Molecular Pharmacology and Antitumor Activity of Palmarumycin-Based Inhibitors of Thioredoxin Reductase

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Molecular pharmacology and antitumor activity of palmarumycin-based inhibitors of thioredoxin reductase

Garth Powis,1 Peter Wipf,2 Stephen M. Lynch,2 Introduction 3 4 Anne Birmingham, and D. Lynn Kirkpatrick The cytosolic thioredoxin redox system is composed of thioredoxin-1 and thioredoxin reductase-1 reductase, 1Department of Experimental Therapeutics, M.D. Anderson Cancer Center, Houston, Texas; 2Department of Chemistry, which catalyzes the NADPH-dependent reduction of University of Pittsburgh, Pittsburgh, Pennsylvania; 3Arizona thioredoxin-1. Thioredoxin reductase-1 is an important Cancer Center; and 4ProlX Pharmaceuticals, Tucson, Arizona regulator of cancer cell growth and survival (1, 2). Thioredoxin-1 acting with peroxiredoxin-1 is an antioxi- dant that scavenges H2O2 (3). Thioredoxins are also able to Abstract reduce buried oxidized thiol residues in proteins (4) and The cytosolic thioredoxin redox system composed of regulate the activity of redox-sensitive transcription factors, thioredoxin-1 and the NADPH-dependent thioredoxin including p53 (5), nuclear factor-nB (6), the glucocorticoid reductase-1 reductase is an important regulator of cell receptor (7), activator protein-1 (8), hypoxia-inducible growth and survival. Thioredoxin-1 is overexpressed in factor-1 (HIF-1; ref. 9), Sp1 (10), and Nrf2 (11). Thiore- many human tumors where it is associated with doxin-1 also binds and inhibits the activity of the apoptosis- increased cell proliferation, decreased apoptosis, and inducing proteins, apoptosis signal-regulating kinase-1 (12) decreased patient survival. We hypothesized that thio- and, the tumor suppressor phosphatase and tensin homo- redoxin reductase-1 provides a target to inhibit the logue deleted on chromosome 10 (13), thus inhibiting activity of overexpressed thioredoxin-1 for the develop- apoptosis. Thioredoxin-1 is overexpressed in many human ment of novel anticancer agents. We found that the tumors (14–17) where it is associated with increased cell naphthoquinone spiroketal fungal metabolite palmarumy- proliferation, decreased apoptosis (18), and poor patient cin CP1 is a potent inhibitor of thioredoxin reductase-1, survival (19, 20). Thioredoxin reductase thus provides a but attempts to exploit the activity of palmarumycin CP1 target to regulate the activity of overexpressed thioredoxin- analogues as antitumor agents in vivo were hampered 1 (21, 22). by their insolubility. We have therefore developed PX- Thioredoxin reductase-1 is a selenocysteine-containing 916, a water-soluble prodrug of a palmarumycin CP1 flavoprotein with broad substrate specificity because of the analogue. PX-916 rapidly releases the parent compound ready accessibility of its COOH-terminal redox active site, at physiologic pH and in plasma but is stable at acid pH, which contains an essential selenocysteine residue (21). allowing its i.v. administration. PX-916 is a potent There are three thioredoxin reductase isoforms: the inhibitor of purified human thioredoxin reductase-1 and canonical cytoplasmic thioredoxin reductase-1, a mitochon- of thioredoxin reductase-1 activity in cells and tumor drial thioredoxin reductase-2, and a testes-specific thiore- xenografts when given to mice and inhibits the down- doxin reductase/glutathione reductase. The cellular level stream targets of thioredoxin-1 signaling, hypoxia- of thioredoxin reductase-1 is subject to complex regulation inducible factor-1A, and vascular endothelial growth (reviewed in ref. 23). The core promoter of the thioredoxin factor in tumors. PX-916 showed excellent antitumor reductase-1 gene contains several transcription factor activity against several animal tumor models with some activation sites, including those for the redox-sensitive cures. Thus, the study shows that water-soluble inhib- factors Oct-1 and Sp1 as well as others. Differential splicing itors of thioredoxin reductase-1, such as PX-916, can and alternative transcription start sites result in multiple block thioredoxin-1 signaling in tumors producing marked formsoftheenzyme.Post-transcriptionalregulation inhibition of tumor growth. [Mol Cancer Ther 2006; involving a selenocysteine insertion sequence element in 5(3):630–6] the 3V-untranslated region directs selenocysteine incorpo- ration, which is necessary for enzyme activity; thus, selenium supplementation can lead to increased thioredoxin reductase-1 activity in cell culture and in selenium- deficient animals (24). Thioredoxin reductase-1 is necessary Received 11/28/05; revised 12/27/05; accepted 1/18/06. for cell proliferation. A thioredoxin reductase-1 knockout is Grant support: NIH grants CA52995 and CA90821. embryonic lethal in mice, and thioredoxin reductase- Requests for reprints: Garth Powis, Department of Experimental 1–deficient fibroblasts derived from the thioredoxin re- Therapeutics, M.D. Anderson Cancer Center, FC-6.3044, 1515 Holcombe Boulevard, Houston, TX 77030-4009. Phone: 713-745-3366; ductase-1 (À/À) embryos do not proliferate in vitro (25). Fax: 713-745-1710. E-mail: [email protected] Furthermore, cancer cell growth is inhibited by thioredoxin Copyright C 2006 American Association for Cancer Research. reductase-1 antisense (26), thioredoxin reductase-1 small doi:10.1158/1535-7163.MCT-05-0487 interfering RNA (27) and by a mutant redox inactive

