Analysis of protease inhibitor combinations in vitro: activity of lopinavir, amprenavir and tipranavir against HIV type 1 wild-type and drug-resistant isolates

Elisabetta Bulgheroni1,*, Paola Citterio1,§, Francesco Croce1, Mirko Lo Cicero1, Ottavia Viganò1, Francesca Soster1, Ting-Chao Chou2, Massimo Galli1 and Stefano Rusconi1,

1 Istituto di Malattie Infettive e Tropicali, Università degli Studi, Ospedale Luigi Sacco, Via GB Grassi 74, 20157 Milano, Italy; 2 Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

Received 13 July 2003; returned 23 October 2003; revised 13 November 2003; accepted 8 December 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background: Despite the increasing number of antiretroviral compounds, the number of useful drug regimens is limited owing to the high frequency of cross-resistance.

Patients and methods: We studied in vitro two-drug combinations using three protease inhibitors (PIs), tipranavir, amprenavir and lopinavir, on isolates (003 and 004) derived from patients with resistance to multiple PIs compared with the drug-susceptible isolate 14aPre in peripheral blood mononuclear cells. Drug interactions were determined by median dose-effect analysis, with the combination index calculated at several inhibitory concentrations (IC).

Results: In 14aPre experiments, the combination tipranavir + lopinavir demonstrated synergy at low concentrations (IC50), an additive effect at IC75 and antagonism at IC90–IC95; tipranavir + amprenavir were antagonistic at all concentrations except IC95, where they were synergic; and the lopinavir + amprenavir combination was always antagonistic. In 003 and 004 infections, tipranavir + lopinavir and tipranavir + amprenavir combinations were antagonistic, and lopinavir + amprenavir were synergic, at all concentrations, with the exception of being additive at IC95.

Conclusions: Our in vitro experiments did not show any advantage in combining second generation PIs as a therapeutic strategy in naive or multi-treatment failure subjects, with the exception of tipranavir + amprenavir at IC95 in infections by a wild-type isolate.

Keywords: HIV-1, protease inhibitors, in vitro combinations, drug resistance, combination index


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Highly active antiretroviral therapy (HAART) has dramatically changed the morbidity and mortality rates associated with HIV type 1 (HIV-1) infection by inhibition of viral replication and restoration of the immune system.13

The incomplete viral suppression or detectable viraemia in a previously suppressed situation, defined as ‘virological failure’, is often observed in clinical practice and is due to many independent factors, such as poor adherence, tolerability and insufficient efficacy of the prescribed therapy and viral drug resistance.48

Different strategies have been adopted to overcome virological failure and are called ‘salvage therapies’. They are based on cocktails of five to nine drug compounds, thus far called MEGAHAART or GIGAHAART, or use of a double protease inhibitor (PI) regimen with the addition of boosted ritonavir as inhibitor of the P-450 cytochrome pathway.916

Data on the combination of two PIs at full dose is still largely unavailable, particularly in vitro studies. A few reports describe dual PI combinations at different doses on susceptible and phenotypically resistant viral isolates and show opposite results, from antagonistic to additive and synergic effects.1722 Use of dual PI combinations provides potential multiple advantages, especially in treatment-experienced patients with resistant virus.15 The pharmacokinetic effects could be the increase in bioavailability, the AUC, Cmin and formation of active metabolites, and the decrease in systemic clearance, Cmax, pharmacokinetic variability and clearance of active metabolites.23

Taking all these issues into consideration, the aim of our study was to best determine the in vitro activity of second generation PI combinations on a susceptible virus isolate and two resistant strains derived from PI-experienced subjects.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Viruses

In our study we evaluated a laboratory-adapted strain (14aPre) and two clinical virus isolates (003 and 004). 14aPre was derived from an HIV-1-infected individual before any antiretroviral therapy (Massachusetts General Hospital, Boston, MA, USA) and was considered a drug-susceptible isolate. Isolates 003 and 004 were from heavily drug-experienced patients at our Institute and were resistant to multiple PIs. The past therapy and the detected protease gene mutations are shown in Table 1.


