ACCELERATED DISCOVERY

Blockage of Drug Resistance In Vitro by Disulfiram, a Drug Used to Treat Alcoholism

Tip W. Loo, David M. Clarke

Affiliation of authors: Medical Research Council Group in Membrane Biology, Departments of Medicine and Biochemistry, University of Toronto, ON, Canada.

Correspondence to: David M. Clarke, Ph.D., Department of Medicine, University of Toronto, Rm. 7342, Medical Sciences Bldg., 1 King's College Circle, Toronto, ON M5S 1A8, Canada (e-mail: david.clarke{at}utoronto.ca).


    ABSTRACT
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 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background: P-glycoprotein (P-gp) pumps a wide range of cytotoxic drugs out of cells. Inhibiting maturation of P-gp would be a novel method for circumventing P-gp-mediated multidrug resistance, which complicates cancer chemotherapy and treatment of patients infected with human immunodeficiency virus. We examined the effect of disulfiram (AntabuseTM) on the maturation and activity of P-gp. Methods: Embryonic kidney cells were transfected with a complementary DNA for the P-pg gene, and the effects of disulfiram on the sensitivity of the transfected cells to cytotoxic agents were determined. Enzyme assays were used to determine the effects of disulfiram on the verapamil-stimulated adenosine triphosphatase (ATPase) activity of P-gp. Disulfiram modifies cysteine residues, and mutant forms of P-gp that lack individual cysteines were used to determine whether particular cysteine residues mediate disulfiram's effects on P-gp activity. Maturation of recombinant P-gp was followed on immunoblots. Results: Disulfiram increased the sensitivity of P-gp-transfected cells to vinblastine and colchicine and inhibited P-gp's verapamil-stimulated ATPase activity. Half-maximal inhibition of ATPase activity occurred at 13.5 µM disulfiram. Disulfiram (at 100 µM) inhibited a P-gp mutant by 43% (95% confidence interval [CI] = 37%–48%) when cysteine was present at position 431 only and by 72% (95% CI = 66%–77%) when cysteine was present at position 1074 only. Treatment of P-gp-transfected cells with 50 nM disulfiram blocked maturation of recombinant P-gp. Conclusions: Disulfiram can potentially reduce P-gp-mediated drug resistance by inhibiting P-gp activity (possibly via cysteine modification) and/or by blocking its maturation. These results suggest that disulfiram has the potential to increase the efficacy of drug therapies for cancer and acquired immunodeficiency syndrome.



    INTRODUCTION
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 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Intrinsic or acquired multidrug resistance during cancer chemotherapy is a major clinical problem (1). The best characterized form of multidrug resistance is that mediated by P-glycoprotein (P-gp), an adenosine triphosphate (ATP)-driven, 170-kd efflux pump that is located in the plasma membrane. P-gp can pump out of the cell a wide range of cytotoxic drugs that have diverse structures and intracellular targets (26).

P-gp-mediated multidrug resistance also complicates treatment with protease inhibitors of patients infected with human immunodeficiency virus (HIV). Oral absorption and penetration of HIV-1 protease inhibitors, such as indinavir, nelfinavir, and saquinavir, into the central nervous system are compromised as a result of relatively high expression of P-gp in the intestinal epithelia and at the blood-brain barrier (7,8).

An important goal during chemotherapy is to inhibit the activity of P-gp. Current protocols for inhibiting P-gp often include a high-affinity P-gp substrate such as cyclosporin A that acts as a competitive inhibitor. This approach has been used successfully for treating pediatric tumors, such as retinoblastoma and neuroblastoma (9). A possible drawback to the use of compounds such as cyclosporin A for inhibiting P-gp is that they tend not to be as effective in protocols where the anticancer drugs also have high affinities for P-gp. The problem is further complicated by the observation that cyclosporin A and modulators of P-gp can increase the level of expression of P-gp at the cell surface because they act as chemical chaperones (10).