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duction Laboratories (Lexington, KY), and rabbit polyclon- al anti-human vascular endothelial growth factor (VEGF) was from Santa Cruz Biotechnology (Santa Cruz, CA). Immunohistochemistry for tumor HIF-1a and VEGF was done as described previously (35).

Figure 1. Compound structures. thioredoxin reductase-1 (28). There are reports that levels of thioredoxin reductase-1 are increased by epidermal growth factor (29) and hypoxia (30) in cancer cells, although tumors show only moderately increased levels of thioredoxin reductase-1 (23, 30). We have identified the naphthoquinone spiroketal fungal metabolite palmarumycin CP1 as a potent inhibitor of thioredoxin reductase-1, but attempts to exploit the activity of palmarumycin CP1 analogues as antitumor agents in vivo were hampered by their insolubility (31). We have recently reported the synthesis of a series of water-soluble palmarumycin CP1 analogue prodrugs (32). We now report the molecular pharmacology and antitumor activity of one of these prodrugs in cancer cells and in human tumor xenografts.

Materials and Methods Materials The palmarumycin CP1 analogues PX-911, PX-916, and PX-960 (Fig. 1) were synthesized as described previously (31, 32). PX-916 was dissolved at 3 mg/mL in 5% ethanol, 10 mmol/L sodium phosphate (pH 4.0) for i.p. and oral administration and in 5% ethanol, 0.9% NaCl, 10 mmol/L sodium phosphate (pH 4.0) for i.v. administration and used within 30 minutes. Human placental thioredoxin reductase- 1, specific activity 33 Amol NADPH oxidized/min/mg protein at room temperature, was prepared as described previously (33). Human recombinant thioredoxin-1 was prepared as described previously (34). Mouse monoclonal anti-human HIF-1a antibody was purchased from Trans-