View this table:
[in this window]
[in a new window]
 
Table 1. Patient treatments and genotypic data
 
Genotypic analysis and sequence of HIV-1 RNA

The RNA was extracted by kit QIAamp HIV (Qiagen, Inc., Chatsworth, CA, USA), according to the manufacturer’s recommendations, and converted into cDNA using 10 000 HIV-1 copies.

The PRO gene was amplified using 10 µL of cDNA by a nested PCR with specific primers. Outer primers were P7 and P8 and inner primers were PRO-F and PRO-R.24,25

Unincorporated primers and nucleotides were removed with QIAquick spin columns (Qiagen) and PCR products were directly sequenced using internal primers and ABI sequencing kit reagents with dye-labelled dideoxy terminators (Applied Biosystems, Inc., Foster City, CA, USA). An automatic DNA sequencer (ABI PRISM 377; Applied Biosystems, Inc.) was used.

Sequences were edited by Factura software and aligned with the HIV-1 IIIB consensus sequence by the Sequence Navigator software program (Applied Biosystems).

Viral isolation

Peripheral blood mononuclear cells (PBMC) taken from the patients were cultivated with PBMC from healthy donors according to the method described by Johnson et al.26 Cell-free supernatants were assayed twice a week by a p24 antigen-production ELISA (NEN Research Products, Boston, MA, USA).

Viral titration was carried out in PBMC, and the viral titre, measured as the 50% tissue-culture infectious dose (TCID50/mL), was calculated using the method of Reed & Muench.27

Compounds

Three PIs and one nucleoside reverse transcriptase inhibitor (NRTI) were used in our experiments.

Lopinavir was obtained from Abbott Laboratories (Abbott Park, IL, USA), tipranavir was originally provided by Pharmacia-Upjohn (Kalamazoo, MI, USA), and amprenavir and abacavir were provided by Glaxo Wellcome Research & Development (Stevenage, UK).

Compounds were diluted in dimethyl sulphoxide at a concentration of 10 mM and stored at –20°C until use.

Mathematical analysis of single-agent inhibitory concentration (IC) and combined drug interaction index (CI)

The concentrations giving half-maximal inhibition (IC50) of each single-agent therapy were determined by dose-effect analysis, using the CalcuSyn for Windows computer software program by T.-C. Chou & M. Hayball (Biosoft, Cambridge, UK).28

The multiple drug effect equation of Chou & Talalay, based on the median dose-effect principle and the isobologram technique, was used to analyse combined drug effects.29 This method involves plotting dose–response curves for each agent and for fixed-ratio combinations of the agents, as described previously.29 The CI values were based on the median dose-effect equation and the combination index equation. Mean CI values were determined, as well as S.E.M.

In this study, we defined a combination as synergic when the CI value was <1 (i.e. greater than the expected additive effect when two agents are combined), additive when CI = 1 and antagonistic when CI > 1 (i.e. less than the expected additive effect when two agents are combined).

Experimental design

In each drug study, 3- or 4-day phytohaemoagglutinin (PHA)-stimulated PBMC were exposed to the HIV-1 inoculum (1000 TCID50/mL per 106 cells) without a subsequent wash. Drugs were added simultaneously. Cells were suspended in a 1.0 mL final volume of R-20 medium supplemented with 10% interleukin-2 in 24-well tissue culture plates, and incubated in a humidified atmosphere with 5% CO2 at 37°C. In all experiments culture medium was changed twice weekly so that 0.5 mL of cell suspension was resususpended in 1.0 mL of fresh medium that contained the original drug concentration(s).

The drug concentrations that inhibited the viruses were evaluated in PBMC according to the method described previously.30,31 Each single drug and each combination was tested in duplicate, and each experiment was repeated at least twice. In addition, uninfected drug-treated toxicity controls were maintained at the highest concentration for each agent studied (either alone or in combination). We also maintained viruses without cells or drugs for the entire duration of the experiments in order to take into account the viral carryover.

