Eijkman-Winkler Center, Hp G04.614, University Medical Center Utrecht, Heidelberglaan 100, NL-3584 CX Utrecht, The Netherlands
Received 24 September 2002; returned 17 December 2002; revised 23 December 2002; accepted 25 January 2003
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials and methods: HIV-infected peripheral blood mononuclear cells (PBMC) were treated for 7 days with different combinations of APHS and RTIs or PIs. The MT-2 cell line was infected with different HIV-1 strains and treated with APHS for 5 days.
Results: APHS showed synergic interactions with the RTIs zidovudine, lamivudine and efavirenz and with the PIs indinavir and ritonavir. The 50% inhibitory concentration (IC50) of APHS in this assay dropped from 13 µM when used alone, to 5 µM after combination with an RTI or PI. In combination with APHS the IC50 of the RTI and PI drugs tested also dropped. APHS inhibits the replication of HIV-1 strains resistant to zidovudine, lamivudine, stavudine, didanosine, zalcitabine and ritonavir.
Conclusions: These results indicate that APHS can be combined with RTIs and PIs and can inhibit several NRTI and PI-resistant HIV-1 strains.
Keywords: o-(acetoxyphenyl)hept-2-ynyl sulphide, Calcusyn, peripheral blood mononuclear cells, MT-2 cell line
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Current anti-HIV therapy consists of combinations of three or more antiretroviral drugs, the so-called highly active antiretroviral therapy (HAART). This therapy is more efficient in suppressing viral production than single drug therapy.1317 Also, synergic interaction, defined as a combined effect greater than expected from the additive effect of the individual drugs,18 has been shown for different combinations of anti-HIV drugs.1923 However, drug combination can also have undesirable side-effects such as emergence of cross-resistance mutations, combined toxicity and, as a consequence, poor adherence to the treatment and frequent alteration in the panel of agents used.10,2429
Intervention at more than a single step in the HIV replication cycle will probably be more efficient in suppressing viral replication and avoiding resistance. Therefore, in the last few years several anti-oxidative compounds, anti-proliferative compounds, anti-inflammatory compounds and compounds that target new viral and cellular factors involved in virus replication have been proposed as anti-HIV agents.1,3,4,3035 In addition to having good antiviral activity, new drugs should also be pharmacologically compatible with other anti-HIV drugs, show minimal toxicity and inhibit HIV strains that are resistant to clinically available drugs.3
We have previously found that o-(acetoxyphenyl)hept-2-ynyl sulphide (APHS) (Figure 1), a selective non-steroidal anti-inflammatory drug (NSAID),36 can inhibit the replication of several HIV-1 strains (Ba-L, HXB2 and AT) in primary cells (peripheral blood mononuclear cells, monocyte-derived macrophages and peripheral blood lymphocytes) in vitro by interfering with the reverse transcription process.37 APHS 50% inhibitory concentration (IC50) for HIV-1 was 6 µM, whereas its 50% toxic concentration for primary cells was 105 µM.
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Donor peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood from HIV-1-, HIV-2- and hepatitis B-seronegative donors, and obtained on Ficoll-Isopaque density gradients. To prepare a PBMC mixed batch, PBMCs isolated from six donors were pooled together in RPMI 1640 medium (Gibco/Invitrogen, Paisley, UK) supplemented with 10% dimethyl sulphoxide (DMSO; Merck, Darmstadt, Germany), 20% fetal calf serum (FCS; Invitrogen) and 10 µg/mL gentamicin (Invitrogen) and frozen at 140°C. Cells were thawed and cultured for 4 days before the experiment in RPMI 1640 medium supplemented with 10% FCS, and 10 µg/mL gentamicin containing 2 µg/mL phytohaemagglutinin (PHA) lectin from Phaseolus vulgaris (Sigma Chemie, Zwijndrecht, The Netherlands) at 37°C and 5% CO2.
Compounds
APHS was supplied by Dr L. J. Marnett. Synthesis details are described by Kalgutkar et al.38 APHS was diluted in aliquots in 100% ethanol, topped with argon gas and stored at 20°C. The concentration of ethanol during incubations never exceeded 0.1%. At this concentration, ethanol did not affect HIV-1 replication or cellular viability (data not shown). Zidovudine (Sigma), indinavir (Merck) and ritonavir (Abbott Laboratories S.A., Baar, Switzerland) were diluted in DMSO and lamivudine (GlaxoSmithKline, Middlesex, UK) and efavirenz (Merck) were diluted in water. The concentration of DMSO during incubations never exceeded 0.001% and no effect on HIV-1 replication or cellular viability was observed at this concentration (data not shown).
