A phase IB trial of 24-hour intravenous PX-12, a thioredoxin-1 inhibitor, in patients with advanced gastrointestinal cancers.

We investigated the safety, pharmacokinetics, and pharmacodynamics of PX-12, a thioredoxin-1 (Trx-1) inhibitor, administered as a 24-hour infusion every 7 or 14 days in patients with gastrointestinal malignancies. PX-12 is the first Trx-1 inhibitor to undergo clinical development. The first Phase 1 study of PX-12 demonstrated promising clinical activity, but the 1 and 3 hour-infusion schedules investigated were associated with a strong and irritating odor due to exhalation of one of its metabolites, 2-butanethiol. In an effort to achieve tolerability and achieve a drug exposure level necessary for biological activity, the current study was undertaken. While the maximally tolerated dose was estimated to be 300 mg/m(2) /24 h once a week as the 2-butanethiol expirate was tolerable at that dose level, no evidence of clinical activity was observed. Pharmacokinetic studies of the parent compound PX-12 demonstrated rapid, irreversible binding to plasma components, resulting in low (ng/ml) peak plasma concentrations of non-bound PX-12 during infusion. DCE-MRI was performed pre-and post-infusion in three patients. There were no significant trends observed in changes in plasma Trx-1, vascular endothelial growth factor (VEGF), or beta fibroblast growth factor (FGF-2) pre- or post-treatment. However, there was a trend for a decrease in circulating Trx-1 during the first four PX-12 treatment cycles in patients that had a Trx-1 baseline level >18 ng/mL. Aggregate clinical trial results suggest that further clinical development of PX-12, as an intravenous infusion, is not feasible. However, the Trx-1 pathway remains a target of interest in patients with gastrointestinal malignancies.

Utilization of distillers dried grains with solubles and phytase in sow lactation diets to meet the phosphorus requirement of the sow and reduce fecal phosphorus concentration.

Two experiments were completed to determine the potential for using distillers dried grains with solubles (DDGS) in diets with or without phytase to provide available P, energy, and protein to highly productive lactating sows without increasing their fecal P. In Exp. 1, the dietary treatments were as follows: (1) corn and soybean meal with 5% beet pulp (BP) or (2) corn and soybean meal with 15% DDGS (DDGS). Besides containing similar amounts of fiber, diets were isonitrogenous (21% CP, 1.2% Lys) and isophosphorus (0.8% P). Sixty-one sows were allotted to dietary treatments at approximately 110 d of gestation (when they were placed in farrowing crates) based on genetics, parity, and date of farrowing. Sows were gradually transitioned to their lactation diet. On d 2 of lactation, litters were cross-fostered to achieve 11 pigs/litter. Sows and litters were weighed on d 2 and 18. Fecal grab samples were collected on d 7, 14, and 18 of lactation. Dietary treatment did not affect the number of pigs weaned (10.9 vs. 10.8) or litter weaning weight. On d 14, DDGS sows had less fecal P concentration than BP sows (28.3 vs. 32.8 mg/g; P = 0.04). Fecal Ca of sows fed DDGS decreased for d 7, 14, and 18 (55.6, 51.4, and 47.1 mg/g of DM, respectively; P = 0.05) but not for BP sows. In Exp. 2, the dietary treatments were as follows: (1) corn and soybean meal (CON), (2) CON + 500 phytase units of Natuphos/kg diet, as fed (CON + PHY), (3) corn and soybean meal with 15% DDGS and no phytase (DDGS), or (4) DDGS + 500 FTU of Natuphos/kg of diet, as fed (DDGS + PHY). Sows (n = 87) were managed as described for Exp 1. Litter BW gain (46.0, 46.3, 42.1, and 42.2 kg; P = 0.25) and sow BW loss (8.1, 7.2, 7.4, and 6.3 kg for CON, CON + PHY, DDGS, and DDGS + PHY, respectively; P = 0.97) were not affected by dietary treatment. Fecal P concentration did not differ among dietary treatments but was reduced at d 14 and 18 compared with d 7 (P = 0.001). However, fecal phytate P concentration was decreased by the addition of DDGS when DDGS and DDGS + PHY were compared with the CON sows except on d 7 (P < 0.05). Sows fed CON diet had greater fecal phytate P than sows fed DDGS, and sows fed DDGS + PHY had less fecal phytate P than sows fed DDGS with no phytase (P = 0.001). Although these experiments were only carried out for 1 lactation, these results indicate that highly productive sows can sustain lactation performance with reduced fecal phytate P when fed DDGS and phytase in lactation diets.