Inhibition of P-gp would ideally involve several approaches. A novel strategy to avoid the effects of P-gp would be to use chemicals as antichaperones to prevent P-gp maturation and transport to the cell surface. While screening for compounds that affect maturation of P-gp, we found that disulfiram (AntabuseTM), a drug used to treat alcoholism, may have antichaperone activity. We have investigated the effect of disulfiram on P-gp-mediated drug resistance and P-gp maturation in human embryonic kidney cells transfected with complementary DNA (cDNA) for P-gp. Since administration of disulfiram to patients results in inactivation of aldehyde dehydrogenase through modification of cysteines, we also examined the effect of disulfiram on the drug-stimulated adenosine triphosphatase (ATPase) activity of wild-type P-gp and nine mutant proteins with altered numbers of cysteine residues.


    METHODS
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 Abstract
 Introduction
 Methods
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 Discussion
 References
 
Measurement of Drug Resistance

HEK 293 (EBNA-1) cells were transfected with pREP4 vector (Invitrogen Corp., Carlsbad, CA) or pREP4 vector containing cDNA coding for P-gp-(His)10 (see below) and cultured in Dulbecco's modified Eagle medium (MEM) supplemented with 0.1 mM MEM nonessential amino acids (Life Technologies, Inc. [GIBCO BRL], Burlington, ON, Canada), 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% (vol/vol) fetal calf serum (Life Technologies, Inc.), under 5% CO2 at 37°C. After 18–24 hours, the medium was replaced with fresh medium containing 0.4 mg/mL hygromycin B (Alexis Biochemicals, San Diego, CA) or 0.4 mg/mL hygromycin B and 100 nM disulfiram (Research Biochemicals International, Natick, MA). (Nontransfected cells are killed by hygromycin B.) After 24 hours, the medium was replaced with fresh medium containing hygromycin B or hygromycin B plus disulfiram (as above) and various concentrations of either vinblastine (0–400 nM) or colchicine (0–1000 nM). One week later, the concentration of drug that inhibited cell growth by 50% (D50) was determined as described previously (11). Briefly, the medium was removed and replaced with OPTI-MEM medium (Life Technologies, Inc.) containing 0.2 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma–Aldrich, Oakville, ON, Canada). After 2 hours, 4 volumes of dimethyl sulfoxide was added to each sample, and the amount of MTT–formazan was measured by absorbance at 540 nm. The amount of MTT–formazan is an indirect measure of the number of viable cells.

Expression and Purification of P-gp Protein

HEK 293 cells were transfected with a cDNA (11) coding for human P-gp with 10 tandem histidine residues at its carboxy-terminus [P-gp-(His)10]. The cells were lysed with 1% (wt/vol) n-dodecyl-ß-D-maltoside (Sigma–Aldrich), and P-gp-(His)10 was purified by nickel–chelate chromatography (Ni-NTA columns; Qiagen, Inc., Mississauga, ON, Canada) (12).

Construction of P-gp Mutants

Human P-gp has cysteine residues at positions 137, 431, 717, 956, 1074, 1125, and 1227. None of these is essential for activity because mutation of all cysteines to alanines (to give a protein we refer to as "Cys-less P-gp") resulted in an active molecule (13). To determine whether specific cysteine residues were sensitive to the presence of disulfiram, mutants that would produce P-gp-(His)10 proteins with only one cysteine residue were constructed (11). Briefly, fragments of P-gp cDNA (<500 base pairs) were ligated into the polylinker region of the Bluescript vector (Stratagene Cloning Systems, La Jolla, CA) for site-directed mutagenesis by the method of Kunkel (14). Oligodeoxynucleotides in which the codon for cysteine was changed to one for alanine were obtained commercially (Sigma–Aldrich, Oakville, ON, Canada). The fragment containing each mutation was subcloned back into its original position to yield a full-length cDNA and then inserted into the mammalian expression vector pMT21 (Genetics Institute, Boston, MA). The integrity of the mutated segment of cDNA was confirmed by sequencing the entire fragment by the dideoxynucleotide chain-termination method (15). A mutant P-gp with cysteine residues at positions 431 and 1074 was also produced. The mutant proteins were expressed and purified as above.