Each compound was tested at four different concentrations: tipranavir ranging from 0.01 to 0.32 µM, amprenavir from 0.01 to 0.64 µM, lopinavir from 0.000625 to 0.32 µM and abacavir from 0.3 to 4.8 µM. The two-drug combination assays were performed with the following concentrations of each drug: 0.25 x IC50, 0.5 x IC50, IC50 and 2 x IC50.

On day 7, the cultures were ended and cell-free supernatants were harvested for p24 antigen-production ELISA, for measurement of virus replication. Cell proliferation and viability were assessed by the Trypan Blue exclusion method.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
None of the drugs tested reached 50% cytotoxicity (CC50) at the maximal concentrations used. Wild-type (14aPre) and resistant (003 and 004) viruses were first tested for each PI (amprenavir, lopinavir and tipranavir) and for abacavir, an NRTI. Results from 003 and 004 were considered as derived from a unique resistant strain, since we noted a constant overlapping between the results of PI combination experiments from these two isolates. Mean values (± S.E.M.) of single target IC50 obtained for 14aPre and for the two PI-resistant isolates are reported in Table 2. As described previously, isolates 003 and 004 were resistant to indinavir, ritonavir and nelfinavir. In these experiments, the two PI-resistant isolates also showed a reduced susceptibility to the other PIs and abacavir compared with 14aPre. The increase in IC50 was as follows: 3.95-fold for tipranavir, 48.5-fold for lopinavir, 4.48-fold for amprenavir and 2.33-fold for abacavir.


View this table:
[in this window]
[in a new window]
 
Table 2. Mean values (± S.E.M.) of single drug IC50s
 
In total, three two-PI combinations were evaluated: tipranavir + lopinavir, tipranavir + amprenavir and lopinavir + amprenavir. An NRTI (abacavir) was tested in combination with amprenavir as reference. The results are shown in Figure 1. In this study we defined a combination as synergic when the CI value was <1, additive when it was 1 and antagonistic when it was >1.



View larger version (15K):
[in this window]
[in a new window]
 
Figure 1. CIs (mean ± S.E.M., minimum three experiments) at different drug concentrations (IC50, IC75, IC90 and IC95) with 14aPre, and 003 and 004 infections. White bars, tipranavir + lopinavir; black bars, tipranavir + amprenavir; grey bars, lopinavir + amprenavir.

 
The drug combinations showed discordant results in infections mediated by 14aPre. Tipranavir + lopinavir exhibited synergy at lower concentrations, were additive at IC75 and were antagonistic at IC90 and IC95. Combinations using tipranavir + amprenavir were antagonistic at all concentrations, with the exception of IC95, where synergy was shown. Lopinavir + amprenavir were antagonistic at both low and high concentrations (Table 3).


View this table:
[in this window]
[in a new window]
 
Table 3. CIs for different drug combinations
 
Drug associations tested in infections mediated by resistant viruses presented a linear pattern in all combinations tested with the four effective doses. Tipranavir + lopinavir and tipranavir + amprenavir were always antagonist. Lopinavir + amprenavir were synergic at IC50, IC75 and IC90, and additive at IC95.

Indinavir + saquinavir served as a control combination and were mildly antagonistic at all inhibitory concentrations, whereas the combination abacavir + amprenavir was synergic using four effective concentrations both in infections with 14aPre and the multidrug-resistant isolates 003 and 004 (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The development of HIV-1 PIs and the use of combination therapy regimens have ushered in a new era in antiretroviral therapeutics. The prospect of sustained viral suppression, together with an improved quality of life, has arisen due to these recent successes. Optimism for the potential of antiretroviral combination therapies against HIV-1 must be tempered by past experience with other human pathogens (e.g. tuberculosis) when complacency led to the eventual emergence of multidrug-resistant isolates.3236

The scarce adherence to complex regimens, the tolerability and the long-term toxicity of antiretrovirals led to the incomplete suppression of viral replication, thus to the emergence of drug resistance as a direct consequence of HIV-1 variability. Effective therapeutical strategies are limited by the frequent occurrence of cross-resistance notwithstanding the increasing number of antiretroviral compounds. Taking this into consideration, our in vitro experiments tested the antiretroviral and cytotoxic effect of two-drug combinations including second generation PIs at full doses, such as amprenavir, lopinavir and tipranavir, both in wild-type and multidrug-resistant isolates of HIV-1.