Table 1. Inhibition of thioredoxin reductase-1 and cell growth by palmarumycin CP1 analogues Figure 2. Inhibition of cellular and tumor thioredoxin reductase by PX- 916. MCF-7 human breast cancer cells grown in medium containing Compound Inhibition of Inhibition of 1 Amol/L selenium for 7 d were treated with PX-916 and cellular human MCF-7 breast thioredoxin reductase activity was measured. A, time course of the thioredoxin cancer cell inhibition of thioredoxin reductase on exposure to 1 Amol/L PX-916. B, concentration dependence of the inhibition of thioredoxin reductase on reductase-1 IC50 growth IC50 exposure to various concentrations of PX-916 for 17 h. Points, mean of (Amol/L) (Amol/L) three determinations; bars, SE. *, P V 0.05; **, P V 0.01, compared with control. C, MCF-7 human breast cancer xenografts were grown in female Palmarumycin CP1 0.35 1.0 scid mice implanted with 90-d 17h-estradiol slow release pellets until they f 3 PX-911 3.2 9.2 were 300 mm . The mice were given a single dose of PX-916 (25 mg/kg i.v.) and tumors were harvested at various times. Thioredoxin PX-916 0.28 3.1 reductase activity was measured in tumor homogenates. Points, mean of PX-960 0.27 4.1 three mice at each time point; bars, SE. **, P < 0.01, compared with pretreatment value.

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MCF-7 human breast cancer cells were obtained from the American Tissue Type Collection (Manassas, VA). All cells were tested to be Mycoplasma free using a PCR ELISA kit (Roche Diagnostics, Inc., Indianapolis, IN) and grown in j 95% humidified air with 5% CO2 at 37 C in McCoy’s 5A medium supplemented with 10% fetal bovine serum. For studies of effects of the palmarumycin CP1 analogues on cellular thioredoxin reductase activity, MCF-7 human

breast cancer cells were grown in medium containing 1 Amol/L selenium for 7 days, which increased cellular thioredoxin reductase activity by f5-fold as reported previously (36).

Figure 3. Antitumor activity of PX-916. A, female Beige nude mice were inoculated s.c. with 107 A673 rhabdomyosarcoma cells. When tumors were 100 mm3 on day 8 (arrow), dosing was begun with vehicle alone (o) or 30 mg/kg/d i.p. PX-916 for five doses (E). Points, mean of six mice per group; bars, SE. B, female scid mice were inoculated s.c. with 107 SHP-77 human small cell lung cancer cells. When tumors were 150 mm3 on day 17 (arrow), dosing was begun with vehicle alone (o), 10 mg/kg/d i.v. PX-916 for eight doses (n), or 25 mg/kg/d i.v. PX-916 for five doses (E). Points, mean of eight mice per group; bars, SE. C, female scid mice implanted 1 d previously with a 90-d 17h-estradiol slow release pellet were inoculated s.c. with 107 MCF-7 human breast cancer cells. When tumors were 175 mm3 on day 8 (arrow), dosing was begun with vehicle alone (o), 22.5 mg/kg/d i.v. PX-916 for five doses (y), 27.5 mg/kg i.v. PX-916 every other day for five doses (E), and 22.5 mg/kg/d orally PX-916 for five doses (n). Points, mean of eight mice per group; bars, SE. Figure 4. Inhibition of tumor HIF-1a, VEGF, and thioredoxin reductase by repeated administration of PX-916. Female scid mice implanted 1 d previously with a 90-d 17h-estradiol slow release pellet were inoculated Cells s.c. with 107 MCF-7 human breast cancer cells. When the tumors were 3 A673 human rhabdomyosarcoma cells were provided by 300 mm , vehicle or 25 mg/kg/d PX-916 was given i.v. for five doses. Twenty-four hours later, the tumors were removed and stained by Dr. Peter Houghton (St. Jude Children’s Hospital, Mem- immunohistochemistry for HIF-1a and VEGF (A) or assayed for thioredoxin phis, TN). SHP-77 human small cell lung cancer cells and reductase activity (B). Columns, mean of four mice; bars, SE. *, P V 0.05.

