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Antimicrobial Agents and Chemotherapy, May 2000, p. 1328-1332, Vol. 44, No. 5
Istituto di Malattie Infettive e Tropicali,
Universitá di Milano, Ospedale Luigi Sacco, Milan, Italy
Received 1 June 1999/Returned for modification 14 December
1999/Accepted 18 January 2000
In our study we examined the anti-human immunodeficiency virus type
1 (anti-HIV-1) activity of a novel HIV-1 protease inhibitor, PNU-140690
(tipranavir), against patient-derived isolates resistant to multiple
other protease inhibitors (PIs). The aim of our experiments was to
investigate the genotypes and the in vitro phenotypes of drug
resistance of PNU-140690. We carried out drug susceptibility tests with
peripheral blood mononuclear cells and a fixed amount of infectious
virus (1,000 50% tissue culture infective doses) to determine the 50%
inhibitory concentration (IC50) and IC90, PCR
assays for the detection of drug resistance mutations in RNA in plasma,
and direct sequencing of PCR products. Phenotypic resistance to PIs was
invariably related to genotypic mutations. The substitutions among the
amino acid residues of the protease included L10I, K20R, L24I, M36I,
N37D, G48V, I54V, L63P, I64V, A71V, V77I, V82A, I84V, and L90M.
Isolates from all of the patients had developed a maximal degree of
resistance to indinavir, ritonavir, and nelfinavir (IC50s, >0.1 µM). We also compared these mutations with the amino acid changes previously described in association with in vivo tipranavir administration. The mutations included the following: I15V, E35D, N37D,
R41K, D60E, and A71T. Infections with IIIB, 14aPre, and N70 were
inhibited by an average drug IC90 of 0.18 ± 0.02 µM
in multiple experiments. The average mean ± standard error of
mean IC90 for the entire group of multidrug-resistant
isolates derived from the mean values for two culture wells with p24
antigen supernatant appeared to be 0.619 ± 0.055 µM (range,
0.31 to 0.86 µM). Tipranavir retained a sustained antiviral activity
against PI-MDR clinical isolates and might be useful in combination
regimens with other antiretroviral agents for patients who have already
failed other PI-containing therapies.
Protease inhibitors (PIs) have
significantly changed the prognosis of human immunodeficiency virus
(HIV) type 1 (HIV-1) infection. Their use is, however, associated with
the appearance of a pattern of many mutations in the protease gene
which favor the development of resistance to multiple other compounds
within the same class of drugs (for reviews, see references
1 and 6). Condra and colleagues
(4) gave the first virological demonstration of cross-resistance to PIs among patients subjected to indinavir (IDV)
monotherapy. Together with the progressive accumulation of mutations
selected by IDV monotherapy, these patients harbored viruses that
became resistant to other PIs, such as saquinavir (SQV) and amprenavir
(VX-478 or 141W94) (4). The follow-up to that study
evidenced that the extent of IDV resistance and the cross-resistance to
other PIs depended upon the single amino acid substitutions, their
numbers, and the combination in which they appeared (5).
Ritonavir (RTV) had also been described as potentially inducing a large
extent of unique resistance or cross-resistance within the PI class
(17).
The complex picture of the emergence of cross-resistance to PIs was
reported in several studies performed both in vitro (12, 20)
and in vivo (13, 16; V. Miller, K. Hertogs, M.-P. de Bethune, R. Pauwels, T. Ivens, H. Azijn, C. Schlecht, B. Nolde, M. Sturmer, B. Morgenstern, M. Kortenbusch, and S. Staszewski, Abstr. Int. Workshop HIV Drug Resistance, Treatment
Strategies Eradication, abstr. 82, p. 75-76, 1997). Additional data on
PIs-related resistance following sequential treatment with several
compounds within this class of drugs are becoming available. Winters
and colleagues (21) have recently reported that an
initial treatment with SQV may cause a full development of
cross-resistance to IDV and nelfinavir (NFV) in patients who receive
such regimens as part of an active anti-HIV-1 combination therapy.
