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Pharmacyclics, Inc., Sunnyvale, California 94085 [R. A. M., K. W. W., Q. F., I. L., D. M., D. M.], and Stanford University School of Medicine, Division of Oncology, Stanford, California 94305 [G. D., B. S.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10-fold using 100 µM MGd. In clonogenic assays using Rif-1 cells, MGd enhanced the activity of Bleo 1000-fold. This effect was shown to be mediated, in part, by MGd inhibition of potentially lethal damage repair. MGd enhanced the tumor response to bleomycin and Dox in vivo. MGd had no significant effect on the systemic exposure to Dox (expressed in terms of the plasma area under the curve, 0–24 h) and did not increase Dox myelosuppression. MGd enhanced the effectiveness of the redox active drugs, Bleo and Dox. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The texaphyrins are metal cation-binding macrocycles that bear strong resemblance to naturally occurring porphyrins. For instance, it has now been demonstrated in both animal tumor models and human clinical trials that texaphyrins accumulate selectively in cancers, similar to certain porphyrins (4, 5, 6, 7) . However, texaphyrins are far easier to reduce (more electron affinity) than typical metalloporphyrins with a half-wave potential of approximately -50 mV (versus normal hydrogen electrode; Ref. 8) . These properties have led to the consideration that metallotexaphyrins might function as radiation-enhancing agents. Animal tumor model studies have demonstrated that the texaphyrin, MGd,2 selectively accumulates in cancers and renders tumors more responsive to ionizing radiation (4 , 7 , 9) . Promising Phase II clinical results also have been reported in patients receiving radiation therapy for brain metastases (6 , 10) . In human Phase I and II studies, selective tumor localization of MGd has been confirmed using magnetic resonance imaging, which can detect the drug based on its paramagnetic properties (5 , 6 , 10) .
Biological redox reactions are central to metabolism and cellular energy production. Recent studies with MGd have indicated that it catalytically reacts with various intracellular reducing metabolites (antioxidants), such as ascorbate and NADPH, to produce hydrogen peroxide and other reactive oxygen species (11) . We hypothesize that the depletion of intracellular reducing metabolites and resulting bioenergetic disruption because of futile redox cycling would lead to increased tumor response to both radiation therapy and chemotherapy. This could explain the results of Bernhard et al. (12) , who failed to observe radiation enhancement with MGd. In this report, we describe the effects of MGd on tumor response to two chemotherapy drugs, Bleo and Dox. These chemotherapeutic agents were selected because they are redox active and known to generate reactive oxygen species within cells (13 , 14) .
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The development and characterization of the human cell line, MES-SA, derived from sarcoma elements of a uterine mixed Müllerian tumor, have been described previously (15) . Monolayer cultures of MES-SA cells were grown in McCoy’s 5A medium supplemented with 10% FCS, 25 mM HEPES (Life Technologies, Inc., Grand Island, NY), 2 mM L-glutamine, and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin; Sigma). Murine EMT-6 mammary sarcoma and Rif-1 radiation-induced fibrosarcoma cell lines were maintained through established in vivo/in vitro propagation procedures (16) . Rif-1 cells were grown in RPMI 1640 supplemented with 15% FCS and penicillin/streptomycin. EMT-6 cells were grown in Waymouth’s medium (MB752/1; Life Technologies, Inc.) supplemented with 15% FCS and penicillin/streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.
In Vitro MTT Cytotoxicity Assays.
MES-SA cells were allowed to adhere to 96-well microtiter plates (4000 cells/well) overnight in 180 µl of McCoy’s 5A medium. A freshly prepared stock solution of Bleo in medium (60 µl) was serially diluted (1:4) down the plate, discarding the final 60 µl. Stock solutions of MGd (1 mM, 750 µM, 500 µM, or 250 µM diluted in medium and 5% mannitol) were added to the plates to give a final volume of 200 µl. Final mannitol concentration was 0.25% in all wells. The plates were incubated at 37°C in a 5% CO2 in air atmosphere. Medium was exchanged after 24 h, and the plates were incubated an additional 48 h prior to analysis for viability using the tetrazolium dye MTT (17)
. In brief, a 5-mg/ml solution of dye in PBS (20 µl/well) was added, and after 2 h the medium was removed and replaced with 100 µl of isopropanol to dissolve the formazan crystals formed by the cells. The plate was read at a test wavelength of 570 nm and a reference wavelength of 650 nm on a multiwell spectrophotometer (ThermoMax; Molecular Devices, Sunnyvale, CA). Each concentration of drug was tested in quadruplicate. The percentage of survival was defined as the percentage of the absorbance of the drug-treated cells to that of the control. Data were adjusted to normalize for effect of MGd treatment alone, which produced 15–45% cytotoxicity in the range of MGd concentrations tested. Experiments with Dox were carried out by serial dilution as above, an exchange of medium after 8 h, followed by addition of MGd for 13 h. Medium was exchanged, and cells were incubated for an additional 2 days as above.
