In vitro activities of novel antifolate drug combinations against Plasmodium falciparum and human granulocyte CFUs

The potency of antimalarial dihydrofolate reductase inhibitors, alone and in synergistic combination with dihydropteroate synthetase inhibitors, against the Kenyan K39 strain of Plasmodium falciparum (pyrimethamine resistant) and against normal replicating human bone marrow cells in in vitro culture has been studied. Therapeutic indices and rank order of synergistic potency were derived. Trimethoprim, pyrimethamine, and the quinazolines WR159412 and WR158122 had the smallest therapeutic indices (1.39, 4.38, 2.56, and 90.0, respectively), while the three triazines clociguanil, WR99210, and chlorcycloguanil had the largest (3,562, 3,000, and 2,000, respectively). In rank order of decreasing activity against P. falciparum, the six most potent drug combinations were WR99210-dapsone, chlorcycloguanil-dapsone, WR158122-dapsone, WR159412-dapsone, WR159412-sulfamethoxazole, and chlorcycloguanil-sulfamethoxazole; pyrimethamine-sulfadoxine was the least potent combination. These experiments form a basis for the selection of rapidly eliminated antifolate combinations for further clinical testing.     

Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil

Increasing resistance of Plasmodium falciparum malaria parasites to chloroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have sparked renewed interest in the antimalarial drugs WR99210 and proguanil, the cycloguanil precursor. To investigate suggestions that WR99210 and proguanil act against a target other than the reductase moiety of the P. falciparum bifunctional DHFR-thymidylate synthase enzyme, we have transformed P. falciparum with a variant form of human DHFR selectable by methotrexate. Human DHFR was found to fully negate the antiparasitic effect of WR99210, thus demonstrating that the only significant action of WR99210 is against parasite DHFR. Although the human enzyme also resulted in greater resistance to cycloguanil, no decrease was found in the level of susceptibility of transformed parasites to proguanil, thus providing evidence of intrinsic activity of this parent compound against a target other than DHFR. The transformation system described here has the advantage that P. falciparum drug-resistant lines are uniformly sensitive to methotrexate and will complement transformation with existing pyrimethamine-resistance markers in functional studies of P. falciparum genes. This system also provides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations against malaria.     

Cycloguanil and its parent compound proguanil demonstrate distinct activities against Plasmodium falciparum malaria parasites transformed with human dihydrofolate reductase

The lack of suitable antimalarial agents to replace chloroquine and pyrimethamine/sulfadoxine threatens efforts to control the spread of drug-resistant strains of the malaria parasite Plasmodium falciparum. Here we describe a transformation system, involving WR99210 selection of parasites transformed with either wild-type or methotrexate-resistant human dihydrofolate reductase (DHFR), that has application for the screening of P. falciparum-specific DHFR inhibitors that are active against drug-resistant parasites. Using this system, we have found that the prophylactic drug cycloguanil has a mode of pharmacological action distinct from the activity of its parent compound proguanil. Complementation assays demonstrate that cycloguanil acts specifically on P. falciparum DHFR and has no other significant target. The target of proguanil itself is separate from DHFR. We propose a strategy of combination chemotherapy incorporating the use of multiple parasite-specific inhibitors that act at the same molecular target and thereby maintain, in combination, their effectiveness against alternative forms of resistance that arise from different sets of point mutations in the target. This approach could be combined with traditional forms of combination chemotherapy in which two or more compounds are used against separate targets.     