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Thioredoxin Reductase-1Assay Pharmacokinetic Studies Thioredoxin reductase-1 activity was measured as the Male C57Bl/6 mice were given PX-916 i.v. at 25 mg/kg. NADPH-dependent colorimetric reduction of dinitrothio- The mice were killed at various times, blood was collected benzoate as described previously (37). Thioredoxin reduc- into heparinized tubes, and plasma was prepared. Plasma tase-1, NADPH, and palmarumycin CP1 analogues were (0.2 mL) was immediately mixed with an equal volume of incubated for 15 minutes before adding dinitrothioben- 0.25 mol/L sodium phosphate (pH 4.0) and extracted for 1 zoate. Thioredoxin reductase activity in cell lysates was hour by inversion with 4 mL ethyl acetate. After centrifu- measured as the thioredoxin-1-dependent reduction of gation, the organic layer (3.8 mL) was removed and insulin by NADPH as described previously (37). Thiore- evaporated under N2 and the residue was dried on a doxin reductase activity in tumor tissue homogenates was lyophilizer. Chromatographic separation was achieved measured also as described previously (24). with a Waters Symmetry C-18 3.9 Â 150 mm column Antitumor Studies (Waters, Milford, PA) with a mobile phase of 0.1% A673 rhabdomyosarcoma, SHP-77 small cell lung cancer, trifluoroactetic acid in 60% methanol at a flow rate of or MCF-7 breast cancer cells (f107) in log cell growth were L/min with detection at 254 nm. For the assay, the sample injected s.c. in 0.1 mL PBS into the flanks of female nude residue was dissolved in 100 AL mobile phase and Beige mice (A673 cells) or female severe combined immu- centrifuged at 15,000 Â g for 5 minutes at 4jC. The limit nodeficient (scid) mice (SHP-77 and MCF-7 cells). Female of detection of the assay for all the compounds from 0.2 mL mice that were to receive the estrogen-dependent MCF-7 mouse plasma was 0.1 Ag/mL. breast cancer cell line were implanted s.c. in the back with a 90-day 17h-estradiol release pellet (Innovative Research of America, Sarasota, FL) a day before the tumor cells. The Results animals were weighed weekly and tumor diameters were Stability measured twice weekly at right angles (dshort and dlong) with PX-916 was stable as a stock solution in ethanol at electronic calipers and converted to volume by the formula: room temperature with a half-life of >5 days. However, 2 volume = [(dshort) (dlong)] / 2; ref. 38. When the tumors in 0.1 mol/L sodium phosphate, PX-916 showed pH- reached volumes between 100 and 170 mm3, the mice were dependent degradation with a half-life of 37 hours at pH stratified into groups of eight animals having approximately 4.0, 1 hour at pH 7.0, and V1 hour at pH 10.0 (data not equal mean tumor volumes and administration of PX-916 shown). Therefore, PX-916 was used as a stock solution was started. When the tumor volume reached z2,000 mm3 in ethanol for in vitro studies and made fresh in vehicle or became necrotic, the animals were euthanized. Tumor (pH 4.0) for in vivo studies. growth rate was calculated from the linear portion of the Inhibition of Thioredoxin Reductase-1 least-squares regression of the cube root of the tumor volume. PX-916 was a potent inhibitor of purified human One-way ANOVA using the General Linear Model (39) was thioredoxin reductase-1 with an IC50 of 0.28 Amol/L, used to test for the effect of treatment on tumor growth rate. which is similar to that of palmarumycin CP1 (Table 1). Toxicity Studies However, unlike palmarumycin CP1, which is almost PX-916 was given i.v. at 25 mg/kg/d for 5 days to female insoluble in aqueous medium, PX-916 is soluble with a scid mice. The mice were killed 24 hours after the last dose maximum solubility in water of f10 mg/mL. Based on and changes in body weight from the start of the study, the observation that PX-916 was rapidly converted to PX- blood lymphocyte, neutrophil, RBC and platelet counts, 960 in aqueous solution, we measured the ability of PX- and aspartate aminotransferase and alanine aminotransfer- 960 to inhibit purified human thioredoxin reductase-1 ase were measured. In separate studies, the maximum and found it to be similar to that of PX-916 (Table 1). tolerated dose was determined as the dose that caused 4 g PX-916 was a selective inhibitor of human thioredoxin body weight loss or death of at least one of three animals. reductase-1 compared with two other NADPH-dependent Pharmacodynamic Studies reductases with an IC50 for human glutathione reductase MCF-7 breast cancer cells (107) were injected s.c. into of >50 Amol/L (>200-fold selectivity for thioredoxin the flanks of female scid mice previously implanted 1 day reductase-1) and 29.2 Amol/L for human cytochrome earlier with a 90-day 17h-estradiol release pellet and P450 reductase (104-fold selectivity for thioredoxin tumors were allowed to grow to f300 mm3. Mice were reductase). given a single i.v. dose of 25 mg/kg PX-916 or vehicle In vitro Activity alone. Mice were killed at various times and the tumors Cell growth inhibition of MCF-7 human breast cancer were removed and immediately frozen in liquid N2 for cells by palmarumycin CP1, PX-916, and PX-960 occurred the thioredoxin reductase assay. In a separate study, at similar concentrations of 1 to 3 Amol/L (Table 1). female scid mice with 300 mm3 MCF-7 tumor xenografts When MCF-7 cells were exposed to 1 Amol/L PX-916, were treated with 25 mg/kg/d i.v. PX-916 or vehicle there was a time-dependent inhibition of cellular thio- alone for 5 days. Twenty-four hours after the last redoxin reductase that was maximum at 24 hours dose, the tumors were removed and fixed for immuno- (Fig. 2A). The IC50 for inhibition of cellular thioredoxin histochemistry or frozen for the thioredoxin reductase reductase by PX-916 was 0.25 Amol/L and maximum assay. inhibition occurred at 0.5 Amol/L (Fig. 2B). Thus,