Disruption of a single copy of the SERCA2 gene results in altered Ca2+ homeostasis and cardiomyocyte function.

A mouse model carrying a null mutation in one copy of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase isoform 2 (SERCA2) gene, in which SERCA2 protein levels are reduced by approximately 35%, was used to investigate the effects of decreased SERCA2 level on intracellular Ca(2+) homeostasis and contractile properties in isolated cardiomyocytes. When compared with wild-type controls, SR Ca(2+) stores and Ca(2+) release in myocytes of SERCA2 heterozygous mice were decreased by approximately 40-60% and approximately 30-40%, respectively, and the rate of myocyte shortening and relengthening were each decreased by approximately 40%. However, the rate of Ca(2+) transient decline (tau) was not altered significantly, suggesting that compensation was occurring in the removal of Ca(2+) from the cytosol. Phospholamban, which inhibits SERCA2, was decreased by approximately 40% in heterozygous hearts, and basal phosphorylation of Ser-16 and Thr-17, which relieves the inhibition, was increased approximately 2- and 2.1-fold. These results indicate that reduced expression and increased phosphorylation of phospholamban provides compensation for decreased SERCA2 protein levels in heterozygous heart. Furthermore, both expression and current density of the sarcolemmal Na(+)-Ca(2+) exchanger were up-regulated. These results demonstrate that a decrease in SERCA2 levels can directly modify intracellular Ca(2+) homeostasis and myocyte contractility. However, the resulting deficit is partially compensated by alterations in phospholamban/SERCA2 interactions and by up-regulation of the Na(+)-Ca(2+) exchanger.

Transgenic overexpression of constitutively active protein kinase C epsilon causes concentric cardiac hypertrophy.

To test the hypothesis that activation of the protein kinase C (PKC) epsilon isoform leads to cardiac hypertrophy without failure, we studied transgenic mice with cardiac-specific overexpression of a constitutively active mutant of the PKCepsilon isoform driven by an alpha-myosin heavy chain promoter. In transgenic mice, the protein level of PKCepsilon in heart tissue was increased 9-fold. There was a 6-fold increase of the membrane/cytosol ratio, and PKC activity in the membrane fraction was 4.2-fold compared with wild-type mice. The heart weight was increased by 28%, and upregulation of the mRNA for beta-myosin heavy chain and alpha-skeletal actin was observed in transgenic mouse hearts. Echocardiography demonstrated increased anterior and posterior wall thickness with normal left ventricular function and dimensions, indicating concentric cardiac hypertrophy. Isolated cardiomyocyte mechanical function was slightly decreased, and Ca(2+) signals were markedly depressed in transgenic mice, suggesting that myofilament sensitivity to Ca(2+) was increased. No differences were observed in either the levels of cardiac Ca(2+)-handling proteins or the degree of cardiac regulatory protein phosphorylation between wild-type and transgenic mice. Unlike mice with PKCbeta(2) overexpression, transgenic mice with cardiac-specific overexpression of the active PKCepsilon mutant demonstrated concentric hypertrophy with normal in vivo cardiac function. Thus, PKC isoforms may play differential functional roles in cardiac hypertrophy and failure.

Alterations in Ca2+ cycling proteins and G alpha q signaling after left ventricular assist device support in failing human hearts.