Intron-exon structure of the MET gene and cloning of an alternatively-spliced Met isoform reveals frequent exon-skipping of a single large internal exon

BACKGROUND AND AIMS: the purine nucleoside analogues cladribine (CdA), fludarabine (F-Ara-AMP) and pentostatin (dCf), are effective therapy for a range of T- and B-cell lymphoid malignancies. The effects upon nucleotide metabolism in human CCRF-CEM T-cell leukaemia and Raji B-cell lymphoma cell lines of these drugs have been compared to assess possible mechanisms of cytotoxicity. METHODS: Leukaemia cells were exposed to a purine nucleoside analogue and perchloric acid extracts were analysed by HPLC for 2'-deoxynucleoside-5'-triphosphates (dNTPs), nucleoside-5'-triphosphates (NTPs) and drug metabolites. RESULTS: After addition of a purine nucleoside analogue, CdA-TP and F-Ara-ATP accumulate in cells while the levels of dCf-TP formed were not detectable by ultra-violet absorbance. In response to accumulating concentrations of drug triphosphate, the cellular levels of dNTPs initially decrease (0-4 h), then accumulate above their initial levels (4-10 h) before slowly declining beyond 10 h. NTPs also accumulate during the period 4-10 h before declining at later times. CONCLUSION: The temporal effects on the levels of dNTPs and NTPs of the 3 purine nucleoside analogues are similar against CCRF-CEM and Raji cells. However, CdA induces major depletions of dTTP, dGTP and dATP in CCRF-CEM cells and F-Ara-A induces a major accumulation of dATP in Raji cells.     

Allosteric regulation of Trypanosoma brucei ribonucleotide reductase studied in vitro and in vivo

Trypanosoma brucei is the causative agent for African sleeping sickness. We have made in vitro and in vivo studies on the allosteric regulation of the trypanosome ribonucleotide reductase, a key enzyme in the production of dNTPs needed for DNA synthesis. Results with the isolated recombinant trypanosome ribonucleotide reductase showed that dATP specifically directs pyrimidine ribonucleotide reduction instead of being a general negative effector as in other related ribonucleotide reductases, whereas dTTP and dGTP directed GDP and ADP reduction, respectively. Pool measurements of NDPs, NTPs, and dNTPs in the cultivated bloodstream form of trypanosomes exposed to deoxyribonucleosides or inhibited by hydroxyurea confirmed our in vitro allosteric regulation model of ribonucleotide reductase. Interestingly, the trypanosomes had extremely low CDP and CTP pools, whereas the dCTP pool was comparable with that of other dNTPs. The trypanosome ribonucleotide reductase seems adapted to this situation by having a high affinity for the CDP/UDP-specific effector dATP and a high catalytic efficiency, Kcat/Km, for CDP reduction. Thymidine and deoxyadenosine were readily taken up and phosphorylated to dTTP and dATP, respectively, the latter in a nonsaturating manner. This uncontrolled uptake of deoxyadenosine strongly inhibited trypanosome proliferation, a valuable observation in the search for new trypanocidal nucleoside analogues.     

In vitro activities of several diaminomethylpyridopyrimidines against Mycobacterium avium complex

Three recently synthesized dihydrofolate reductase (DHFR) inhibitors designated SoRI 8890, 8895, and 8897 were evaluated for their in vitro activities against 25 isolates of Mycobacterium avium complex. The MICs at which 50 and 90% of isolates were inhibited were 1 and 2, 4 and 8, and 4 and 8 microgram/ml for SoRI 8890, 8895, and 8897, respectively. Although the addition of dapsone at 0.5 microgram/ml did not significantly enhance the in vitro activities of these compounds, their activities alone were comparable to, if not better than, results seen with other DHFR inhibitors, such as pyrimethamine or WR99210.     

Cloning and analysis of the human complement factor H gene promoter

The compound WR 238605 is a primaquine analog being developed by the U.S. Army as an antimalarial drug. Currently, there is no established treatment for Plasmodium vivax parasitemias that are not cured by chloroquine. This study tested WR 238605, chloroquine, and their combinations against a chloroquine-resistant strain of P. vivax (AMRU 1) in Aotus monkeys. A total dose of 3 mg/kg of WR 238605 given at a dosage of 1 mg/kg/day for three days cleared patent parasites in all eight monkeys but recrudescence of parasitemia occurred 15-25 days after initiation of treatment. A total dose of 9 mg/kg of WR 238605 over a three-day period cured all three monkeys of their infections. A total dose of 30 mg/kg of chloroquine did not clear patent infections in three monkeys, whereas a total dose of 60 mg/kg generally (two of three) cleared patent parasitemia but did not cure. Whereas total doses of 30 mg/kg of chloroquine or 3 mg/kg of WR 238605 given alone failed to cure, both drugs given in combination at these dosages cured two of three infections. These results indicate that WR 238605 may be an alternative treatment for chloroquine-resistant vivax malaria.     