HIV-1 infection of PBMCs
PHA-stimulated PBMCs were washed twice to remove PHA and incubated for 7 days at a concentration of 5 x 105 cells/mL with HIV-1Ba-L at a multiplicity of infection (MOI) of 0.0025, in the presence of APHS and/or other drugs and 10 U/mL recombinant interleukin-2 (IL-2) (Roche Diagnostics Nederland B. V., Almere, the Netherlands), at 37°C and 5% CO2. To correct for the input virus, an extra control consisting of medium containing the same amount of input virus as added to the cells was included in the experiment. The amount of p24 in this control was subsequently subtracted from the p24 values of the samples in order to obtain the exact amount of p24 produced by the cells.
p24-core antigen quantification by ELISA
After 7 days incubation, samples of the supernatants were collected, inactivated by addition of Empigen (Calbiochem, La Jolla, CA, USA) and by heat inactivation at 56°C for 30 min. p24-core antigen concentration was determined by an ELISA (AMPAK, DAKO, Cambridgeshire, UK) as described previously.39,40 The absorbance values were converted into p24 concentration (ng/mL) with the use of a calibration curve made by serial dilutions of recombinant p24 protein (NIBSC, UK) that was submitted to the same treatment as the samples.
Determination of viability of PBMCs
After 7 days incubation, the metabolic activity of PBMCs from the HIV-1 infection model was assessed by a cellular viability assay, as described previously.41 Briefly, 150 µg/mL tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) (Sigma) was added to the cells. During the subsequent 2 h incubation period, metabolically active cells convert MTT into blue formazan crystals. Afterwards, three-quarters of the supernatant was gently removed and substituted by stop buffer consisting of 90% 2-propanol, 10% Triton X-100 and 0.4% HCl (Merck). When all formazan crystals were dissolved absorbance was measured at 550 nm using 630 nm as reference.
Determination of IC50 of the compounds
The ability of each compound to decrease p24 antigen was expressed as IC50. IC50 was calculated using the computer software program CalcuSyn for Windows (Biosoft, Cambridge, UK) according to the method of Chou & Talalay.18,42 This program uses the median-effect equation to produce doseeffect curves:
fa = 1/[1 + (Dm/D)m] (Equation 1)
where fa represents the fraction affected by the dose (reduction of p24 at a certain drug concentration expressed in decimals), Dm is the median effect dose (same as IC50), D is the dose of the drug and m is the sigmoidicity coefficient of the doseeffect curve. Data were accepted when the linear correlation coefficient of the median-effect plot based on experimental data was >0.90.
Combination of HIV-1 inhibitors
In the combination experiments, PBMCs were infected as described above and seeded in the presence of multiple-diluted fixed ratios of a combination of APHS and the clinically available drugs zidovudine, lamivudine, efavirenz, indinavir and ritonavir, in duplicate wells, according to the chequerboard design (Table ). Before the experiments were carried out, IC50 values of all drugs were determined as described before in order to choose the appropriate dosage ranges for the combinations with APHS. Concentrations of APHS, zidovudine, lamivudine, efavirenz, indinavir and ritonavir were in the range 0.7524 µM, 0.6310 nM, 0.6310 nM, 37.5600 pM, 0.6310 nM and 0.8814 nM, respectively. After 7 days of incubation, p24 antigen in culture supernatant was quantified by ELISA and cellular viability was assessed by a cytotoxicity assay. fa was calculated for each drug combination after p24 quantification. Using the CalcuSyn program, doseeffect curves were plotted for each drug combination, i.e. each dose was plotted against the corresponding fa value. Using the multiple drug-effect equation, which is based on the median-effect equation and the isobologram method, IC50 was calculated for both drugs alone and in combination at their equipotent ratio (IC50 APHS/IC50 drug). Data were accepted when the linear correlation coefficient of the median-effect plot based on experimental data was >0.90. The combination index (CI), a quantitative measure of drug interaction, was calculated for each fa at three different drug ratios (1:2, 1:1 and 2:1) based on the IC50 values of each drug used alone [e.g. 1:1 drug ratio represents the equipotent ratio (IC50 APHS:IC50 drug)], according to the following equation:
|
(Equation 2)
where CIx stands for the combination index at x% inhibition, (D)1 and (D)2 are the doses of drug 1 and 2 used when inhibiting x% in combination and (Dx)1 and (Dx)2 are the doses of drug 1 and 2 alone, inhibiting x% (calculated from equation 1). A drug combination is additive when CI 1, synergic when CI < 1 (combined effect is greater than the additive effect) and antagonistic when CI > 1 (combined effect is less than the additive effect). As the range of CI << 1, the degree of synergic interaction gets stronger.18 CI values were calculated using the mutually non-exclusive assumption.