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Table 2. Toxicity of PX-916 in scid mice

Schedule Dose (mg/kg) DBody Alanine Aspartate WBC weight (g) aminotransferase (units/L) aminotransferase (units/L) (K/AL)

Control À1.2 F 1.5 30.3 F 10 154.1 F 62.4 3.0 F 0.6 QD Â 5 i.v. 25 À0.6 F 0.3 39.8 F 12.8 166.9 F 29.8 1.3 F 0.6*

NOTE: PX-916 was given to female scid mice at 25 mg/kg/d i.v. for 5 days, and mice were killed 24 hours after the last dose. There were four mice per group (mean F SE). *P < 0.05.

inhibition of purified human thioredoxin reductase-1, daily doses of PX-916 of 30 mg/kg i.v. The major toxicities MCF-7 cellular thioredoxin reductase, and cell growth observed 24 hours after five daily doses of PX-916 of 25 mg/ inhibition of MCF-7 cells occurred at about the same kg i.v. were neutropenia and thrombocytopenia, with no concentration of PX-916. increase in plasma liver enzymes and no significant weight In vivo Inhibition of TumorThioredoxin Reductase and loss (Table 2). No other gross toxicities were observed. Antitumor Activity Pharmacokinetics A single i.v. dose of PX-916 of 25 mg/kg inhibited MCF-7 When incubated with fresh mouse plasma at room human tumor xenograft thioredoxin reductase-1 up to 60% temperature, PX-960 had a half-life of 31 minutes at room at 24 hours and the inhibition was maintained for at least 48 temperature, whereas PX-916 rapidly disappeared and was hours (Fig. 2C). The growth of A673 human rhabdomyo- converted to PX-960 with a half-life of <2.0 minutes. Not sarcoma xenografts (mean F SE, n = 6 mice) was decreased surprisingly, when PX-916 was given to mice at 25 mg/kg from 153 F 35 mm3/d in the vehicle control group to 5 F i.v., it could not be detected in plasma 5 minutes after 3mm3/d for 5 days after dosing with PX-916 at 30 mg/ administration and only a very small peak of the parent kg/d i.p. for five doses (97% inhibition; P < 0.01; Fig. 3A). compound PX-960 could be detected at 5 minutes but not at PX-916 given i.v. also showed good antitumor activity 30 minutes. Two unidentified metabolite peaks could be against the SHP-77 small cell lung cancer with a decrease in seen at 5 minutes, but these had disappeared by 30 minutes tumor growth rate 5 days after the end of dosing (mean F (data not shown). SE, n = 8 mice) from 150 F 48 mm3/d in the vehicle control group to 27 F 14 mm3/d when given at 25 mg/kg/d i.v. for Discussion five doses (82% inhibition; P < 0.05; Fig. 3B). In this study, Thioredoxin-1 is overexpressed in many human tumors three of eight mice had no histologically detectable tumor (14–17) where it is associated with increased cell proliferation, when the study was terminated on day 42. When PX-916 decreased apoptosis (18), and decreased patient survival