly driven by its superior extravascular penetration (Fig. 3B; Supplementary Fig. S2) and higher cytotoxic potency under hypoxia (Fig. 2B-D), whereas for SN29751, improved systemic (plasma) PK (Fig. 4A) makes a larger contribution to its higher activity against xenografts in mice. The improved extravascular penetration of SN30000 is consistent with its higher lipophilicity (Table 1) and removal of the H-bond donor 3-NH₂ group, these features being the major determinants of MCL diffusion coefficients for TPZ analogues (38). The higher hypoxic cytotoxicity potency of SN30000 presumably reflects its 57-mV higher one-electron reduction potential⁵

Studies with three xenograft models show a substantial (∼3-fold) therapeutic gain for SN30000 and SN29751 over TPZ with single-dose radiation (Fig. 5A), and SR-PKPD model predictions based on plasma PK achievable in rats also indicate a large therapeutic advantage for the analogues over TPZ in this second species (Fig. 4D). SN30000 and SN29751 are also clearly superior to TPZ in combination with fractionated radiation (Fig. 5A, B, and D), although the magnitude of improvement with HT29 tumors seems to be less than for single dose radiation (Fig. 5A). This might reflect a lesser penetration problem for bioreductive prodrugs in combination with fractionated radiation if, as suggested (31), moderately hypoxic cells closer to blood vessels make a larger contribution to outcome than severely hypoxic cells in this setting.

The present study shows that the mechanism of action of SN30000 and SN29751 is similar to that of TPZ. All three compounds are metabolized under hypoxia to the corresponding nontoxic 1-oxides (Fig. 1D), with an indistinguishable cell line dependence as hypoxic cytotoxins (Fig. 2B). Consistent with this, we have recently shown a strong correlation between TPZ and SN30000 reductive metabolism under hypoxia in a panel of 16 cell lines (48). In addition, the relationship between γH2AX formation and clonogenic cell killing is the same for TPZ, SN30000, and SN29751, and Chinese hamster ovary cell lines defective in homologous recombination repair show similar hypersensitivity to all three compounds,⁶

PK evaluation of anticancer drugs during clinical development is typically limited to describing concentration-time profiles in plasma, rather than in the effect compartment within tumors. The spatially resolved PK model described here provides a unique opportunity to simulate PK in hypoxic regions of tumors during the phase I trial of SN30000 and to compare this with an analogous simulation for TPZ based on its reported plasma PK at MTD in humans (50). We plan to link this to PD prediction in humans using the cellular PKPD model described here, essentially as for the virtual therapeutic trial in rats represented by Fig. 4D. This will provide an early indication, independent of response biomarkers, whether SN30000 offers a therapeutic gain over TPZ as a hypoxic cytotoxin for use in humans.

W.R. Wilson, J.M. Brown, and W.A. Denny: consultants, Proacta, Inc.; M.P. Hay, K.O. Hicks, F.B. Pruijn, B.G. Siim, W.A. Denny, and W.R. Wilson: inventors on patents related to SN30000 and SN29751; W.R. Wilson and W.A. Denny: commercial research grant, Proacta, Inc.

We thank Dianne Ferry for assistance with bioanalysis and Drs. Jingli Wang and Thorsten Melcher for critical comments on the manuscript.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.