Effects of cytosine arabinoside on human leukemia cells

Cytosine arabinoside (Ara-C) is used to treat leukemias, with complete remission induced by combination chemotherapy in approximately 70% of cases of acute myelogenous leukemia (AML). Ara-CTP acts as a competitive inhibitor of DNA polymerase and may also be incorporated into DNA. Accumulation of deoxyribonucleoside triphosphates (dNTPs) induced by Ara-C may indicate disruption of DNA synthesis in susceptible leukemia cells. A procedure has been developed for the quantification of Ara-CTP and dNTPs from small samples of leukaemia cells from patients (4 x 10(7) cells) activated with concanavalin A (10 micrograms/ml, 48 hr) and grown in the presence of [32P]orthophosphate (1.1 microM, 9 x 10(6) Ci/mol, 16 hr). The susceptibilities to Ara-C of the human leukemia cell lines CCRF-CEM (IC50 = 6.30 nM), CCRF-HSB-2 (IC50 = 10.4 nM) and MOLT-4 (IC50 = 10.0 nM) may be correlated with their abilities to accumulate high concentrations of Ara-CTP (> 1000 amol/cell) with increases of between 1.3- and 3.4-fold in dATP, dGTP and dTTP for the four cell lines, while dCTP decreased between 0.23- and 0.78-fold. By contrast, an Ara-C-resistant derivative of HL-60 cells (IC50 = 400 nM) accumulated only low concentrations of Ara-CTP (71 amol/cell) without significant changes in dNTPs. High concentrations of Ara-CTP in leukemia cells induce accumulations of dATP, dGTP and dTTP due to inhibition of DNA synthesis, and depletion of dCTP. This imbalance in the pools of the four dNTPs could lead to genetic miscoding and cell death.     

Testing candidate genes that may affect susceptibility to leprosy

Due to increased chloroquine resistance, the antifolate/sulpha drug combinations are becoming increasingly important in the chemotherapy of falciparum malaria. However, point mutations in the dihydrofolate reductase gene lead to resistance to the antifolate drugs. We therefore investigated the prevalence of the 6 reported point mutations in this gene among field isolates of Plasmodium falciparum from Kenya, to determine if the mutations correlated with resistance to pyrimethamine and the biguanides cycloguanil and chlorcycloguanil. We used a mutation-specific polymerase chain reaction technique to test for these reported mutations in 21 Kenyan isolates and 4 reference lines. We also amplified and directly sequenced the dihydrofolate reductase coding sequence from these parasites to confirm the results and test for other possible mutations. Of the reported mutations, we found S108N, which is the central mutation of pyrimethamine resistance, and mutations N51I and C59R, which modulate the levels of resistance and may confer decreases in response to cycloguanil that are folate and p-aminobenzoic acid dependent. No isolate possessed the paired point mutations S108T and A16V, or I164L and S108N, which have been associated with cycloguanil resistance in previous studies. These results provided supportive evidence for the combined use of a cycloguanil-class drug (e.g., chlorproguanil) and a sulpha drug (e.g., dapsone) against P.falciparum malaria in Kenya.     