Measurement of Verapamil-Stimulated ATPase Activity

An aliquot of solution that contained 50 ng of purified P-gp-(His)10 in 10 mM Tris–HCl (pH 7.5), 500 mM NaCl, 300 mM imidazole (pH 7.0), 0.1% (wt/vol) n-dodecyl-ß-D-maltoside, and 10% (vol/vol) glycerol was mixed with an equal volume of a solution containing sheep brain phosphatidylethanolamine (Type II-S; Sigma–Aldrich), 10 mM Tris–HCl (pH 7.5), and 150 mM NaCl and sonicated (Branson Ultrasonic, Danbury, CT) for 45 seconds on ice. The enzymatic reaction was initiated by the addition of an equal volume of a solution containing 100 mM Tris–HCl (pH 7.5), 100 mM NaCl, 20 mM MgCl2, 10 mM ATP, and 2 mM verapamil (Sigma–Aldrich) (12). After 30 minutes at 37°C, the reaction was stopped by the addition of an equal volume of 12% (wt/vol) sodium dodecyl sulfate (SDS). To determine the amount of inorganic phosphate produced during the enzymatic reaction, an equal volume of solution containing 3% (wt/vol) L-ascorbic acid, 0.5% (wt/vol) ammonium molybdate, 2.5% (wt/vol) SDS, and 0.5 M HCl was added, followed by 0.75 volume of a solution containing 2% (wt/vol) sodium citrate, 2% (wt/vol) sodium meta-arsenite, and 2% (vol/vol) acetic acid (final volume = 105 µL). The mixture was incubated for 10 minutes at 37°C, and the absorbance was measured at 850 nm. The amount of phosphate released was calculated from a phosphate standard curve (16).

The effect of disulfiram on verapamil-stimulated ATPase activity was determined by incubating a P-gp–lipid mixture (prepared as described above) with various concentrations (0.5–1000 µM) of disulfiram for 5 minutes at room temperature. The enzymatic reaction was carried out, and the amount of inorganic phosphate released was determined as described above. To determine the effect of disulfiram on specific cysteines, the P-gp-(His)10 mutants with single cysteine residues (and the mutant with two cysteine residues) were treated identically, except that they were incubated only with 100 µM disulfiram, and activities were expressed relative to those of control samples that were not incubated with disulfiram. To measure stimulation of P-gp's ATPase activity by drug substrates, Cys-less P-gp with 10 tandem histidine residues at its carboxy-terminus [Cys-less P-gp-(His)10] was used, and the solution used to initiate the enzymatic reaction contained 2 mM disulfiram, 2 mM verapamil, 100 µM vinblastine, 2 mM colchicine, or no drug substrate.

Western Blot Analysis

HEK 293 cells were transfected with cDNAs coding for rabbit SERCA1 Ca2+-ATPase, wild-type P-gp with the epitope for monoclonal antibody A52 at its carboxy-terminus (P-gp-A52), Cys-less P-gp with the same carboxy-terminal epitope (Cys-less P-gp-A52), or cystic fibrosis transmembrane conductance regulator (CFTR). The A52 epitope tag was derived from rabbit SERCA1 Ca2+-ATPase (11). Twenty-four hours after transfection, the cells were incubated with various concentrations of disulfiram (0–800 nM). After another 24 hours, the cells were lysed with an equal volume of SDS sample buffer (i.e., 125 mM Tris–HCl [pH 6.8], 20% [vol/vol] glycerol, 4% [wt/vol] SDS, and 2% [vol/vol] 2-mercaptoethanol), and equivalent amounts of the whole cell extracts were subjected to 5.5% (wt/vol) SDS–polyacrylamide gel electrophoresis, blotted onto a sheet of nitrocellulose, and incubated with 1 µg/mL solutions of either monoclonal antibody A52 (from D. H. MacLennan, University of Toronto, ON, Canada) or M3A7 (from J. R. Riordan, Mayo Clinic, Scottsdale, AZ). The blots were developed with peroxidase-conjugated goat anti-mouse antibody (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD) and visualized by use of an enhanced chemiluminescence system (Pierce Chemical Co., Rockford, IL). Prestained molecular-weight markers (Sigma–Aldrich) were used to estimate the molecular masses of the immunoreactive proteins.