In Vitro Clonogenic and PLDR Assays.
Rif-1 cells (200 to 105) were plated into 25-cm2 plastic tissue culture flasks and allowed to adhere for 24 h. Bleo and MGd were added to final concentrations of 0.88–4.4 and 50 µM, respectively. After a 4- or 24-h drug exposure, the cells were washed twice with Hank’s solution, replenished with complete medium, and returned to the incubator for an additional 6–10 days. The colonies were fixed with 10% buffered formalin and stained with crystal violet for counting (plating efficiencies were 43–50%). To evaluate the effects on PLDR, cultures of Rif-1 (4 x 105 cells/flask) were treated with Bleo and MGd for 18 h, washed with fresh medium, and then treated with trypsin either immediately or after incubation in Hank’s solution at room temperature for a period of 4 h. Cells were counted and replated, and colonies were determined after further incubation as above (plating efficiencies were 34–46%). To study the effect of drug sequence, Rif-1 cells in medium were exposed under ambient conditions to the following conditions: (a) Bleo for 4 h, rinsed, and then treated with MGd for 4 h; (b) MGd for 4 h, rinsed, and then treated with Bleo for 4 h; (c) both drugs together for 4 h; (d) Bleo alone in medium containing 0.125% mannitol (drug vehicle) for 4 h; (e) Bleo alone for 4 h, rinsed, and then treated with medium containing 0.125% mannitol for 4 h. Colonies were determined after further incubation as above (plating efficiencies were 32–44%). MGd alone had no effect on plating efficiency.
In Vivo Tumor Models.
All animals received care in compliance with Guide for the Care and Use of Laboratory Animals (NIH Publication, 1996). Female BALB/c mice, 7–8 weeks of age, were obtained from Simonsen Laboratories (Gilroy, CA). EMT-6 cells (5–7 x 105) were injected s.c. into the right hind flanks. Animals were studied 10–14 days after inoculation with tumor cells when the tumors had reached 5–7 mm diameter and a depth of 2.5–3.5 mm. Tumor volume, measured three times/week using a caliper, was calculated assuming the conformation of a hemiellipsoid (16)
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The dose of Dox was selected to produce an 50% reduction in tumor growth compared with untreated controls. Dox (7.5 mg/kg), Bleo (10 or 20 units/kg), and/or MGd (0.5–40 µmol/kg) were administered i.v. on days 0, 7, and 14. Control animals received either no treatment, Dox, Bleo, or MGd. Kaplan-Meier analyses were performed by scoring events as the time for the tumor to reach four times the pretreatment volume. A log-rank test was used to determine statistical significance.
Pharmacokinetic Measurements.
The plasma pharmacokinetics of MGd and Dox was evaluated in 11-week-old male Sprague Dawley rats (Simonsen Laboratories) weighing 295–358 g. Each rat received injections i.v. via the tail vein with either a single administration of 20 µmol/kg MGd, a single administration of 1.3 mg/kg Dox, or an administration of 1.3 mg/kg Dox, followed by an administration of 20 µmol/kg MGd 1 h later. The Dox and MGd infusions were delivered over 30 s and 1 min, respectively. Blood was drawn into EDTA collection tubes by retroorbital bleeding under methoxyflurane anesthesia (Schering-Plough, Kenilworth, NJ). For each drug or drug combination, the combined data from three subgroups of rats (4 rats/subgroup) was used to characterize the 48-h postdosing period.
MGd and Dox concentrations in plasma were measured by high-performance liquid chromatography. MGd was analyzed using a modified technique based on the method of Parise et al. (18) . Dox was assayed using a modified procedure based on the method of Robert (19) , Camaggi et al. (20) , and Pfieffer et al. (21) . Standards for both assays were prepared by adding known amounts of analyte to blank rat plasma. For both MGd and Dox, plasma concentrations at 48 h were below the lower limit of quantitation.
Hematological Toxicity.
BALB/c mice receiving Dox (7.5 mg/kg) and MGd (40 µmol/kg) administered on days 0, 7, and 14 were compared with untreated animals and animals receiving Dox alone. There were 8 animals in each group, and complete blood counts were measured on days 14 and 28 by the Cell-dyn system (Abbott Laboratories).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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using an MTT assay. The IC50 (50% inhibitory concentration) for Bleo was reduced by 1 log using 100 µM MGd. These results demonstrate dose-dependent MGd enhancement of the in vitro cytotoxicity of Bleo to MES-SA cells. Similar results were obtained using Rif-1 cells (data not shown).