An international randomized study with long-term follow-up of single versus combination chemotherapy of multibacillary leprosy

A total of 307 patients with lepromatous leprosy and borderline lepromatous leprosy were randomized to dapsone monotherapy or to one of two types of drug combinations. A 3-year treatment phase was followed by a 5-year observation phase. The evaluation included 233 patients for whom together there were 1,404 years of observation. A total of 1,956 blinded histopathological specimens were processed centrally. When entering the trial isolates from 13 patients (5.6%) showed dapsone resistance in the mouse footpad test, and these patients were evaluated separately. Dapsone monotherapy (68 patients) had the same frequency of cure as the combination of dapsone and rifampin (77 patients) or the four-drug regimen consisting of dapsone, rifampin, isoniazid, and prothionamide (75 patients). We did not find a significant difference in the clearance of bacteria either between the monotherapy and the two-drug combination or the monotherapy and the four-drug combination. Six months after the initiation of treatment, disease in 15% of the patients who received dapsone monotherapy but none of the patients who received combined treatment were clinically progressive. After another 1 to 9 months of treatment the disease in all patients was stable or regressive. There was no difference in the type or frequency of reactions. Only after the end of the scheduled observation phase three relapses were reported. All three treatment regimens well tolerated. Dapsone monotherapy is highly effective in the treatment of multibacillary leprosy under the conditions of well-controlled treatment. Combination regimens seem only to accelerate the regression of the active disease when they are compared with monotherapy with dapsone. The mouse footpad test does not reflect the clinical resistance and cannot be recommended for use in making therapeutic decisions.     

Iodoquinol associated seizures and radiopacity

With the recent observations of efflux of chloroquine from Plasmodium falciparum and modulation of chloroquine resistance by calcium channel blockers, such as verapamil, a great deal of attention has been focused on the development of new modulators that can potentiate the efficacy of chloroquine. We report a new compound, WR268954, that has weak intrinsic antimalarial activity compared to chloroquine. In vitro, it increased the susceptibilities of chloroquine-resistant P. falciparum strains to chloroquine and quinine, but did not affect the chloroquine-susceptible strains. In the presence of 2,000 nM of WR268954, the 50% inhibitory concentration of chloroquine for drug-resistant P. falciparum decreased 90-fold in comparison with the control (chloroquine only). The same concentration of WR268954 increased the potentiation of chloroquine in resistant strains to a level approximately equivalent to that observed for the sensitive strain. This compound also potentiates the efficacy of quinine in drug-resistant parasites. However, WR268954 did not enhance the efficacy of mefloquine in the mefloquine-resistant parasites. In this report, the data show the synergistic effect of WR268954 on the antimalarial activity of chloroquine in drug-resistant strains of P. falciparum, but only an additive effect on drug-sensitive strains of parasites. Compound WR268954 belongs to a pyrrolidino alkane amine class whose in vitro chloroquine resistance modulator activity supports the basis for the synthesis of this class of compounds.     

Research on new antimalarial drugs and the use of drugs in combination at the Bangkok Hospital for Tropical Diseases

With the emergence of multidrug resistant falciparum malaria in Thailand, various approaches have been taken. Research on new antimalarial drugs and the use of existing available drugs with modification are urgently needed. New drugs and drugs in combination such as pyronaridine, WR 238605, arteether, dihydroartemisinin, benflumetol atovaquone/proguanil are being evaluated. Drug combinations for the treatment of patients suffering from uncomplicated falciparum malaria include quinine-tetracycline for 7 days, or sequential treatment of artesunate (600 mg given over 5 days) followed by mefloquine (1,250 mg divided into 2 doses 6 hours apart) are recommended. The sequential treatment is highly recommended for those who failed other treatment regimens. Other combinations such as a short course sequential treatment of artesunate (300 mg given over 2.5 days) followed by a single dose of 750 mg mefloquine, or a combination of mefloquine 1,250 mg together with tetracycline 1 g per day or doxycycline 200 mg per day for 7 days are alternative treatment regimens with acceptable cure rates. The simultaneous administration of artesunate and mefloquine, in various doses and duration of treatment, is currently being investigated. Until proven otherwise, the drug combinations are still recommended for all adult patients suffering from acute uncomplicated falciparum malaria contracted in multidrug resistant areas. In severe malaria and malaria in children, the drug combinations need further investigation.     