Drug susceptibility assay
MT-2 cells (Medical Research Council, London, UK) were cultured in RPMI 1640 medium supplemented with 10% FCS and 10 µg/mL gentamicin, and passaged once a week until a maximum of 20 passages. The cells were maintained at 37°C, 5% CO2. One day before the experiments, the cells were passaged 1:1 in a concentration of 1 x 106 cells/mL.
HXB2 is the molecular clone of the first laboratory HIV-1 isolate. The genes that contain the drug resistance mutations were excised from the clinical isolates and cloned into the genetic background of HXB2. Thus all HIV-1 strains tested have the same genetic background. 41 + 215Y is a zidovudine-resistant HIV-1 strain as described by Jeeninga et al.43 This strain contains one amino acid (aa) change at codon 41 (Met is substituted by Leu) and one aa change at codon 215 (Thr is substituted by Tyr). 184V is a lamivudine-resistant HIV-1 strain as described by Schuurman et al.5 This strain contains one aa change at codon 184 (Met is substituted by Val). Strain 3096 is an RTI-resistant HIV-1 strain as described by de Jong et al.27 This strain contains an insertion of two aa between codons 68 and 69 of RT as well as an aa change at codon 67. Phenotypic resistance analysis showed high levels of resistance to zidovudine, lamivudine and stavudine, and moderate levels of resistance to didanosine and zalcitabine. Strain 4602 is a ritonavir-resistant HIV-1 strain as described by Nijhuis et al.9 It contains the following four mutations: 36I, 54V, 71V and 82T.
The susceptibility of these HIV-1 strains to APHS was analysed in a cell-killing assay (MTT assay) as described previously.44 Briefly, MT-2 cells in a concentration of 0.4 x 106 cells/mL were seeded with or without HIV-1 wild-type or drug-resistant mutants at an MOI of 0.002 or 0.006 and in the presence of increasing concentrations of APHS and maintained at 37°C, 5% CO2. After 5 days of incubation, a cellular viability assay was carried out as described above. In this case, the amount of formazan reflects the number of cells protected by the drug against killing by the virus and is used as a read out for drug susceptibility. The IC50 of APHS for the wild-type and for each of the virus mutants was determined using a computer software program.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antiviral activity of APHS in combination with the RTIs zidovudine, lamivudine and efavirenz, and the PIs indinavir and ritonavir, was studied. PBMCs, infected with HIV-1Ba-L, were incubated with increasing concentrations of APHS and drugs, and diluted according to the chequerboard design (Table ). After 7 days, viral production and cellular viability were quantified. All fixed ratio combinations of drugs showed no cytotoxicity as assessed by a cytotoxicity assay (data not shown). IC50 values for individual drugs and their combinations were determined (Table
). The IC50 of APHS alone varied from 10 to 15 µM (mean 13 µM). When used alone, the IC50s of zidovudine, lamivudine, efavirenz, indinavir and ritonavir were 3, 10, 1, 11 and 6 nM, respectively. IC50s of these clinically available drugs were comparable to those previously described in the literature.45 The IC50 of APHS dropped to 4, 5 and 6 µM (mean 5 µM) when used in combination with another drug. The IC50s of zidovudine, lamivudine, efavirenz, indinavir and ritonavir dropped to 2, 3, 0.2, 5 and 3 nM, respectively. Although a definite trend was evident, the only statistically significant decreases in IC50s were observed for indinavir when used in combination with APHS and for APHS when used in combination with efavirenz (P < 0.05 according to the Students t-test).