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. Plated cells were incubated with MGd for 4 or 24 h with increasing concentrations of Bleo. These data indicated that MGd enhanced the activity of Bleo 1000-fold in this assay. In another experiment, cultures were incubated with MGd and Bleo and then treated with trypsin and plated, either immediately or after a 4-h "recovery" period. Improved survival was observed in control cultures allowed to recover for 4 h but not in cultures treated with MGd (Fig. 3)
. This suggests that PLDR was inhibited by MGd. In other experiments, drug treatment sequence was studied by treating Rif-1 cultures with Bleo and MGd for 4 h together or sequentially, after medium exchange (Fig. 4)
. No change in survival was observed when MGd was added after Bleo treatment. Conversely, the surviving fraction decreased considerably (2 logs) when MGd was added 4 h before Bleo, although not to as great a degree as when the two drugs were added simultaneously (3 logs).
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. This effect was not seen when MGd was added after Dox. Similar results were found using Rif-1 cells (data not shown). The origins of this in vitro antagonism are unknown but could arise from interactions involving Dox and MGd directly or through the intermediacy of reducing metabolites present in the tissue culture medium (thiols, ascorbate, NADPH, and others). An analogous dependence on the scheduling of drug administration was not observed in vivo (see below).
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, animals receiving 20 µmol/kg of MGd with each of three weekly doses of Bleo showed significant delay in EMT-6 tumor growth compared with animals receiving Bleo alone. The median time required for tumors to grow to four times their original volume was increased to 17.7 days in animals receiving MGd in combination with a Bleo dose of 10 units/kg compared with 12.3 days for animals receiving Bleo alone (P = 0.008). MGd combined with 20 units/kg Bleo delayed tumor growth to 20.8 days (P = 0.015, compared with 20 units/kg Bleo alone).
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. No toxicity was observed with the MGd doses used.
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, a dose of 5 µmol/kg of MGd enhanced the tumor response to Dox when given either before or after Dox.
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A, a 20-µmol/kg i.v. dose of MGd given 1 h after administration of Dox (1.3 mg/kg i.v.) resulted in a transient increase in the plasma Dox concentration measured 2 min after MGd administration. At all other time points, Dox concentrations in the Dox plus MGd group were similar to concentrations in the corresponding Dox-alone group. The geometric mean plasma concentration at each time point was used to calculate area under the curve (0–24 h) using linear trapezoidal integration for the Dox-alone and Dox-plus-MGd groups, resulting in values of 204 and 222 ng/h/ml, respectively. Therefore, the transient elevation of Dox concentration seen at 2 min resulted in only a 9% increase in systemic Dox exposure when expressed in terms of area under the curve. MGd concentrations at all time points were similar in the Dox-plus-MGd and MGd-alone groups (Fig. 7
B), suggesting that a prior administration of Dox did not affect MGd plasma pharmacokinetics under the conditions of this study.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MGd enters tumor cells and enhances tumor response to ionizing radiation (4 , 7 , 9) . Ionizing radiation induces the formation of hydroxyl radicals, placing the cells under oxidative stress (30) . These radicals damage DNA, which is believed to be the culprit cytotoxic event. The depletion of reducing metabolites and production of reactive oxygen species caused by MGd may render the cells more susceptible to oxidative damage and lower the threshold for apoptosis.
Cytotoxic chemotherapy agents may lead to the generation of reactive oxygen species within cells and cause oxidative stress. Bleo and Dox are two agents known to generate reactive oxygen species (13 , 14) . Bleo is a glycopeptide that chelates iron inside the cell and binds to DNA. In reactions involving Fe(II) and oxygen, an "activated" Bleo species is formed that damages DNA through free radical intermediates (31) . Superoxide and hydrogen peroxide can also react with Fe(II) or Fe(III) bleomycin, respectively, to produce the activated form of the drug. DNA damage from Bleo and ionizing radiation is similar both in induction and repair (32) . Dox has several modes of action, including DNA double- and single-strand breakage associated with DNA intercalation and inhibition of topoisomerase II (33) . Dox can also form complexes with iron and copper and produce cytotoxic reactive oxygen species (14) . In the presence of reducing metabolites, Dox can undergo a one-electron reduction to the semiquinone radical. This radical can rapidly react with oxygen to form superoxide and, in the presence of Fe(II), highly reactive hydroxyl radicals (34) . The generation of free radicals is also associated with mitochrondrial membrane damage (35) .