was given i.v. at 10 mg/kg/d i.v. for eight doses, tumor (19, 20). The redox activity of thioredoxin-1 is necessary for F 3 growth was decreased to only 91 24 mm /d (39% its biological effects in stimulating cancer cell growth and inhibition; P < 0.05) by 5 days after the end of dosing. inhibiting apoptosis (12, 13, 34). Although thioredoxin The growth of MCF-7 human breast cancer xenografts was reductase-1 is necessary for the reduction of thioredoxin-1, F decreased 5 days after the end of dosing from 47 its levels are only moderately increased in tumors (23, 30). 3 F 3 10 mm /d in the vehicle control group to 22 4mm/d by We hypothesized that preventing the reduction of thio- PX-916 at 22.5 mg/kg/d i.v. for five doses (52% inhibition; redoxin-1 by inhibiting tumor thioredoxin reductase-1 F 3 P < 0.05), 22 8mm/d by PX-916 at 27.5 mg/kg i.v. every could be a critical point at which to block the redox- other day for five doses (52% inhibition; P > 0.05), and 18.5 dependent biological effects of thioredoxin-1 leading to F 3 8mm/d by PX-916 at 27.5 mg/kg/d orally for five inhibition of tumor growth. doses (62% inhibition; P < 0.05; Fig. 3C). We have reported previously that palmarumycin CP1 Tu m o r H IF -1 Aand VEGF and some of its analogues are potent inhibitors of Levels of the HIF-a transcription factor and its down- thioredoxin reductase-1 (31). However, all prior com- stream target VEGF are increased by thioredoxin-1 expres- pounds were very insoluble and could not be given to sion (9). We therefore examined the effect of PX-916 animals. PX-916 was synthesized as a water-soluble administration on tumor HIF-1a and VEGF levels (Fig. 4A). prodrug of the palmarumycin CP1 derivative PX-960 and Twenty-four hours after the last dose of five daily doses was found to retain the ability to inhibit purified of PX-916 of 25 mg/kg, there was a decrease in MCF-7 thioredoxin reductase-1 with an IC50 of 0.28 Amol/L. PX- xenograft staining for both HIF-1a and VEGF. At the same 916 also inhibited thioredoxin reductase-1 activity in MCF- time, levels of tumor thioredoxin reductase activity were 7 human breast cancer cells with an IC50 of 0.25 Amol/L decreased by 75% (Fig. 4B). and was an inhibitor of MCF-7 cell growth with an IC50 of Tox i c i t y 3.1 Amol/L. As with other reported inhibitors of thiore- The maximum tolerated single i.v. dose of PX-916 to doxin reductase-1 (40), PX-916 was an NADPH and time female scid mice was 50 mg/kg, and the mice tolerated five dependent, apparently irreversible inhibitor of thioredoxin

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Table 2. Toxicity of PX-916 in scid mice (Cont’d)

Neutrophils Lymphocytes Monocytes RBC Hemoglobin Platelets (K/AL) (K/AL) (K/AL) (mol/L/AL) (g/dL) (K/AL)