Fluvoxamine inhibits the CYP2C19-catalysed metabolism of proguanil in vitro

OBJECTIVE: The potent CYP1A2 inhibitor fluvoxamine has recently been shown also to be an effective inhibitor of the CYP2C19-mediated metabolism of the antimalarial drug proguanil in vivo. The purpose of the present study was to confirm this interaction in vitro. METHODS: A high-performance liquid chromatography (HPLC) method was developed to assay 4-chlorophenylbiguanide (4-CPBG) and cycloguanil formed from proguanil by microsomes prepared from human liver. The limit of detection was 0.08 nmol mg-'. h-I. RESULTS: The formation of 4-CPBG and cycloguanil could be described by one-enzyme kinetics, indicating that the formation of the two metabolites is almost exclusively catalysed by a single enzyme, i.e. CYP2C19 within the concentration range used, or that the contribution of an alternative low-affinity enzyme, probably CYP3A4, is very low. This notion was confirmed by the lack of potent inhibition by four CYP3A4 inhibitors: ketoconazole, bromocriptine, midazolam and dihydroergotamine. Fluvoxamine was a very effective inhibitor of the oxidation of proguanil, displaying Ki values of 0.69 micromol x l(-1) for the inhibition of cycloguanil formation and 4.7 micromol x l(-1) for the inhibition of 4-CPBG formation. As expected, the CYP2C19 substrate omeprazole inhibited the formation of both metabolites with an IC50 of 10 micromol x l(-1). Norfluoxetine and sulfaphenazole inhibited proguanil oxidation with Ki values of 7.3-16 micromol x l(-1), suggesting that the two compounds are moderate inhibitors of CYP2C19. CONCLUSIONS: Fluvoxamine is a fairly potent inhibitor of CYP2C19 and it has the potential for causing drug-drug interactions with substrates for CYP2C19 such as imipramine, clomipramine, amitriptyline and diazepam. The combination of fluvoxamine and proguanil can not be recommended.     

Interaction of combinations of drugs, chemosensitizers, and peptides with the P-glycoprotein multidrug transporter

P-Glycoprotein functions as an ATP-driven efflux pump for hydrophobic natural products and peptides, and gives rise to resistance to multiple chemotherapeutic drugs. The inhibition of colchicine transport via P-glycoprotein by various compounds was determined in a plasma membrane vesicle model system. A chemotherapeutic drug (vinblastine) and several chemosensitizers (verapamil, reserpine, cyclosporin A) and hydrophobic peptides (N-acetyl-leucyl-leucyl-methioninal, leupeptin, pepstatin A, valinomycin) were examined, both as individual species and as combinations of compounds. The median effect analysis was used to determine the concentration of each combination required to produce a median effect, Dm, as well as the sigmoidicity of the concentration-effect plot, m. The combination of cyclosporin A and verapamil was the only one established to be mutually nonexclusive, whereas several mutually exclusive pairs of compounds were identified. The combination index, CI, was calculated for several combinations of drugs, chemosensitizers, and peptides, and used to ascertain whether effects were synergistic, antagonistic, or additive. Some combinations (vinblastine/verapamil; verapamil/valinomycin) showed antagonism over the entire concentration range. Other combinations (valinomycin/N-acetyl-leucyl-leucyl-methioninal; cyclosporin A/verapamil) displayed both synergism and antagonism over different regions of the CI plot. Many combinations of compounds displayed additive interactions over most of the CI plot. The median effect analysis may be helpful in identifying potentially useful additive or synergistic combinations of compounds for reversal of Pgp-mediated drug resistance.     