Combinations with two PIs were analysed in PBMC with a 1:1 ratio between the compounds. Drug concentrations of equal activity were paired (i.e. 0.25 x IC50, 0.5 x IC50, IC50 and 2 x IC50). Indinavir + saquinavir served as a control combination with two first-generation PIs, whereas abacavir + amprenavir was used as a combination between drugs directed against two different enzymatic targets. No cellular toxicity was observed during the entire duration of the experiments.

Susceptibility tests confirmed the data derived from the genotypic analysis and showed a fold change in drug susceptibility of mutated versus wild-type isolate ranging from 3.95- to 48.5-fold for antiretroviral resistance. Our resistant isolates showed resistant phenotypes compared with a previous report in the literature regarding lopinavir.22 With regard to amprenavir, Petropoulos et al.37 defined as resistant an amprenavir IC50 > 3.0 µM in viruses showing amprenavir genotypic resistance. Recently, McCallister et al.38 reported a lack of clinical response for tipranavir IC50 greater than two-fold wild-type virus in isolates derived from heavily-treated individuals enrolled in the BI 1182.52 Phase II trial. After examining these phenotypic profiles, our resistant isolates met the definition of being phenotypically resistant.

With regard to combination experiments, tipranavir + amprenavir exhibited synergy at high drug concentrations (IC95) in 14aPre infections. Moreover, our results demonstrated the efficacy of the lopinavir + amprenavir combination against the two resistant isolates we tested. This was probably due to the fact that the two PIs have favourable molecular interactions within the PI binding site; thus the enzyme function is blocked with higher efficacy. In contrast, the antagonism seen in infections with the multidrug-resistant isolates using tipranavir + lopinavir and tipranavir + amprenavir could derive from the competition of the two compounds for the catalytic region of the viral protease, or competitive cellular uptake or partial overlapping binding of inactivation.

Previous in vitro experiments by Molla et al.22 showed additive effects using the tipranavir + lopinavir and lopinavir + amprenavir combinations. Our results were slightly different. This could be due to two main reasons, the first being the use of a different cellular target (MT4 cell line in Molla’s experiments and PBMC in ours) and the second being that we examined both wild-type and resistant clinical isolates, compared with a single wild-type laboratory strain in Molla’s experiments. Recently, Doyon et al.39 showed an additive effect of tipranavir + lopinavir and tipranavir + amprenavir combinations in C8166 cells using wild-type and resistant recombinant viruses.

Although in our experiments the lower concentrations of each compound caused some additive or synergic interactions in some conditions (i.e. tipranavir + lopinavir showed synergy at IC50 and an additive effect at IC75 in 14aPre infections), it should be restated that suboptimal drug dosages could increase the likelihood of the emergence of multidrug-resistant variants, and the therapeutic objectives should demand high effects at high doses.

The results of our study indicate caution for the use of dual PI therapy in HIV-1-infected individuals who have failed multiple regimens. The antagonism we observed in vitro (e.g. tipranavir + lopinavir and tipranavir + amprenavir) suggests that these two two-PI combinations would exhibit a lower efficacy than expected. Our in vitro experiments did not evidence any advantage in combining second-generation PIs as a therapeutic strategy in naive or multi-failed subjects, with the exception of tipranavir + amprenavir at IC95 in infections mediated by the wild-type isolate.