|
|
To determine the susceptibility of wild-type HIV-1 and several NRTI- or PI-resistant HIV-1 strains for APHS, a drug susceptibility assay was carried out. This assay gives information not only about antiviral activity of APHS but also about its cytotoxicity patterns. The IC50s of APHS for wild-type HIV-1 and drug-resistant strains are depicted in Table . The IC50 of APHS for the wild-type strain HXB2 was 10 µM. The IC50 of APHS for the zidovudine-resistant strain 41 + 215Y was 2 µM. The IC50 of APHS for the lamivudine-resistant strain 184V was 5 µM. The IC50 of APHS for the zidovudine-, lamivudine-, stavudine-, didanosine- and zalcitabine-resistant strain 3096 was 4 µM. The IC50 of APHS for the ritonavir-resistant strain 4602 was 5 µM. APHS did not show any significant toxicity for MT-2 cells at the concentrations tested and therefore it can be concluded that APHS was capable of inhibiting the replication of all four HIV-1 strains tested.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is important to investigate the interaction between new drugs and currently used drugs, especially the determination of the possible synergic interactions and the possible adverse effects arising from the combination. In this study, there was a clear trend towards a decrease in IC50 of the drugs when used in combination than when used alone. This means that the same effect can be obtained with lower doses of the individual drugs and, in this way, toxic side-effects are also minimized.
There are several methods described in the literature to determine synergic interaction.46 We used the computer program CalcuSyn,42 which is based on the median-effect principle, as described before.18 This program is simple to use, is not limited by the number, effect and interactions of the drugs tested, and only requires a minimum of data points to analyse drug interaction. CalcuSyn is often used in combination studies of anti-HIV agents.20,45,4749 It has been shown that the results obtained by this method are comparable to those of other methods such as MacSynergy, especially if more than one drug ratio is employed.20,48
Although combinations of APHS with the RTIs zidovudine, lamivudine and efavirenz showed, for all drug ratios, slight antagonistic to synergic interactions at the 50% inhibition level, moderate synergic to strong synergic interactions were observed at the 95% inhibition level. The fact that synergic interactions between APHS and RTIs become stronger at higher percentages of inhibition is important for in vivo therapeutics when a higher percentage of inhibition is desired. The strongest synergic interactions were found between APHS and lamivudine. Moderate antagonistic to synergic interactions were found between APHS and the PIs indinavir and ritonavir. No correlation was found between the percentage of inhibition level and the degree of interaction between APHS and PI. Slightly different effects were observed at different drug ratios for all combinations tested. This reflects the importance of choosing the most effective drug ratio for a combination of antiviral drugs.
The strong synergic interactions between APHS and RTIs are probably because APHS also inhibits the reverse transcription process. There are many studies that report a benefit arising from the combination of NRTIs and NNRTIs.19,5054 Although the mechanism for synergy seen with the use of NNRTIs and NRTIs is not known, it is conceivable that the interaction of the NNRTI with the RT at the NNRTI-binding site, which has been shown to cause conformational distortion of the catalytic aspartate triad,55 may allow improved incorporation of the NRTI into the growing DNA molecule, leading to more efficient chain termination or decreased rates of phosphorolytic removal of the terminator. APHS can also interact synergically with PIs. In acutely infected cell cultures, individual cells are present at different stages of the HIV-1 life cycle. Several other RTIs have shown synergic interactions with PIs.20,23,49,50,56,57 Combination of antiviral compounds with different targets can inhibit HIV-1 replication in a greater proportion of cells at different stages of viral replication, resulting in an increased viral suppression.56,20,23 Importantly, no strong antagonism was found between APHS and RTIs or PIs. Although in vivo studies will have to be conducted, these data indicate that APHS can safely be used in combination with RTIs and PIs.
New drugs should not only be pharmacologically compatible with currently available drugs but also be able to inhibit the replication of drug-resistant HIV strains. Some compounds with this characteristic are being developed.45,48,5860 In this study, APHS inhibited the replication of both wild-type and several NRTI and PI-resistant HIV-1 strains. Since the mutations tested are very common in vivo, APHS may prove to be very useful against HIV-1 strains that have acquired resistance to clinically available anti-HIV-1 drugs.