A dose-response effect was observed for MGd enhancement of tumor responsiveness to Bleo and Dox. The dose-response effect could be readily measured in vitro with Bleo (Figs. 1
2
4
) assayed by either MTT viability or clonogenic survival using MES-SA or Rif-1 cells, respectively. The observed enhancement of Bleo activity may partly be attributable to inhibition of PLDR in cells oxidatively stressed by MGd. This assertion is supported by the data presented in Fig. 3
, which show that the survival benefit attributable to delayed trypsin treatment was abrogated in MGd-treated cultures. However, the magnitude of the Bleo enhancement suggests that additional mechanisms must be operative that are unique to this agent. One possibility is that reactive oxygen species generated in the presence of MGd contribute to the formation of activated Bleo within cells, either by electron transfer or via the release of Fe(II) ions from intracellular storage sites (31)
. Another possible mechanism is increased membrane permeabilization by MGd. Electroporation of cells in culture has been reported to enhance Bleo activity by 3 orders of magnitude (36)
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Interestingly, activity was enhanced when MGd was added before but not after exposure of cultures to Bleo (Fig. 4)
. This finding suggests that it may be possible to use the selective tumor localization of MGd, obtained by its prior administration, to improve the therapeutic index of Bleo. It is important to note that differences in PLDR would not be detected in this experiment, because cells remain in growth phase. Consistent with the above results, administration of MGd and Bleo (MGd within 5 min of Bleo) led to increased tumor responsiveness in vivo (Fig. 6)
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Dox activity was antagonized when MGd was added to cells before or simultaneously with Dox (Fig. 5)
. Addition of MGd after Dox had no effect on activity in vitro (also observed with Bleo). The source of antagonism is currently unclear. These findings could be explained based on a direct interaction between MGd and Dox or interference with cellular uptake in vitro. Alternatively, cells grown in tissue culture may not accurately represent tumor cell concentrations of redox active molecules such as thiols and ascorbate. These differences may mask the effects of redox active drugs such as MGd.
Indeed, as is shown in Table 2
, MGd could be given before or after Dox administration in vivo. This suggests that the metabolic perturbations induced by MGd do not need to precede the cellular damages caused by Dox. MGd has been found to accumulate in cells and is retained there for long periods of time (6)
. These findings are consistent with the hypothesis that MGd exerts its effects by depleting intracellular reducing metabolites (11)
. Xu et al. (9)
have shown recently, using 31P nuclear magnetic resonance spectroscopy, that MGd treatment leads to a decrease in high-energy phosphates within tumors in mice. This reduction in energy metabolites correlated with increased tumor response to radiation treatment.
Dox is metabolized in the liver. Concomitant use with MGd did not appear to significantly change its plasma pharmacokinetics and did not appear to augment the known myelosuppressive side effects of Dox. Although MGd appeared to cause a transient increase in plasma Dox levels at 2 min, there were no significant effects at other time points or in the area under the curve (Fig. 7
A). Future studies must evaluate the potential for enhanced cardiotoxicity with combined use of Dox and MGd.
These findings suggest important potential clinical uses for MGd as a chemotherapy enhancer. MGd selectively accumulates in tumor cells, suggesting the possibility that chemotherapy activity could be enhanced at the tumor but not in normal tissue, thereby increasing the therapeutic margin. Preclinical studies have shown that MGd is excreted primarily by the liver (4) . In human clinical trials conducted to date, MGd has shown dose-limiting renal toxicity at 26 µmol/kg in a single dose escalation Phase I trial (5) . In a Phase Ib/II trial in patients receiving whole brain radiation therapy for brain metastases, dose-limiting hepatocellular toxicity has been seen at a dose of 8.4 mg/kg given daily for 10 days (6 , 10) . It is likely that chemotherapy drugs with nonoverlapping toxicities will be best suited for use in combination with MGd.
MGd is a novel redox active drug that may have diverse applications in cancer therapy. It enhances tumor responsiveness to radiation and certain chemotherapy drugs such as Bleo and Dox. Other drugs are currently being tested for use in combination with MGd, and the potential toxicity of combined use will need to be examined for each agent. The studies reported here support redox modulation and inhibition of PLDR as important mechanisms of action for MGd as an enhancer of radiation and chemotherapy. Moreover, these studies and the putative mechanism provide potential new directions for discovering other novel cancer drugs with unique mechanisms of action involving redox reactions in biological systems.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Pharmacyclics, Inc., 995 East Arques Avenue, Sunnyvale, CA 94085. Phone: (408) 774-0330; Fax: (408) 774-0340. 
2 The abbreviations used are: MGd, motexafin gadolinium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Bleo, bleomycin; Dox, doxorubicin; PLDR, potentially lethal damage repair.
Received 3/ 1/01; revised 5/22/01; accepted 6/ 1/01.
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