Therapeutic decision-making should depend on sound clinical trial evidence. It is likely that in vitro combination drug-effect analyses such as these, coupled with pharmacokinetic data, may be of assistance to clinicians in choosing therapies. It should be noted that the present studies have examined combined anti-HIV-1 ‘efficacy’ of PIs in terms of synergy or antagonism in vitro. In clinical settings, the combined ‘toxicity’ toward the host in terms of synergy or antagonism is also important. This may be particularly useful for individuals breaking through antiretroviral therapy who may have viral isolates demonstrating multidrug-resistance, and for whom only anecdotal clinical information is available. Our studies suggest that the design of appropriate regimens for such patients will remain a challenge.


    Acknowledgements
 
This work was supported by an AIDS research grant (III and IV AIDS Project) from the Istituto Superiore di Sanità, Rome (40C.80 and 40D.74) to S.R., ‘Progetto Giovani’ Ministero Università e Ricerca Scientifica Tecnologica 2001 to E.B. and P.C., and Anlaids ONLUS Sez. Lombarda, Milan, Italy. We thank Elizabeth L. Kaplan for language editing and continuous support, Professor Mauro Moroni and Dr Giulia Marchetti for the critical review of the manuscript, and Professor Martin S. Hirsch for his valuable expert advice. This work was presented in part at the Sixth International Congress on Drug Therapy in HIV Infection, Glasgow, UK, 17–21 November 2002 (Abstract P230).


    Footnotes
 
* E. Bulgheroni and P. Citterio contributed equally to this work. Back

§ E. Bulgheroni and P. Citterio contributed equally to this work. Back

Corresponding author. Tel: + 39-02-39043350; Fax: + 39-02-3560805; E-mail: stefano.rusconi{at}unimi.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Palella, F. J., Jr, Delaney, K. M., Moorman, A. C. 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, 853–60.[Abstract/Free Full Text]

2 . Hogg, R. S., Heath, K. V., Yip, B. et al. (1998). Improved survival among HIV-infected individuals following initiation of antiretroviral therapy. Journal of the American Medical Association 279, 450–4.[Abstract/Free Full Text]

3 . Mocroft, A., Katlama, C., Johnson, A. M. et al. (2000). AIDS across Europe, 1994–98: the EuroSIDA Study. Lancet 356, 291–6.[CrossRef][ISI][Medline]

4 . Paterson, D. L., Swindell, S., Mohr, J. et al. (2000). Adherence to protease inhibitor therapy and outcomes in patients with HIV infection. Annals of Internal Medicine 133, 21–30.[Free Full Text]

5 . Stone, V. E., Clark, J., Lovell, J. et al. (1998). HIV/AIDS patients’ perspectives on adhering to regimens containing protease inhibitors. Journal of General Internal Medicine 13, 586–93.[CrossRef][ISI][Medline]

6 . Williams, A. & Friedland, G. (1997). Adherence, compliance and HAART. AIDS Clinical Care 9, 51–8.[Medline]

7 . Yeh, K. C., Deutsch, P. J., Haddix, H. et al. (1998). Single-dose pharmacokinetics of indinavir and the effect of food. Antimicrobial Agents and Chemotherapy 42, 332–8.[Abstract/Free Full Text]

8 . D’Aquila, R. T., Shapiro, J. M., Brun-Vezinet, F. et al. (2000). Drug resistance mutations in HIV-1. Topics in HIV Medicine 10, 11–5.

9 . Montaner, J. S. G., Harrigan, P. R., Jahnke, N. et al. (2001). Multiple drug rescue therapy for HIV-infected individuals with prior virologic failure to multiple regimens. AIDS 15, 61–9.[CrossRef][ISI][Medline]

10 . Staszewski, S., Gute, P., Carlebach, A. et al. (1998). Virological and immunological response to MEGA-HAART salvage therapy after failure of multiple antiretroviral regimens. AIDS 12, S40.