OBJECTIVE: Left ventricular assist device support mechanically unloads the failing ventricle with resultant improvement in cardiac geometry and function in patients with end-stage heart failure. Activation of the G alpha q signaling pathway, including protein kinase C, appears to be involved in the progression of heart failure. Similarly down-regulation of Ca2+ cycling proteins may contribute to contractile depression in this clinical syndrome. Thus we examined whether protein kinase C activation and decreased Ca2+ cycling protein levels could be reversed by left ventricular assist device support. METHODS: Left ventricular myocardial specimens were obtained from seven patients during placement of left ventricular assist device and heart transplantation. We examined changes in protein levels of G alpha q, phospholipase C beta 1, regulators of G protein signaling (RGS), sarcoplasmic reticulum Ca2+ ATPase, phospholamban and translocation of protein kinase C isoforms (alpha, beta 1, and beta 2). RESULTS: The paired pre- and post-left ventricular assist device samples revealed that RGS2, a selective inhibitor of G alpha q, was decreased (P < 0.01), while the status of G alpha q, phospholipase C beta 1, RGS3 and RGS4 were unchanged after left ventricular assist device implantation. Translocation of protein kinase C isoforms remained unchanged. Left ventricular assist device support increased sarcoplasmic reticulum Ca2+ ATPase protein level (P < 0.01), while phospholamban abundance was unchanged. CONCLUSIONS: We conclude that altered protein expression and stoichiometry of the major cardiomyocyte Ca2+ cycling proteins rather than reduced phospholipase C beta 1 activation may contribute to improved mechanical function produced by left ventricular assist device support in human heart failure.

Structure-based drug design: combinatorial chemistry and molecular modeling.

Drug discovery efforts are shifting to include the rapid synthetic procedures of combinatorial chemistry and the elegance of rational library design. The wealth of computational methods which explore both the receptor structure and the ultimate pharmacophore complementarity, provide novel avenues for chemists to discover new lead compounds or design virtual libraries for screening prior to the synthetic stage. This mini-review provides an overview of a few current methodologies of library generation, highlighting docking procedures which have utility in both the discovery and optimization stages of drug development. Three specific examples of different approaches to the use of docking are provided. These describe the development of inhibitors to the human A3 adenosine receptor and HIV-1 protease, and the evaluation of the activity of novel inhibitors of the redox regulator protein, human thioredoxin.

Effect of angiotensin-converting enzyme inhibition on protein kinase C and SR proteins in heart failure.

We tested the hypothesis that activation of protein kinase C (PKC) isoforms in pressure-overload heart failure was prevented by angiotensin-converting enzyme (ACE) inhibition, resulting in normalization of cardiac sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) 2a and phospholamban protein levels and improvement in intracellular Ca2+ handling. Aortic-banded and control guinea pigs were given ramipril (5 mg. kg-1. day-1) or placebo for 8 wk. Ramipril-treated banded animals had lower left ventricular (LV) and lung weight, improved survival, increased isovolumic LV mechanics, and improved cardiomyocyte Ca2+ transients compared with placebo-treated banded animals. This was associated with maintenance of SERCA2a and phospholamban protein expression. Translocation of PKC-alpha and -epsilon was increased in placebo-treated banded guinea pigs compared with controls and was attenuated significantly by treatment with ramipril. We conclude that ACE inhibition attenuates PKC translocation and prevents downregulation of Ca2+ cycling protein expression in pressure-overload hypertrophy. This represents a mechanism for the beneficial effects of this therapy on LV function and survival in heart failure.

Parallel syntheses of disulfide inhibitors of the thioredoxin redox system as potential antitumor agents.