    RESULTS
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 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Effect of Disulfiram on P-gp-Mediated Drug Resistance

Any compound that reduces the ability of P-gp-expressing cells to grow in the presence of cytotoxic agents, such as vinblastine and colchicine, is a potential inhibitor of P-gp (17). We found that the D50s for vinblastine and colchicine were 62 nM (95% confidence interval [CI] = 47–76) and 49 nM (95% CI = 28–68), respectively, in transfected cells expressing P-gp-(His)10 (Fig. 1Go). In control cells that did not express P-gp-(His)10, the D50 values were 1.7 nM (95% CI = 0.4–2.8) for vinblastine and 5.8 nM (95% CI = 1.5–9.7) for colchicine. Inclusion of 100 nM disulfiram during incubation of P-gp-(His)10-expressing cells with vinblastine or colchicine decreased the D50 values to 4.2 nM (95% CI = 0.9–7.1) and 10 nM (95% CI = 4.3–15), respectively, values that are not statistically significantly different from those obtained in similar experiments that used control cells.



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Fig. 1. Effect of disulfiram on P-glycoprotein (P-gp)-mediated drug resistance. HEK 293 (EBNA-1) cells transfected with P-gp or vector (Control) were incubated for 7 days with (+ Disulfiram) or without (- Disulfiram) 100 nM disulfiram in the presence of various concentrations of vinblastine or colchicine. The medium was then removed and replaced with OPTI-MEM medium containing 0.2 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). After 2 hours, 4 volumes of dimethyl sulfoxide was added to each sample, and the amount of MTT–formazan was measured spectrophotometrically to assess the number of viable cells. The concentration of drug substrate that inhibited cell growth by 50% (D50) was determined. Error bars are 95% confidence intervals for three experiments.

 
Effect of Disulfiram on Drug-Stimulated ATPase Activity of P-gp

To determine whether disulfiram directly inhibited the activity of P-gp, we assayed purified P-gp-(His)10 for drug-stimulated ATPase activity in the presence of disulfiram. P-gp activity was inhibited by disulfiram, with half-maximal inhibition occurring at 13.5 µM disulfiram (Fig. 2, AGo). We then tested whether disulfiram could inhibit the activity of nine mutant P-gp-(His)10s: Cys-less P-gp-(His)10 (18), seven in which only one cysteine was present (the other six cysteines were replaced with alanine), and one in which two cysteines were present. Fig. 2, BGo, shows that 100 µM disulfiram inhibited P-gp-(His)10 mutants with single cysteines by 43% (95% CI = 37%–48%) (relative to that of a sample that was not treated with disulfiram) when cysteine was present at position 431 only and by 72% (95% CI = 66%–77%) when it was at position 1074 only. Mutant P-gp-(His)10s with a single cysteine at any of the other positions in which it occurs in the wild-type protein and the Cys-less P-gp-(His)10 showed essentially no inhibition of drug-stimulated ATPase activity in the presence of 100 µM disulfiram. Inhibition of a P-gp-(His)10 mutant with cysteines at both position 431 and position 1074 was not statistically significantly different from that of wild-type P-gp-(His)10.



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Fig. 2. Effect of disulfiram and other drug substrates on adenosine triphosphatase (ATPase) activity of P-glycoprotein (P-gp). Panel A: Purified wild-type P-gp with 10 tandem histidine residues at its carboxy-terminus [P-gp-(His)10] was assayed for ATPase activity in the presence of 2 mM verapamil and various concentrations of disulfiram, as described in the text. The % ATPase Activity is relative to that without disulfiram. Panel B: Wild-type P-gp-(His)10 (Wild) and P-gp-(His)10 mutants in which all cysteines were replaced with alanine (Cys-less); all cysteines but one were replaced with alanine (C137, C431, C717, C956, C1074, C1125, and C1227; the number indicates the position of the single cysteine); or all cysteines except those at positions 431 and 1074 were replaced with alanine (C431/1074) were incubated with 100 µM disulfiram for 5 minutes at room temperature and then assayed for verapamil-stimulated ATPase activity. The % ATPase Activity is relative to that without disulfiram. Panel C: P-gp-(His)10 was assayed for ATPase activity in the presence of 1 mM disulfiram (Dis), 1 mM verapamil (Ver), 50 µM vinblastine (Vin), or 1 mM colchicine (Colch). Fold-stimulation is the ATPase activity in the presence of drug substrate relative to that without drug. Error bars are 95% confidence intervals for three experiments.