Since APHS is a derivative of aspirin and since previous studies in a rat air pouch model indicated that APHS concentrations up to 100 mg/kg are not toxic,36 it is reasonable to believe that it will be safe to use APHS in vivo. In conclusion, since previous studies have shown that APHS has minimal toxicity in vivo and a good anti-HIV activity, and since this study showed that APHS acts synergically with clinically available RTIs and PIs and is able to inhibit both wild-type and RTI- and PI-resistant HIV-1 strains, APHS is a very promising candidate for anti-HIV therapy in vivo.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Tavel, J. A., Miller, K. D. & Masur, H. (1999). Guide to major clinical trials of antiretroviral therapy in human immunodeficiency virus-infected patients: protease inhibitors, non-nucleoside reverse transcriptase inhibitors, and nucleotide reverse transcriptase inhibitors. Clinical Infectious Diseases 28, 64376.[ISI][Medline]
3 . Richman, D. D. (2001). HIV chemotherapy. Nature 410, 9951001.[CrossRef][ISI][Medline]
4 . Baba, M. (1997). Cellular factors as alternative targets for inhibition of HIV-1. Antiviral Research 33, 14152.[CrossRef][ISI][Medline]
5 . Schuurman, R., Nijhuis, M., van Leeuwen, R., Schipper, P., de Jong, D., Collis, P. et al. (1995). Rapid changes in human immunodeficiency virus type 1 RNA load and appearance of drug-resistant virus populations in persons treated with lamivudine (3TC). Journal of Infectious Diseases 171, 14119.[ISI][Medline]
6 . Maxeiner, H. G., Keulen, W., Schuurman, R., Bijen, M., de Graaf, L., van Wijk, A. et al. (2002). Selection of zidovudine resistance mutations and escape of human immunodeficiency virus type 1 from antiretroviral pressure in stavudine-treated pediatric patients. Journal of Infectious Diseases 185, 10706.[CrossRef][ISI][Medline]
7 . Carr, A., Samaras, K., Burton, S., Law, M., Freund, J., Chisholm, D. J. et al. (1998). A syndrome of peripheral lipodystrophy, hyperlipidaemia and insulin resistance in patients receiving HIV protease inhibitors. AIDS 12, F518.[CrossRef][ISI][Medline]
8
.
Huang, H., Chopra, R., Verdine, G. L. & Harrison, S. C. (1998). Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science 282, 166975.
9 . Nijhuis, M., Schuurman, R., de Jong, D., Erickson, J., Gustchina, E., Albert, J. et al. (1999). Increased fitness of drug resistant HIV-1 protease as a result of acquisition of compensatory mutations during suboptimal therapy. AIDS 13, 234959.[CrossRef][ISI][Medline]
10 . Notermans, D. W., van Leeuwen, R. & Lange, J. M. (1996). Treatment of HIV infection. Tolerability of commonly used antiretroviral agents. Drug Safety 15, 17687.[ISI][Medline]
11 . Mansky, L. M. (1996). The mutation rate of human immunodeficiency virus type 1 is influenced by the vpr gene. Virology 222, 391400.[CrossRef][ISI][Medline]
12 . Perelson, A. S., Neumann, A. U., Markowitz, M., Leonard, J. M. & Ho, D. D. (1996). HIV-1 dynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time. Science 271, 15826.[Abstract]
13 . Lipsky, J. J. (1996). Antiretroviral drugs for AIDS. Lancet 348, 8003.[CrossRef][ISI][Medline]
14
.
Hammer, S. M., Squires, K. E., Hughes, M. D., Grimes, J. M., Demeter, L. M., Currier, J. S. et al. (1997). A controlled trial of two nucleoside analogues plus indinavir in persons with human immunodeficiency virus infection and CD4 cell counts of 200 per cubic millimeter or less. AIDS Clinical Trials Group 320 Study Team. New England Journal of Medicine 337, 72533.
15 . Mocroft, A., Vella, S., Benfield, T. L., Chiesi, A., Miller, V., Gargalianos, P. et al. (1998). Changing patterns of mortality across Europe in patients infected with HIV-1. EuroSIDA Study Group. Lancet 352, 172530.[CrossRef][ISI][Medline]
16
.
Palella, F. J., Jr, Delaney, K. M., Moorman, A. C., Loveless, M. O., Fuhrer, J., Satten, G. A. et al. (1998). Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. New England Journal of Medicine 338, 85360.
17 . Henry, K., Erice, A., Tierney, C., Balfour, H. H., Jr, Fischl, M. A., Kmack, A. et al. (1998). A randomized, controlled, double-blind study comparing the survival benefit of four different reverse transcriptase inhibitor therapies (three-drug, two-drug, and alternating drug) for the treatment of advanced AIDS. AIDS Clinical Trial Group 193A Study Team. Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology 19, 33949.[Medline]
18 . Chou, T. C. & Talalay, P. (1984). Quantitative analysis of doseeffect relationships: the combined effects of multiple drugs or enzyme inhibitors. Advances in Enzyme Regulation 22, 2755.[CrossRef][ISI][Medline]
19
.