11 . Katlama, C., Divivier, C., Monroux, M. et al. (1999). GIGHAART: a rescue therapy for HIV patients with multiple HAART failures. Antiviral Therapy 4, Suppl. 1, 77.

12 . Katlama, C., Dominguez, S., Duvivier, C. et al. (2003). Long-term benefit of treatment interruption in salvage therapy (GIGHAART ANRS 097). In Program and Abstracts of the Tenth Conference on Retroviruses and Opportunistic Infections, Boston, MA, 2003. Abstract 68, p. 81. Foundation for Retrovirology and Human Health, Alexandria, VA, USA.

13 . De Truchis, P., Force, G., Zucman, D. et al. (1998). Effects of a "salvage" combination therapy with ritonavir + saquinavir in HIV-infected patients previously treated with protease inhibitors (PI). In Program and Abstracts of the Fifth Conference on Retroviruses and Opportunistic Infections, Chicago, IL, 1998. Abstract 425, p. 159. Foundation for Retrovirology and Human Health, Alexandria, VA, USA.

14 . Tebas, P., Kane, E., Klenbert, M. et al. (1998). Virologic responses to ritonavir/saquinavir containing regimen in patients who have previously failed nelfinavir. In Program and Abstracts of the Fifth Conference on Retroviruses and Opportunistic Infections, Chicago, IL, 1998. Abstract 510, p. 175. Foundation for Retrovirology and Human Health, Alexandria, VA, USA.

15 . Hammer, S. M., Vaida, F., Bennett, K. K. et al. (2002). Dual vs single protease inhibitor therapy following antiretroviral treatment failure. A randomized trial. Journal of the American Medical Association 288, 169–80.[Abstract/Free Full Text]

16 . Moyle, G. J. & Back, D. (2001). Principles and practice of HIV-protease inhibitor pharmacoenhancement. HIV Medicine 2, 105–13.[CrossRef][Medline]

17 . Deminie, C. A., Bechtold, C. M., Stock, D. et al. (1996). Evaluation of reverse transcriptase and protease inhibitors in two-drug combinations against human immunodeficiency virus replication. Antimicrobial Agents and Chemotherapy 40, 1346–51.[Abstract]

18 . Patick, A. K., Boritzki, T. J. & Bloom, L. A. (1997). Activities of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor nelfinavir mesylate in combination with reverse transcriptase and protease inhibitors against acute HIV-1 infection in vitro. Antimicrobial Agents and Chemotherapy 41, 2159–64.[Abstract]

19 . 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, 2367–73.[Abstract]

20 . Merrill, D. P., Manion, D. J., Chou, T.-C. et al. (1997). Antagonism between human immunodeficiency virus type 1 protease inhibitors indinavir and saquinavir in vitro. Journal of Infectious Diseases 176, 265–8.[ISI][Medline]

21 . Nascimbeni, M., Lamotte, C., Peytavin, G. et al. (1999). Kinetics of antiviral activity and intracellular pharmacokinetics of human immunodeficiency virus type 1 protease inhibitors in tissue culture. Antimicrobial Agents and Chemotherapy 43, 2629–34.[Abstract/Free Full Text]

22 . Molla, A., Mo, H., Vasavanonda, S. et al. (2002). In vitro antiviral interaction of lopinavir with other protease inhibitors. Antimicrobial Agents and Chemotherapy 46, 2249–53.[Abstract/Free Full Text]

23 . Flexner, C. (2000). Dual protease inhibitor therapy in HIV-infected patients: pharmacologic rationale and clinical benefits. Annual Review of Pharmacology and Toxicology 40, 651–76.[CrossRef]

24 . Drusano, G. L., Bilello, J. A., Stein, D. S. et al. (1998). Factors influencing the emergence of resistance to indinavir: role of virologic, immunologic and pharmacologic variables. Journal of Infectious Diseases 178, 360–7.[ISI][Medline]