Dulioust and colleagues (9) observed that previous therapy
with SQV followed by therapy with IDV allowed a viral evolution that
maintained the initial selection of drug resistance and adapted the
viral population to the IDV pressure. These results suggest that HIV-1 quasispecies would likely adapt to the presently available drugs which
target the same enzyme.
Taking into account the rapid occurrence of PI cross-resistance,
clinicians who are treating patients with HIV-1 infection will need new
active PIs in the near future.
In the present study we examined the in vitro anti-HIV-1 activity of a
novel HIV-1 PI, PNU-140690 (tipranavir) (15), which retained
potent activity against RTV-resistant clinical isolates (2).
We used isolates from patients with resistance to multiple other PIs:
SQV and/or NFV, in addition to IDV and RTV. We have analyzed the
genotypic pattern and the phenotypic drug resistance of the new
compound in vitro.
(This study was presented in part at the 3rd International Workshop on
HIV Drug Resistance and Treatment Strategies, 23 to 26 June 1999, San
Diego, Calif., abstr. 17, p. 11.)
Sampling and analytical protocol.
We studied a number of
PI-experienced subjects who had failed their current treatment (defined
as a 1 log10 increase in viral load with or without
clinical worsening or a decrease in the CD4+ cell count of
Cells.
Peripheral blood mononuclear cells (PBMCs) from
HIV-1-seronegative donors were obtained by Ficoll-Hypaque
density-gradient centrifugation of heparinized venous blood. The PBMCs
were treated with phytohemagglutinin (PHA-P; 2 µg/ml; Difco
Laboratories, Detroit, Mich.), propagated in R-20 medium (RPMI 1640 medium supplemented with 20% heat-inactivated fetal calf serum [Sigma
Chemical, St. Louis, Mo.], 50 U of penicillin per ml, 50 µg of
streptomycin per ml, 2 mM L-glutamine, and 10 mM HEPES
buffer) supplemented with 10% interleukin-2 (Human T-Stim;
Collaborative Research Inc., Bedford, Mass.), and incubated at 37°C
in 5% CO2. MT-2 cells, a lymphoblastoid cell line, were
provided by R. C. Gallo (Institute of Human Virology, Baltimore,
Md.) and were maintained in R-10 medium (which is identical to R-20
medium except that it contains only 10% heat-inactivated fetal calf serum).
Viruses.
We evaluated several drug-resistant isolates
derived from patients who were undergoing anti-HIV-1 combination
therapy. Different clinical isolates and laboratory-adapted strains of
HIV-1 were used as controls. 14aPre was derived from an
HIV-1-seropositive individual before any therapy (Massachusetts General
Hospital, Boston, Mass.); N70 was an isolate derived from a clinically
asymptomatic patient and was a gift from D. D. Ho (Aaron Diamond
AIDS Research Center, New York, N.Y.). The IIIB prototype strain of
lymphotropic HIV-1 was obtained from R. C. Gallo (Institute of
Human Virology). Viral titers ranged from 2 × 104 to
4.5 × 104 50% tissue culture infective doses
(TCID50s)/106 PBMCs for the new clinical
isolates and from 1.5 × 105 to 3 × 105 TCID50s/106 million PBMCs for
the control isolates.
Compounds.
The sulfonamide-containing nonpeptidic PI
PNU-140690 was provided by Pharmacia-Upjohn (Kalamazoo, Mich.). IDV was
obtained from Merck Research Laboratories (West Point, Pa.). RTV was
obtained from Abbott Laboratories (Abbott Park, Ill.). NFV was obtained from Agouron Pharmaceuticals, Inc. (La Jolla, Calif.).
Drug susceptibility test.