Maga, G., Hubscher, U., Pregnolato, M., Ubiali, D., Gosselin, G. & Spadari, S. (2001). Potentiation of inhibition of wild-type and mutant human immunodeficiency virus type 1 reverse transcriptases by combinations of nonnucleoside inhibitors and D- and L-(beta)-dideoxynucleoside triphosphate analogs. Antimicrobial Agents and Chemotherapy 45, 1192200.
20 . Deminie, C. A., Bechtold, C. M., Stock, D., Alam, M., Djang, F., Balch, A. H. et al. (1996). Evaluation of reverse transcriptase and protease inhibitors in two-drug combinations against human immunodeficiency virus replication. Antimicrobial Agents and Chemotherapy 40, 134651.[Abstract]
21 . Villahermosa, M. L., Martinez-Irujo, J. J., Cabodevilla, F. & Santiago, E. (1997). Synergistic inhibition of HIV-1 reverse transcriptase by combinations of chain-terminating nucleotides. Biochemistry 36, 1322331.[CrossRef][ISI][Medline]
22 . Tremblay, C., Merrill, D. P., Chou, T. C. & Hirsch, M. S. (1999). Interactions among combinations of two and three protease inhibitors against drug-susceptible and drug-resistant HIV-1 isolates. Journal of Acquired Immune Deficiency Syndromes 22, 4306.[ISI][Medline]
23
.
Snyder, S., DArgenio, D. Z., Weislow, O., Bilello, J. A. & Drusano, G. L. (2000). The triple combination indinavirzidovudinelamivudine is highly synergistic. Antimicrobial Agents and Chemotherapy 44, 10518.
24 . Carr, A., Miller, J., Law, M. & Cooper, D. A. (2000). A syndrome of lipoatrophy, lactic acidaemia and liver dysfunction associated with HIV nucleoside analogue therapy: contribution to protease inhibitor-related lipodystrophy syndrome. AIDS 14, F2532.[CrossRef][ISI][Medline]
25 . Max, B. & Sherer, R. (2000). Management of the adverse effects of antiretroviral therapy and medication adherence. Clinical Infectious Diseases 30, Suppl. 2, S96116.[CrossRef][ISI][Medline]
26 . Monno, L., Appice, A., Cavaliere, R., Scarabaggio, T. & Angarano, G. (1999). Highly active antiretroviral therapy failure and protease and reverse transcriptase human immunodeficiency virus type 1 gene mutations. Journal of Infectious Diseases 180, 56871.[CrossRef][ISI][Medline]
27 . de Jong, J. J., Goudsmit, J., Lukashov, V. V., Hillebrand, M. E., Baan, E., Huismans, R. et al. (1999). Insertion of two amino acids combined with changes in reverse transcriptase containing tyrosine-215 of HIV-1 resistant to multiple nucleoside analogs. AIDS 13, 7580.[CrossRef][ISI][Medline]
28 . Rousseau, M. N., Vergne, L., Montes, B., Peeters, M., Reynes, J., Delaporte, E. et al. (2001). Patterns of resistance mutations to antiretroviral drugs in extensively treated HIV-1-infected patients with failure of highly active antiretroviral therapy. Journal of Acquired Immune Deficiency Syndromes 26, 3643.[ISI][Medline]
29 . Maga, G. & Spadari, S. (2002). Combinations against combinations: associations of anti-HIV 1 reverse transcriptase drugs challenged by constellations of drug resistance mutations. Current Drug Metabolism 3, 7395.[ISI][Medline]
30 . Daelemans, D., Vandamme, A. M. & De Clercq, E. (1999). Human immunodeficiency virus gene regulation as a target for antiviral chemotherapy. Antiviral Chemistry and Chemotherapy 10, 114.[ISI][Medline]
31 . Lori, F., Jessen, H., Lieberman, J., Clerici, M., Tinelli, C. & Lisziewicz, J. (1999). Immune restoration by combination of a cytostatic drug (hydroxyurea) and anti-HIV drugs (didanosine and indinavir). AIDS Research and Human Retroviruses 15, 61924.[CrossRef][ISI][Medline]
32
.