 
P-gp exhibits drug substrate-stimulated ATPase activity (19). We measured the stimulatory effect of saturating concentrations (determined in preliminary experiments) of disulfiram and three drugs that are known to be P-gp substrates. Cys-less P-gp-(His)10 was used because disulfiram may inhibit wild-type P-gp by modifying Cys431 and Cys1074 (see below). The ATPase activity of Cys-less P-gp-(His)10 was stimulated 7.6-fold (95% CI = 5.9–9.1) by 1 mM disulfiram, 12.3-fold (95% CI = 9.9–14.3) by 1 mM verapamil, sixfold (95% CI = 5.1–6.7) by 50 µM vinblastine, and fivefold (95% CI = 4.3–5.7) by 1 mM colchicine.

Effect of Disulfiram on P-gp Expression.

Agents that affect the activity of P-gp can act as chemical chaperones (20,21) to aid the folding of P-gp (10). Compounds, such as cyclosporin A, verapamil, and vinblastine, induce maturation of processing mutants of P-gp. A processing mutant of P-gp is one that is retained in the endoplasmic reticulum as core glycosylated biosynthetic intermediates (apparent mass of 150 kd) and is rapidly degraded. In the presence of drug substrates, the mutant P-gp matures (apparent mass of 170 kd) and is transported to the cell surface where it is active (10).

To test whether disulfiram had chemical chaperone-like activity, cells transfected with P-gp-A52 cDNA were incubated with various concentrations of disulfiram, and expression of a recombinant P-gp with the epitope for monoclonal antibody A52 was detected by western blot analysis. In the absence of disulfiram, both mature 170-kd and immature 150-kd forms of the recombinant P-gp were detected (Fig. 3, AGo). Disulfiram at concentrations of 50–800 nM almost completely inhibited the maturation of the protein; the major products were the 150-kd immature protein and a smaller protein that may be a degradation product. Inhibition of maturation was not due to covalent modification of cysteine, since maturation of Cys-less P-gp was also blocked by disulfiram. In contrast, 2 µM N-ethylmaleimide, another compound that modifies sulfhydryl groups, did not affect the maturation of the recombinant wild-type or Cys-less P-gp (data not shown). Inhibition of P-gp maturation was not due to cell death because the cells were still viable in the presence of 10 µM disulfiram (data not shown).



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Fig. 3. Effect of disulfiram on P-glycoprotein (P-gp) expression. A) HEK 293 cells expressing wild-type P-gp with the epitope for monoclonal antibody A52 at its carboxy-terminus, a mutant P-gp with its cysteines replaced with alanine and the same carboxy-terminal epitope (Cys-less P-gp), rabbit SERCA1 Ca2+-adenosine triphosphatase, or cystic fibrosis transmembrane conductance regulator (CFTR) were incubated with various concentrations (0–800 nM) of disulfiram. After 24 hours, whole-cell extracts from equal numbers of cells were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblot analysis as described in the text. The molecular masses of the immunoreactive proteins (110, 150, 160, 170, and 190 kd) were calculated by comparing their relative mobilities with those of prestained molecular weight markers. B) HEK 293 cells expressing Cys-less P-gp (as above) were grown in Dulbecco's modified Eagle medium (Control) or medium supplemented with 10 µM cyclosporin A (Cyc), 200 nM disulfiram (Dis), or 10 µM cyclosporin A and 200 nM disulfiram (Cyc+Dis). After 18 hours, extracts from equal numbers of cells were analyzed as above.

 
Disulfiram did not appear to inhibit synthesis of recombinant P-gp, since the total amount of immunoreactive P-gp was similar at all concentrations of disulfiram. Similarly, expression of SERCA1 Ca2+-ATPase, a membrane protein that is found in the endoplasmic reticulum and is not trafficked to the cell surface, was not affected by disulfiram; similar amounts of the 110-kd protein were present in cells treated with up to 800 nM disulfiram. We also tested whether disulfiram affected trafficking of CFTR, another ABC transporter (transporters with ATP-binding cassettes) (4). Only about 25% of synthesized CFTR protein is normally processed to the plasma membrane (22). We found that inhibition of maturation of CFTR required a higher concentration of disulfiram than that required to inhibit maturation of P-gp.

Cyclosporin A can act as a chemical chaperone by stabilizing newly synthesized P-gp for transport to the cell surface (10). We tested whether cyclosporin A could reverse the effects of disulfiram on maturation of recombinant P-gp. Fig. 3, BGo, shows that 10 µM cyclosporin A increased the amounts of both the 150-kd and the 170-kd forms of the protein, while 200 nM disulfiram almost completely inhibited maturation. At a concentration of 10 µM, cyclosporin A almost completely overcame the inhibitory effect of 200 nM disulfiram.