The concentrations of PNU-140690
that inhibited the viruses were evaluated in PBMCs. In all the
experiments, uninfected cell controls were maintained for determination
of drug toxicity. Cell proliferation and viability were assessed by the
trypan blue dye exclusion method. Virus replication were measured in
cell-free culture supernatants by an HIV-1 p24 antigen enzyme-linked
immunosorbent assay (DuPont-NEN Research Products, Boston, Mass.). We
maintained virus without cells or drug for the entire duration of the
experiments in order to take into account the viral carryover.
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Susceptibility to PNU-140690 (Tipranavir) of Human
Immunodeficiency Virus Type 1 Isolates Derived from Patients with
Multidrug Resistance to Other Protease Inhibitors
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
20%) and for whom genotypic characterization of the HIV-1 protease
indicated multiple amino acid substitutions. Then, we conducted the
phenotypic test before the beginning of the PI treatment and at the
time of the sequencing, and at this point susceptibility to PNU-140690
was determined.
PCR assay for drug resistance mutations in RNA. Plasma RNA was extracted with a nucleic acid extraction kit (QUIAamp Viral RNA kit; Quiagen, Inc., Chatsworth, Calif.) according to the manufacturer's recommendations. The pol gene was reverse transcribed with the antisense primer P8 (7). RNA was denatured together with the P8 antisense primer at 70°C for 5 min. The reaction mixture (30 µl) contained 1× reaction buffer, 0.5 mM each deoxynucleoside triphosphate, (dNTP), 1.6 µM P8 primer, 30 U of RNAsin, and Moloney murine leukemia virus reverse transcriptase (200 U). The reverse transcription reaction started with 42°C for 60 min, followed by 95°C for 5 min. The HIV-1 protease-coding sequences were amplified by a PCR assay. The reaction mixture (100 µl) contained 50 mM Tris-HCl (pH 8.3), 25 mM KCl, 1 mM MgCl2, 0.2 mM each dNTP, 2.5 U of Taq DNA polymerase (Perkin-Elmer, Norwalk, Conn.), and 25 pmol of primers P8 and P7 (7). The reaction was initiated with 1 cycle at 95°C for 9 min and then with 35 cycles at 94°C for 30 s, 68°C for 1 min, and 72°C for 2 min, followed by incubation at 72°C for 6 min. The second amplification was performed with 10 µl of the first amplification product. The reaction mixture (100 µl) contained 50 mM Tris-HCl (pH 8.3), 25 mM KCl, 1.5 mM MgCl2, 0.2 mM each dNTP, 2.5 U of Taq DNA polymerase (Perkin-Elmer), and 25 pmol of primers Pro-F (5'-TGTAAAACGACGGCCAGTACAACAACTCCCTCTCA-3') and Pro-R (5'-CAGGAAACAGCTATGACCAATGGCCATTGTTTAAC-3') (the M13 sequence is underlined). The conditions that we used were 95°C for 9 min for 1 cycle, then 94°C for 50 s, 45°C for 50 s, and 72°C for 2 min repeated for 2 cycles, and then 35 cycles at 94°C for 30 s, 55°C for 50 s, and 72°C for 2 min, followed by incubation at 72°C for 10 min. The final PCR products were analyzed on a 2% agarose gel.
Sequencing procedures. Unincorporated primers and nucleotides were removed with the QUIAQUICK Gel Extraction Kit (Quiagen, Inc.), and the PCR products were sequenced directly by using dye-labeled M13 primers and ABI sequencing kit reagents (Applied Biosystems, Inc., Foster City, Calif.) in the presence of 10% glycerol. An automatic DNA sequencer (373A; Applied Biosystems, Inc.) was used. The sequence pattern was confirmed by sequencing both the positive and negative strands and was derived from two independent sequencing reactions. They were aligned with the HIV-1 IIIB consensus sequence and were analyzed by using the Sequence Navigator software program, version 1.0.1 (Applied Biosystems, Inc.).
Nucleotide sequence accession numbers. All sequences have been submitted to GenBank (Bethesda, Md.) and have been given accession nos. AF154954 to AF154963.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Baseline characteristics.