Martin, M., Serradji, N., Dereuddre-Bosquet, N., Le Pavec, G., Fichet, G., Lamouri, A. et al. (2000). PMS-601, a new platelet-activating factor receptor antagonist that inhibits human immunodeficiency virus replication and potentiates zidovudine activity in macrophages. Antimicrobial Agents and Chemotherapy 44, 31504.
33
.
Rozera, C., Carattoli, A., De Marco, A., Amici, C., Giorgi, C. & Santoro, M. G. (1996). Inhibition of HIV-1 replication by cyclopentenone prostaglandins in acutely infected human cells. Evidence for a transcriptional block. Journal of Clinical Investigation 97, 1795803.
34 . Bourinbaiar, A. S. & Lee-Huang, S. (1995). The non-steroidal anti-inflammatory drug, indomethacin, as an inhibitor of HIV replication. FEBS Letters 360, 858.[CrossRef][ISI][Medline]
35 . Macilwain, C. (1993). Aspirin on trial as HIV treatment. Nature 364, 369.[ISI][Medline]
36
.
Kalgutkar, A. S., Crews, B. C., Rowlinson, S. W., Garner, C., Seibert, K. & Marnett, L. J. (1998). Aspirin-like molecules that covalently inactivate cyclooxygenase-2. Science 280, 126870.
37 . Pereira, C. F., Paridaen, J. T. M. L., Rutten, K., Huigen, M. C. D. G., van de Bovenkamp, M., Middel, J. et al. (2003). Aspirin-like molecules that inhibit human immunodeficiency virus-1 replication. Antiviral Research, in press.
38 . Kalgutkar, A. S., Kozak, K. R., Crews, B. C., Hochgesang, G. P. J. & Marnett, L. J. (1998). Covalent modification of cyclooxygenase-2 (COX-2) by 2-acetoxyphenyl alkyl sulfides, a new class of selective COX-2 inactivators. Journal of Medicinal Chemistry 41, 480018.[CrossRef][ISI][Medline]
39 . McKeating, J. A., McKnight, A. & Moore, J. P. (1991). Differential loss of envelope glycoprotein gp120 from virions of human immunodeficiency virus type 1 isolates: effects on infectivity and neutralization. Journal of Virology 65, 85260.[ISI][Medline]
40 . Moore, J. P., McKeating, J. A., Weiss, R. A. & Sattentau, Q. J. (1990). Dissociation of gp120 from HIV-1 virions induced by soluble CD4. Science 250, 113942.[ISI][Medline]
41 . Pauwels, R., Balzarini, J., Baba, M., Snoeck, R., Schols, D., Herdewijn, P. et al. (1988). Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds. Journal of Virological Methods 20, 30921.[CrossRef][ISI][Medline]
42 . Chou, T. C. & Hayball, M. P. (1996). CalcuSyn: Windows Software for Dose Effect Analysis. BIOSOFT, Cambridge, UK.
43 . Jeeninga, R. E., Keulen, W., Boucher, C., Sanders, R. W. & Berkhout, B. (2001). Evolution of AZT resistance in HIV-1: the 4170 intermediate that is not observed in vivo has a replication defect. Virology 283, 294305.[CrossRef][ISI][Medline]
44 . Boucher, C. A., Keulen, W., van Bommel, T., Nijhuis, M., de Jong, D., de Jong, M. D. et al. (1996). Human immunodeficiency virus type 1 drug susceptibility determination by using recombinant viruses generated from patient sera tested in a cell-killing assay. Antimicrobial Agents and Chemotherapy 40, 24049.[Abstract]
45 . Essey, R. J., McDougall, B. R. & Robinson, W. E., Jr (2001). Mismatched double-stranded RNA (polyI-polyC(12)U) is synergistic with multiple anti-HIV drugs and is active against drug-sensitive and drug-resistant HIV-1 in vitro. Antiviral Research 51, 189202.[CrossRef][ISI][Medline]
46 . Greco, W. R., Bravo, G. & Parsons, J. C. (1995). The search for synergy: a critical review from a response surface perspective. Pharmacological Reviews 47, 33185.[ISI][Medline]
47 . Georgiou, N. A., van der Bruggen, T., Jansen, C. A., Oudshoorn, M., Nottet, H. S., Marx, J. J. et al. (2001). The chemotherapeutic agent bleomycin in a two-drug combination with zidovudine, ritonavir or indinavir synergistically inhibits HIV type-1 replication in peripheral blood lymphocytes. International Journal of Antimicrobial Agents 18, 5138.[CrossRef][ISI][Medline]
48 . Chong, K. T. & Pagano, P. J. (1997). In vitro combination of PNU-140690, a human immunodeficiency virus type 1 protease inhibitor, with ritonavir against ritonavir-sensitive and -resistant clinical isolates. Antimicrobial Agents and Chemotherapy 41, 236773.[Abstract]
49 . Lamarre, D., Croteau, G., Wardrop, E., Bourgon, L., Thibeault, D., Clouette, C. et al. (1997). Antiviral properties of palinavir, a potent inhibitor of the human immunodeficiency virus type 1 protease. Antimicrobial Agents and Chemotherapy 41, 96571.[Abstract]
50 . Buckheit, R. W., Jr, Russell, J. D., Xu, Z. Q. & Flavin, M. (2000). Anti-HIV-1 activity of calanolides used in combination with other mechanistically diverse inhibitors of HIV-1 replication. Antiviral Chemistry and Chemotherapy 11, 3217.[ISI][Medline]
51
.