2.5 F 0.5 0.4 F 0.1 0.11 F 0.05 9.4 F 0.5 15.5 F 0.7 773 F 67 0.9 F 0.3* 0.3 F 0.1 005 F 0.01 8.9 F 0.4 14.9 F 0.4 436 F 142*

reductase-1, most likely reacting with the selenocysteine- 20,000 genes on the array, possibly because thioredoxin containing catalytic site. Two other NADPH-dependent reductase-1 activity was only decreased 43% by the reductases, human glutathione reductase and cytochrome treatment (27). The genes that did change, leukotriene B4 P450 reductase, were not inhibited by PX-916 until 12-hydroxydehydrogenase, ubiquitin D, differentiation- concentrations at least 100-fold higher. enhancing factor, fibronectin 1, apolipoprotein 3, pros- Stability studies showed that, at physiologic pH, PX-916 aposin, choline/ethanolamine phosphotransferase, and was converted to its parent PX-960 with a half-life of 1 IFN-a-inducible protein, give little clue to the pathways hour. It was much more stable at pH 4.0 with a half-life of affected by thioredoxin reductase-1. 37 hours and was formulated at this pH for i.v. adminis- Several anticancer agents have been reported to inhibit tration. In mouse plasma, the breakdown of PX-916 to PX- thioredoxin reductase-1, including alkylating agents and 960 was rapid with a half-life of <2 minutes and no PX-916 platinum agents (40), the polyphenol curcumin (43), the could be detected in the plasma after i.v. administration of porphyrin gadolinium macrocycle, motexafin gadolinium PX-916. Thus, the inhibition of thioredoxin reductase-1 in (22), and the quinol NSC 706704 (44). It remains to be tumor xenografts and the antitumor activity is likely to be determined whether concentrations of any of these com- almost completely due to the parent PX-960. PX-960 could pounds that inhibit tumor thioredoxin reductase-1 can be be detected only transiently in mouse plasma after obtained in vivo, and most of these agents have other administration of PX-916 due to rapid metabolism or more mechanisms that are more likely than thioredoxin reduc- likely rapid distribution of the very lipophilic PX-960. tase-1 inhibition to account for their antitumor activity. When PX-916 was given as a single dose of 25 mg/kg i.v. to Although this work has focused on the antitumor mice with MCF-7 breast cancer xenografts, the tumor consequences of the inhibition of thioredoxin reductase, thioredoxin reductase-1 activity was inhibited by up to 60% there are several other diseases where thioredoxin reduc- and remained inhibited for 48 hours. Repeated adminis- tase is thought to play a pathophysiologic role, such as tration of PX-916 for 5 days gave 75% inhibition of tumor diabetic neuropathy, rheumatoid arthritis, Sjogren’s syn- thioredoxin reductase 24 hours after the last dose. drome, AIDS, and reperfusion injury (1, 2, 42). Thioredoxin PX-916 given i.p. or i.v. showed excellent antitumor reductase inhibitors may have therapeutic benefits in these activity against A673 rhabdomyosarcoma, SHP-77 small conditions as well as in cancer. cell lung cancer, and MCF-7 breast cancer. In SHP-77, In summary, we have shown that PX-916, a water-soluble complete tumor regressions were seen in some mice. The prodrug of a palmarumycin CP1 analogue, rapidly releases most active schedule was every other day administration, the parent compound at physiologic pH and in plasma but and tumor growth was inhibited as long as the drug was is stable at acid pH, allowing its i.v. administration. PX-916 given. Significant antitumor activity was not seen following is a potent inhibitor of purified human thioredoxin oral administration at doses that gave i.v. antitumor reductase-1 and of thioredoxin reductase activity in cells activity. We have reported previously that thioredoxin-1 and tumor xenografts when given to mice. PX-916 acts by a redox mechanism to increase HIF-1a levels and exhibited antitumor activity against several animal tumor VEGF formation, associated with an increase in tumor models, with some cures, and blocked the expression of the angiogenesis (9), and this effect was reversed by a downstream targets of thioredoxin-1 signaling, HIF-1a and thioredoxin-1 inhibitor (41). MCF-7 tumor xenografts in VEGF, in the tumors. mice treated with PX-916 showed a decrease in tumor HIF- 1a and VEGF presumably due to the inhibition of thioredoxin-1 redox signaling. Inhibition of thioredoxin References reductase-1 might affect other pathways in the cell. As 1. Powis G, Montfort WR. 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