We have reported previously that unsymmetrical disulfide inhibitors of the human thioredoxin/thioredoxin reductase redox system (hTrx/TR) possess antitumor activity. We have broadened the search for more potent inhibitors and evaluated a large range of mono- and bis-disulfide compounds, prepared using parallel syntheses. Reaction of isothioisourea-HCI salts (R') or bis-salts (R) with aromatic or aryl thiols (R") in wells of 96-well plates produced >450 derivatives with the structures R"SSR' and R"SSRSSR". The excellent yield and purity of the disulfides provided sufficient material for evaluations of enzyme inhibition and cytotoxicity. Selection criteria based on the IC50 values for hTrx/TR inhibition and for cytotoxicities of the disulfides identified agents for subsequent scale-up syntheses and in vivo evaluations of antitumor activity. These scale-up studies confirmed the original activities of agents synthesized in the plates and validated the parallel synthetic approach. Structure-activity information derived from the hTrx/TR IC50 data allow for a number of generalizations. The most potent inhibitors of the Trx system contained two heteroatoms ortho to the disulfide moiety in an aromatic functionality. The thioalkylating moieties had greatest activity with one branch point alpha to the disulfide. In the absence of branching, more potent inhibition was observed with the electron withdrawing functionalities. Bis-disulfides showed patterns of activity which depended on chain length, with optimum activity observed when the disulfide units were separated by 3.9 A, a similar distance to that separating the thioredoxin active site cysteine residues. From the agents selected for scale-up syntheses, three disulfide compounds were studied for their antitumor activity in vivo against human tumor xenografts in scid mice. One of the analogues discovered through the combinatorial syntheses/screening for Trx inhibition, 1-phenylethyl 2-imidazolyl disulfide, N1 (ProlX agent PX-C5), has demonstrated excellent in vivo activity against the MCF-7 human breast cancer and the HL-60 human leukemia, thus validating this approach for novel drug discovery.

Enhanced myocardial contractility and increased Ca2+ transport function in transgenic hearts expressing the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase.

In this study, we investigated whether the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca2+ transport pump (SERCA1a) can functionally substitute the cardiac SERCA2a isoform and how its overexpression affects cardiac contractility. For this purpose, we generated transgenic (TG) mice that specifically overexpress SERCA1a in the heart, using the cardiac-specific alpha-myosin heavy chain promoter. Ectopic expression of SERCA1a resulted in a 2.5-fold increase in the amount of total SERCA protein. At the same time, the level of the endogenous SERCA2a protein was decreased by 50%, whereas the level of other muscle proteins, including calsequestrin, phospholamban, actin, and tropomyosin, remained unchanged. The steady-state level of SERCA phosphoenzyme intermediate was increased 2.5-fold, and the maximal velocity of Ca2+ uptake was increased 1.7-fold in TG hearts, demonstrating that the overexpressed protein is functional. Although the basal cytosolic calcium signal was decreased by 38% in TG cardiomyocytes, the amplitude of cytosolic calcium signal was increased by 71.8%. The rate of calcium resequestration was also increased in TG myocytes, which was reflected by a 51.6% decrease in the normalized time to 80% decay of calcium signal. This resulted in considerably increased peak rates of myocyte shortening and relengthening (50.0% and 66.6%, respectively). Cardiac functional analysis using isolated work-performing heart preparations revealed significantly faster rates of contraction and relaxation in TG hearts (41.9% and 39.5%, respectively). The time to peak pressure and the time to half-relaxation were shorter (29.1% and 32.7%, respectively). In conclusion, our study demonstrates that the SERCA1a pump can functionally substitute endogenous SERCA2a, and its overexpression significantly enhances Ca2+ transport and contractile function of the myocardium. These results also demonstrate that the SERCA pump level is a critical determinant of cardiac contractility.

Thioredoxin redox control of cell growth and death and the effects of inhibitors.

Thioredoxin is a redox protein found over-expressed in some human tumors. Thioredoxin is secreted by tumor cells and stimulates cancer cell growth. Redox activity is essential for growth stimulation by thioredoxin. Cells transfected with thioredoxin cDNA show increased tumor growth and decreased apoptosis in vivo and decreased sensitivity to apoptosis induced by a variety of agents both in vitro and in vivo. Cells transfected with a redox-inactive mutant thioredoxin show inhibited tumor growth in vivo. Thus, thioredoxin offers an attractive target for anticancer drug development. A class of disulfide inhibitors of thioredoxin has been identified. These disulfides inhibit cancer cell growth in culture and have antitumor activity against some human tumor xenografts in animals.