The rationale for combination versus single-entity therapy in hypertension

The rationale behind combination therapy relates to the fact that when two different classes of agents are combined, they may provide complementary, additive, or synergistic antihypertensive effects through different mechanisms. Lower doses of two drugs, which provide blood pressure reduction similar to higher doses of one drug, may enhance tolerability and improve compliance. Investigative efforts have been undertaken to explore fixed-dose combinations of drugs that do not include diuretics. The first nondiuretic fixed-dose combinations are an angiotensin-converting enzyme (ACE) inhibitor-calcium antagonist combination or a beta-blocker-calcium antagonist combination. The rationale for an ACE inhibitor-calcium antagonist combination is based on the fact that both drugs reduce vasoconstriction through different mechanisms. The ACE inhibitor largely attenuates vasoconstriction through augmentation of vasodilatory kinins and reduction of the vasoconstrictive effect of angiotensin II, whereas the calcium antagonists, through attenuating the transmembrane flux of calcium, inhibit calcium-mediated electromechanical coupling in contractile tissue in response to numerous stimuli. Moreover, both classes of drugs facilitate salt and water excretion by the kidney through different mechanisms. The ACE inhibitor restores the renal-adrenal response to salt loading, whereas the calcium antagonist possesses intrinsic natriuretic properties through poorly described mechanisms of inhibiting renal tubular salt and water reabsorption. The combination of a beta-blocker and dihydropyridine calcium antagonist is logical due to the different antihypertensive mechanisms of these drugs without risk of cardiac conduction abnormalities. There is evidence in clinical trials that ACE inhibitors may offset one of the major side effects associated with calcium antagonist therapy: pedal edema. Although the studies are small and the observations subjective, there is consistent evidence that the combination may provide an opportunity to reduce the likelihood of this common clinical problem. There is also evidence of reduced calcium antagonist-associated constipation and headache with this type of drug combination, likely because lower doses of this agent are used in combination with ACE inhibitors. However, there is no published evidence that calcium antagonists reduce the cough associated with the ACE inhibitor.     

Efficacy of artemisinin and mefloquine combinations against Plasmodium falciparum. In vitro simulation of in vivo pharmacokinetics

The activity of artemisinin in combination with mefloquine was tested in vitro against a chloroquine-sensitive (F32) strain of Plasmodium falciparum. A method of repetitive dosing and extending the culture observation period to 28-30 days was used to mimic the in vivo pharmacokinetic situation. Plasmodium falciparum was exposed to artemisinin from 10(-8) to 10(-5) M, mefloquine from 3 x 10(-9) to 10(-5) M and their combinations. The exposure time for artemisinin was 3 hours twice daily and for mefloquine 24 hours. The drug-dosing duration was 3 days. Neither artemisinin nor mefloquine alone provided radical clearance of P. falciparum, even when maximum concentrations (10(-5) M) were applied. The antiparasitic activity of artemisinin and mefloquine were significantly higher when dosed alone. Effective concentrations for different degrees of inhibition (EC 50, 90 and 99) of both artemisinin and mefloquine respectively were significantly lower when used in combination. At concentrations normally reached in vivo, this effect was clearly synergistic (P = 0.016) Our in vitro model of intermittent dosing of artemisinin and mefloquine combinations for 3 days provides significant evidence of positive interaction between the two compounds. Lower combination concentrations around the MIC-values for the individual compounds showed synergistic effect, and high concentrations showed additive effect. This indicates that such drug combinations may provide radical clearance at concentrations lower than those required for single-drug treatment.     