25 . Rusconi, S., La Seta Catamancio, S., Citterio, P. et al. (2000). Susceptibility to PNU-140690 (tipranavir) of human immunodeficiency virus type 1 isolates derived from patients with multidrug resistance to other protease inhibitors. Antimicrobial Agents and Chemotherapy 44, 1328–32.[Abstract/Free Full Text]

26 . Johnson, V. A., Barlow, M. A., Merrill, D. P. et al. (1990). Three-drug synergistic inhibition of HIV-1 replication in vitro by zidovudine, recombinant soluble CD4 and recombinant interferon-alpha A. Journal of Infectious Diseases 161, 1059–67.[ISI][Medline]

27 . Dulbecco, R. (1998). Endpoint methods – measurements of the infectious titer of a viral sample. In Virology (Dulbecco, R. & Ginsberg, H. S., Eds), pp. 22–5. J. P. Lippincott, Philadelphia, PA, USA.

28 . Chou, T.-C. & Talalay, P. (1984). Quantitative analysis of dose-effect relationships: the combined effect of multiple drugs or enzyme inhibitors. Advances in Enzyme Regulation 22, 27–55.[CrossRef][ISI][Medline]

29 . Chou, T.-C. (1991). The median-effect principle and the combination index for quantitation of synergism and antagonism. In Synergism and Antagonism in Chemotherapy (Chou, T.-C. & Rideout, D. C., Eds), pp. 61–102. Academy Press, New York, NY, USA.

30 . Rusconi, S., De Pasquale, M., Milazzo, L. et al. (1997). In vitro effects of continuous pressure with zidovudine (ZDV) and lamivudine on a ZDV-resistant HIV-1 isolate. AIDS 11, 1406–10.[ISI][Medline]

31 . Rusconi, S., De Pasquale, M., Milazzo, L. et al. (1998). Loss of lamivudine resistance in a zidovudine and lamivudine dual-resistant human immunodefiency virus type 1 (HIV-1) isolate after discontinuation of in vitro lamivudine drug pressure. Antiviral Therapy 3, 203–7.[ISI][Medline]

32 . Jacobs, R. F. (1996). Multi-drug resistant tuberculosis. Clinical Infectious Diseases 19, 1–8.

33 . Hecht, F. M., Grant, R. M., Petropoulos, C. J. et al. (1998). Sexual transmission of an HIV-1 variant resistant to multiple reverse-transcriptase and protease inhibitors. New England Journal of Medicine 339, 307–11.[Free Full Text]

34 . Andreoletti, L., Weiss, L., Si-Mohamed, A. et al. (2002). Multidrug-resistant HIV-1 RNA and proviral DNA variants harboring new dipeptide insertions in the reverse transcriptase pol gene. Journal of Acquired Immune Deficiency Syndromes 29, 102–4.[ISI][Medline]

35 . Brenner, B. G., Routy, J. P, Petrella, M. et al. (2002). Persistence and fitness of multidrug-resistant human immunodeficiency virus type 1 acquired in primary infection. Journal of Virology 76, 1753–61.[Abstract/Free Full Text]

36 . Yeni, P. G., Hammer, S. M., Carpenter, C. C. J. et al. (2002). Antiretroviral treatment for adult HIV infection in 2002. Updated recommendations of the International AIDS Society—USA Panel. Journal of the American Medical Association 288, 222–35.[Abstract/Free Full Text]

37 . Petropoulos, C. J., Parkin, N. T., Limoli, K. L. et al. (2000). A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1. Antimicrobial Agents and Chemotherapy 44, 920–8.[Abstract/Free Full Text]

38 . McCallister, S., Kohlbrenner, V., Squires, K. et al. (2003). Characterization of the impact of genotype, phenotype, and inhibitory quotient on antiviral activity of tipranavir in highly treatment-experienced patients. Antiviral Therapy 8, S15.

39 . Doyon, L., Tremblay, S., Wardrop, E. et al. (2003). Characterization of HIV-1 showing decreased susceptibility to tipranavir and their inhibition by tipranavir-containing drug mixtures. Antiviral Therapy 8, S17.