    DISCUSSION
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 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
A major goal during chemotherapy is to block the activity of P-gp. The most common method for blocking the activity of P-gp during chemotherapy is to include the competitive inhibitor cyclosporin A (23). Such an approach has been successfully used in treating pediatric tumors, such as neuroblastoma and retinoblastoma (9). The use of cyclosporin A to inhibit P-gp activity does have disadvantages. Therapeutic compounds whose affinity for P-gp is higher than that of cyclosporin A will be preferentially transported out of the cell. Furthermore, cyclosporin A increases expression of P-gp, suggesting that relatively larger amounts of inhibitors would be needed to inhibit P-gp activity, and cyclosporin A has been reported to cause cancer (24). In contrast, continued use of disulfiram does not appear to cause tumor formation, although it can cause hepatitis (25).

Our results showed that disulfiram inhibited the drug-stimulated ATPase activity of P-gp-(His)10 but not that of Cys-less P-gp-(His)10 and suggest that disulfiram modifies essential cysteine residues in P-gp. The verapamil-stimulated ATPase activities of P-gp-(His)10 mutants that had cysteine only at positions 431 or 1074 were inhibited by disulfiram. These cysteines are found in the homology A (Walker A motifs) nucleotide-binding consensus sequences in the two nucleotide-binding domains of P-gp (26) and can be modified by other compounds such as N-ethylmaleimide (27,28). Covalent modification of cysteine residues of mitochondrial and cytosolic aldehyde dehydrogenase is the molecular basis for use of disulfiram to treat chronic alcoholism. A cysteine residue (Cys302) close to the active site reacts with disulfiram (29). Irreversible inhibition of the aldehyde dehydrogenases also occurred when they were modified by the disulfiram metabolite S-methyl N,N-diethylthiolcarbamate sulfoxide (30). Although we have been able to show an effect of disulfiram on P-gp in vitro, it is possible that its metabolites (31) are responsible for its effect on processing and activity of P-gp in cells grown in culture. In vivo studies are needed to determine if P-gp is irreversibly inhibited by disulfiram metabolites, but many of the disulfiram metabolites have not been chemically synthesized or are not available commercially.

We showed that disulfiram prevented maturation of P-gp with an attached epitope. This would result in less P-gp at the cell surface and may explain why disulfiram-treated cells were more sensitive to vinblastine and colchicine than transfected cells that were not treated with disulfiram. The effect may be transient, since the addition of cyclosporin A partly restored maturation of P-gp even in the presence of disulfiram. This observation suggests that disulfiram does not act mainly by imposing a general secretory pathway block.

Disulfiram is normally taken orally as a daily 500-mg dose, although it may also be implanted subcutaneously as a single 1000-mg dose (32). Pharmacokinetic data suggest that 80%–90% of an ingested dose is absorbed from the gastrointestinal tract and is rapidly distributed to tissues and organs (33). Therefore, the average concentration of an oral dose of disulfiram in a 100-kg person would be expected to be about 20 µM. In practice, however, it has been difficult to measure disulfiram levels in fresh human blood samples due to its rapid conversion to other metabolites (31). Disulfiram metabolites have been detected in fresh blood samples in micromolar concentrations after patients were given daily 300-mg oral doses for 2 weeks.

Our results suggest that drug regimens that include disulfiram should be clinically tested for their potential to improve the efficacy of cancer and acquired immunodeficiency syndrome chemotherapies. Disulfiram is readily available and is relatively inexpensive. The doses—and, consequently, the side effects—of chemotherapeutic drugs may also be decreased by the addition of disulfiram. These results also show that drugs that target maturation of P-gp can be the goal of a search for a new generation of P-gp inhibitors.


    NOTES
 
Supported by Public Health Service grant CA80900 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; and by grants from the Medical Research Council of Canada (MRC) and the Canadian Cystic Fibrosis Foundation. D. M. Clarke is an MRC Scientist.


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 Abstract
 Introduction
 Methods
 Results
 Discussion
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Manuscript received February 2, 2000; revised April 24, 2000; accepted May 3, 2000.


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