Antigonococcal activity of 11 drugs used for therapy or prophylaxis of malaria

Antibacterial agents are often used in malarial endemic areas for antimalarial prophylaxis (such as doxycycline and clindamycin). Gonococcal infections may coexist in the same geographic area, thus becoming suppressed by compounds directed toward malarial parasites. We tested 11 drugs with activity for Plasmodium species against 105 Neisseria gonorrhoeae strains. Traditional or investigational antimalarials such as arteflene (Ro 42-1611), chloroquine, primaquine, pyrimethamine, quinacrine, and quinine were observed to be inactive. Fansidar (sulfadoxine-pyrimethamine) and mefloquine possess marginal action in a minority of gonococcus strains (< 10%). Doxycycline [minimum inhibitory concentration inhibiting 90% of tested strains (MIC 90) 2 micrograms/ml] and azithromycin (MIC 90, 0.5 microgram/ml) among the antibacterials were very active, indicating a dual role as antimalarial and antigonococcal agents. Thus, the gonorrhea and sexually transmitted disease epidemiologic data from geographic regions where doxycycline or newer macrolides may be used for malarial prophylaxis or therapy could be significantly altered.     

Reversal of chloroquine resistance in murine malaria parasites by prostaglandin derivatives

An oligomeric ester of prostaglandin B2 (OC-5186) was found to reverse chloroquine resistance in the murine malarial parasite Plasmodium berghei. When mice were infected with either chloroquine-sensitive or -resistant P. berghei on day 0 (by intraperitoneal injection of 1 x 10(6) parasitized erythrocytes), they died before day 23. When treated with 15 mg/kg/day of chloroquine for the first four days of infection, all mice infected with the sensitive-strain survived, while all those infected with the resistant strain died before day 23. When OC-5186 (3-12 mg/kg/day) was administered in combination with chloroquine for the first four days, 60% of the animals infected with the resistant strain survived. The differences in the survival rate between the group treated with chloroquine only and the group treated with a combination of drugs (chloroquine plus 3-12 mg/kg/day of OC-5186) were significant. There was also a significant inhibition of parasitemia in the group treated with the combination of drugs. The combinations of chloroquine and a monomer ester of prostaglandin B2 (OC-5181) had some antimalarial activity, but the differences between the chloroquine-treated group and the combination treatment group were not significant in terms of both the parasitemia and the survival rate. Another oligomeric ester of prostaglandin E1 (MR-356) as well as unesterified monomer prostaglandins (PGA2 and PGB2) were ineffective by themselves and in combination with chloroquine.     

Modulation of fluorouracil cytotoxicity by interferon-alpha and -gamma

Because interferons (IFN)-alpha and -gamma individually have increased fluorouracil (FUra) cytotoxicity in several in vitro models, we studied the effects of FUra combined with IFN-alpha + gamma in HT29 colon cancer cells. A 96-hr exposure to IFN-alpha (500 units/ml) plus IFN-gamma (10 units/ml) and a 72-hr exposure to 0. 25-1 microM FUra (hr 24-96) inhibited cell growth and colony formation in an additive or more-than-additive fashion. When cells were exposed to IFN-alpha + gamma and FUra, free FdUMP levels became detectable, whereas [3H]FUra-RNA incorporation decreased. Exposure to IFN-alpha + gamma, FUra, or the combination decreased dTTP pools to 58%, 43%, and 17% of control, respectively. A marked increase in the dATP to dTTP ratio was seen with FUra with or without IFN-alpha + gamma. Thymidylate synthase catalytic activity was reduced to 28% and 24% of control with FUra with or without IFN-alpha + gamma, suggesting that the enhanced dTTP depletion must be due to another mechanism. FUra-mediated thymidylate synthase inhibition was accompanied by a 124-fold increase in total deoxyuridylate immunoreactivity and a 31-fold increase in dUTP pools, but the addition of IFN-alpha + gamma attenuated the accumulation. Treatment with IFN-alpha + gamma and FUra individually interfered with nascent DNA chain elongation, whereas the three-drug combination produced the most striking effects. IFN-alpha + gamma plus FUra produced the greatest amount of single-strand breaks in nascent DNA and dramatically decreased net DNA synthesis. IFN-alpha + gamma with or without FUra produced double-strand breaks in parental DNA. These results suggest that dTTP depletion, dATP/dTTP imbalance, pronounced inhibition of DNA synthesis, and damage to nascent and parental DNA contribute to the enhanced cytotoxicity with the triple combination.