Mechanisms of inhibition of the thioredoxin growth factor system by antitumor 2-imidazolyl disulfides.

The interactions of a series of 2-imidazolyl disulfide antitumor compounds with the thioredoxin reductase(TR)/thioredoxin (hTrx) redox system have been studied. Disulfides III-2 (n-butyl 2-mercaptoimidazolyl disulfide) and VI-2 (ethyl 2-mercaptoimidazolyl disulfide) were substrates for reduction by TR with Km values of 43 and 48 microM. Disulfides IV-2 (1-methylpropyl 2-mercaptoimidazolyl disulfide) and DLK-36 (benzyl 2-mercaptoimidazolyl disulfide) were competitive inhibitors of the reduction of hTrx by TR with Ki values of 31 microM. None of the disulfides were substrates for reduction by human glutathione reductase. The disulfides caused reversible thioalkylation of hTrx at the redox catalytic site as shown by the fact that there was no thioalkylation of a mutant hTrx where both the catalytic site Cys32 and Cys35 residues were replaced by Ser. In addition, the disulfides caused a slower irreversible inactivation of hTrx as a substrate for reduction by TR, with half-lives for III-2 of 30 min, for IV-2 of 4 hr, and for IX-2 (t-butyl 2-mercaptoimidazolyl disulfide) of 24 hr. This irreversible inactivation of hTrx occurred at concentrations of the disulfides an order of magnitude below those that inhibited TR, and involved the Cys73 of hTrx, which is outside the conserved redox catalytic site, as shown by the resistance to inactivation of a mutant hTrx where Cys73 was replaced by Ser. Electrophoretic and mass spectral analyses of the products of the reaction between the disulfides and hTrx show that modification of 1-3 Cys residues of the protein occurred in a concentration-dependent fashion. The disulfides inhibited the hTrx-dependent proliferation of MCF-7 breast cancer cells with IC50 values for III-2 and IV-2 of 0.2 and 1.2 microM, respectively. The results show that although the catalytic sites of TR and hTrx are reversibly inhibited by the 2-imidazolyl disulfides, it is the irreversible thioalkylation of Cys73 of hTrx by the disulfides that most probably accounts for the inhibition of thioredoxin-dependent cell growth by the disulfides.

Effects of total replacement of atrial myosin light chain-2 with the ventricular isoform in atrial myocytes of transgenic mice.

BACKGROUND: In contrast to their well-known and critical role in excitation-contraction coupling of vascular smooth muscle, the effects of the myosin light chains on cardiomyocyte mechanics are poorly understood. Accordingly, we designed the present experiment to define the cardiac chamber-specific functional effects of the ventricular isoform of the regulatory myosin light chain (MLC2v). METHODS AND RESULTS: Postnatal transgenic cardiac-specific overexpression of MLC2v was achieved by use of the alpha-myosin heavy chain promoter. Enzymatically disaggregated atrial and ventricular mouse myocytes were field-stimulated at multiple frequencies, and mechanical properties and calcium kinetics were studied by use of video edge detection and FURA 2-AM, respectively. MLC2v overexpression resulted in complete replacement of the atrial with the ventricular isoform of the regulatory myosin light chain at the steady-state mRNA and protein levels in the atria of transgenic mice. Mechanical properties of transgenic atrial myocytes were enhanced to the level of ventricular myocytes of control animals in association with modest decreases in the amplitude of the calcium transient. CONCLUSIONS: MLC2v modulates chamber-specific contractility by enhanced calcium sensitivity and/or improved cross-bridge cycling of the thin and thick filaments of the cardiomyocyte.

Selenium and the thioredoxin redox system: effects on cell growth and death.