King, R. W., Klabe, R. M., Reid, C. D. & Erickson-Viitanen, S. K. (2002). Potency of nonnucleoside reverse transcriptase inhibitors (NNRTIs) used in combination with other human immunodeficiency virus NNRTIs, NRTIs, or protease inhibitors. Antimicrobial Agents and Chemotherapy 46, 16406.
52 . Piras, G., Nakade, K., Yuasa, S. & Baba, M. (1997). Three-drug combination of MKC-442, lamivudine and zidovudine in vitro: potential approach towards effective chemotherapy against HIV-1. AIDS 11, 46975.[ISI][Medline]
53 . Carr, A., Vella, S., de Jong, M. D., Sorice, F., Imrie, A., Boucher, C. A. et al. (1996). A controlled trial of nevirapine plus zidovudine versus zidovudine alone in p24 antigenaemic HIV-infected patients. The Dutch-Italian-Australian Nevirapine Study Group. AIDS 10, 63541.[ISI][Medline]
54 . Brennan, T. M., Taylor, D. L., Bridges, C. G., Leyda, J. P. & Tyms, A. S. (1995). The inhibition of human immunodeficiency virus type 1 in vitro by a non-nucleoside reverse transcriptase inhibitor MKC-442, alone and in combination with other anti-HIV compounds. Antiviral Research 26, 17387.[CrossRef][ISI][Medline]
55 . Esnouf, R., Ren, J., Ross, C., Jones, Y., Stammers, D. & Stuart, D. (1995). Mechanism of inhibition of HIV-1 reverse transcriptase by non-nucleoside inhibitors. Nature Structural Biology 2, 3038.[ISI][Medline]
56
.
Drusano, G. L., DArgenio, D. Z., Symonds, W., Bilello, P. A., McDowell, J., Sadler, B. et al. (1998). Nucleoside analog 1592U89 and human immunodeficiency virus protease inhibitor 141W94 are synergistic in vitro. Antimicrobial Agents and Chemotherapy 42, 21539.
57 . Pagano, P. J. & Chong, K. T. (1995). In vitro inhibition of human immunodeficiency virus type 1 by a combination of delavirdine (U-90152) with protease inhibitor U-75875 or interferon-alpha. Journal of Infectious Diseases 171, 617.[ISI][Medline]
58 . Ludovici, D. W., Kukla, M. J., Grous, P. G., Krishnan, S., Andries, K., de Bethune, M. P. et al. (2001). Evolution of anti-HIV drug candidates. Part 1: From alpha-anilinophenylacetamide (alpha-APA) to imidoyl thiourea (ITU). Bioorganic and Medicinal Chemistry Letters 11, 22258.[CrossRef][Medline]
59 . Ludovici, D. W., Kavash, R. W., Kukla, M. J., Ho, C. Y., Ye, H., De Corte, B. L. et al. (2001). Evolution of anti-HIV drug candidates. Part 2: Diaryltriazine (DATA) analogues. Bioorganic and Medicinal Chemistry Letters 11, 222934.[CrossRef][Medline]
60 . Ludovici, D. W., De Corte, B. L., Kukla, M. J., Ye, H., Ho, C. Y., Lichtenstein, M. A. et al. (2001). Evolution of anti-HIV drug candidates. Part 3: Diarylpyrimidine (DAPY) analogues. Bioorganic and Medicinal Chemistry Letters 11, 22359.[CrossRef][Medline]
|