Thioredoxin is a redox protein found overexpressed in some human tumors. Thioredoxin is secreted by tumor cells and enhances the sensitivity of the cancer cells to other growth factors. Redox activity is essential for stimulation of cell growth by thioredoxin. Cells transfected with thioredoxin cDNA show increased tumor growth and decreased apoptosis in vivo and decreased sensitivity to apoptosis induced by a variety of agents both in vitro and in vivo. Cells transfected with a redox-inactive mutant thioredoxin show inhibited tumor growth in vivo. Dietary selenium has been shown to prevent some forms of human cancer. Selenocysteine is an essential component of thioredoxin reductase, the flavoenzyme that is responsible for the reduction of thioredoxin. Selenium added to the culture medium increases thioredoxin reductase activity due to an increase in thioredoxin reductase protein but mostly due to an increase in the specific activity of the enzyme. Some diaryl chalcogenide (selenium and tellurium) compounds have been studied as inhibitors of thioredoxin reductase. The most active were diaryl tellurium compounds, which were noncompetitive inhibitors of thioredoxin reductase with Ki values of 2-10 microM. Several of the compounds inhibited cancer cell colony formation in vitro with IC50s as low as 2 microM.

Redox active disulfides: the thioredoxin system as a drug target.

Thioredoxin, and particularly extracellular thioredoxin, presents an attractive target for developing novel agents to treat cancer. Our studies have involved the examination of a series of alkyl 2-imidazolyl disulfides as inhibitors of the growth-stimulatory activity of the thioredoxin system. We originally determined the disulfides to be weak reversible inhibitors of thioredoxin reductase. Subsequently, we have shown that alkyl 2-imidazolyl disulfides interact directly with thioredoxin, thioalkylating critical cysteine residues or causing dimerization of the protein leading to its loss of biological activity. One of the analogues that binds to thioredoxin, 1-methylpropyl 2-imidazolyl disulfide (IV-2), selectively inhibits the thioredoxin-dependent growth of tumor cells in culture and has antitumor activity against MCF-7 and HL-60 tumors in vivo. Our work involves the development of a parallel combinatorial synthetic method to produce a large number of disulfide analogues at one time. These analogues, which differ sterically, electronically, and physically, were produced in a 96-well plate. The biological activity of these analogues was evaluated, also in the 96-well plate format. This rapid method of evaluating biological activity is a means to identify agents with specificity for inhibition of the thioredoxin system, and may provide novel antitumor agents with activity against solid tumor cancers.

Cell line-directed screening assay for inhibitors of thioredoxin reductase signaling as potential anti-cancer drugs.

We have used a cell line-directed screening approach (CDSA) to identify novel inhibitors of the thioredoxin reductase signaling pathway which contributes to the transformed phenotype of some human tumors. Two 2-imidazolyl disulfide compounds, previously identified as inhibitors of thioredoxin reductase, were screened for growth inhibitory activity in the National Cancer Institute (NCI) human cancer cell line panel. The COMPARE pattern recognition algorithm was used to identify similar compounds from > 60,000 compounds in the NCI investigational drug database. Of 47 nondiscreet compounds tested in a thioredoxin reductase/thioredoxin insulin reduction assay, 37 (77%) were inhibitors with IC50s < or = 10 micrograms/ml and 15 of those (32%) had IC50s < or = 1 microgram/ml. These compounds were all as selective or more selective for thioredoxin reductase than for glutathione reductase, while three compounds were inhibitors of thioredoxin. In comparison to CDSA, the number of compounds with IC50s < or = 1 microgram/ml identified by screening of 52 compounds from the database whose growth inhibiting activity was unrelated to the activity of the disulfide compounds was only 2%. Screening of 221 randomly selected natural products gave only 3% of compounds with IC50s < or = 1 microgram/ml. Thus, the CDSA using data from the NCI cancer cell panel and known inhibitors of the selected target as seed compounds can greatly increase hit rates, compared with random screening, for identifying novel inhibitors of a target, in this case thioredoxin signaling.

Oxidative inactivation of thioredoxin as a cellular growth factor and protection by a Cys73-->Ser mutation.

Thioredoxin (Trx) is a widely distributed redox protein that regulates several intracellular redox-dependent processes and stimulates the proliferation of both normal and tumor cells. We have found that when stored in the absence of reducing agents, human recombinant Trx undergoes spontaneous oxidation, losing its ability to stimulate cell growth, but is still a substrate for NADPH-dependent reduction by human thioredoxin reductase. There is a slower spontaneous conversion of Trx to a homodimer that is not a substrate for reduction by thioredoxin reductase and that does not stimulate cell proliferation. Both conversions can be induced by chemical oxidants and are reversible by treatment with the thiol reducing agent dithiothreitol. SDS-PAGE suggests that Trx undergoes oxidation to monomeric form(s) preceding dimer formation. We have recently shown by X-ray crystallography that Trx forms a dimer that is stabilized by an intermolecular Cys73-Cys73 disulfide bond. A Cys73-->Ser mutant Trx (C73S) was prepared to determine the role of Cys73 in oxidative stability and growth stimulation. C73S was as effective as Trx in stimulating cell growth and was a comparable substrate for thioredoxin reductase. C73S did not show spontaneous or oxidant-induced loss of activity and did not form a dimer. The results suggest that Trx can exist in monomeric forms, some of which are mediated by Cys73 that do not stimulate cell proliferation but can be reduced by thioredoxin reductase. Cys73 is also involved in formation of an enzymatically inactive homodimer, which occurs on long term storage or by chemical oxidation. Thus, although clearly involved in protein inactivation, Cys73 is not necessary for the growth stimulating activity of Trx.

Synthesis and bioreductive potential of a mono N-oxide derivative of the alkylating agent chlorambucil.

Chlorambucil N-oxide (CHLN-O) was synthesized and evaluated for in vitro bioreductive antitumor activity. A time-dependent hypoxic differential was observed when EMT6 cells were exposed to CHLN-O in the presence of rat liver microsomes and reducing equivalents. The cytotoxicity of the N-oxide was potentiated under hypoxia, and augmented further by a combination of low pH and hypoxia. Metabolic studies were also undertaken, which utilized previously described HPLC methodology for the analysis of CHLN-O loss from biological fluids. These demonstrated the requirement for microsomal enzymes and reducing equivalents, and also illustrated the time-dependent manner of CHLN-O loss from isolated microsomal preparations.

High-performance liquid chromatographic method for the separation of chlorambucil and its N-oxide prodrug.

A reversed-phase high-performance liquid chromatographic method is described to distinguish chlorambucil N-oxide from the parent chlorambucil and quantitate both after separation from biological samples. The influence of solvent pH, alcohol, acid and ion-pairing agent on the separation is described. The stability of chlorambucil and its N-oxide in buffers and alcohols, as well as stability during filtration is discussed with potential application for metabolic studies.

The thioredoxin/thioredoxin reductase redox system and control of cell growth.

Thioredoxin is a redox protein that is important for a variety of intracellular functions, possibly including regulation of transcription factor activity. We have shown that human thioredoxin has the same predicted amino acid sequence as adult T-cell-derived leukemic cell growth factor. Recombinant human thioredoxin stimulates the proliferation of Swiss murine 3T3 fibroblasts with an EC50 of 100 nM and the proliferation of a number of human cancer cells. Site-directed mutagenesis of the active-site cysteines of thioredoxin has shown that redox activity is necessary for the stimulation of cell proliferation. Added 125I-thioredoxin is taken up by cells in culture and could have intracellular action. A series of alkyl 2-imidazolyl disulfides have been shown to be competitive inhibitors of human thioredoxin reductase with Ki values of 3.3 to 8.6 microM. The compounds inhibited Swiss 3T3 serum-dependent proliferation with IC50 values of 2.0 to 4.0 microM, and one compound inhibited Swiss 3T3 thioredoxin-dependent proliferation with an IC50 value of 60 nM.