Eight isolates were obtained from
patients who received IDV, RTV, or SQV (mean ± standard error of
the mean [SEM] treatment duration, 18.25 ± 2.61 months) and
were demonstrated to be both IDV and RTV resistant. These subjects had
failed antiviral treatment, defined as a 1 log10 increase
in viral load with or without clinical worsening or a decreasing
CD4+ cell count of 20%. Two isolates were obtained from
a single patient with a previous exposure to NRTIs and NNRTIs at the
beginning and the end of PI treatment (11 months of therapy). A summary of the patients' features, including the CD4 counts (range, 28 to
1,210/µl) and HIV-1 loads (range, 660 to 490,000 copies/ml), is shown
in Table 1.
|
Genotypic and phenotypic profiles related to previous PI
treatment.
A total of 10 primary HIV-1 isolates obtained from nine
patients treated with SQV, RTV, or IDV were genotypically sequenced and
evaluated for their sensitivities to IDV, RTV, and NFV (Table 2). The results showed that all the
isolates had a reduced sensitivity with a maximal degree of resistance
(IC50, >0.1 µM) to IDV, RTV, and NFV when they were
tested. Four isolates come from patients who had received only IDV, two
were from patients who had received combination therapy with SQV and
RTV, one was from a patient who had received combination therapy with
SQV and NFV, one was from a patient who had received NFV alone, and one
was from a patient who had received RTV alone. Patients 003, 005, 009, and 010 received sequential therapy with different PIs, without
achieving control of their viremia or the maintainance of a stable
CD4+ cell count. All the resistant strains had at least
three mutations, which were localized either at the active site of the
enzyme or outside the known binding cleft.
|
Prevalence of mutations described in association with PNU-140690 treatment. Following the recent report of genotypic resistance to PNU-140690 in a phase II clinical trial by Wang and colleagues (Y. Wang, W. W. Freimuth, C. L. Daenzer, M. T. Borin, C. M. Tutton, A. A. Piergies, R. M. Wurtz, H. I. Li, J. W. Davis, D. J. Crampton, and the PNU-140690 Team, Abstr. 2nd Int. Workshop on HIV Drug Resistance and Treatment Strategies, abstr. 5, p. 5, 1998), we analyzed in our clinical isolates the prevalence of the amino acid changes whose occurrence was previously described during in vivo PNU-140690 administration. The mutations included the following: I15V, E35D, N37D, R41K, D60E, and A71T. Virus from one single patient showed multiple substitutions at positions E35, N37, and D60 on both occasions (pre- and posttherapy [007 and 008, respectively]), and viruses from the other subjects exhibited variable substitution patterns which included various mixtures of mutations at selected codons. Viruses from two patients (patients EB and SU) did not show any PNU-140690-related change. The highest prevalence was represented by the E35D amino acid substitution (60%), followed by the D60E and N37D (30%) and the R41K (20%) changes.
PNU-140690 phenotypic susceptibility.
PNU-140690 suppressed
the replication of either laboratory and clinical viral strains.
Infections with laboratory-adapted viral strains such as IIIB, 14aPre,
and N70 were inhibited by the drug, with an average IC90 of
0.18 ± 0.02 µM in repeat experiments. The IC50s and
the IC90s for the multidrug-resistant clinical isolates are
shown in Table 3. The PNU-140690
IC50s ranged from 0.046 to 0.383 µM, and a dose-response
to the drug was shown for all the clinical isolates. The average
mean ± SEM IC90 for the entire cohort of
multidrug-resistant isolates from the patients was derived from the
mean values for two culture wells with p24 antigen supernatant and
appeared to be 0.619 ± 0.055 µM (range, 0.31 to 0.86 µM). We
did not observe any characteristic pattern in the phenotypic susceptibilities compared to the genotypic resistance profile.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The consecutive use of different PIs, which can also be due to the different times of approval of PIs for clinical use, can be the most frequent reason for the development of resistance and the consequent therapeutic failure and has frequently occurred in HIV-1-infected patients (10, 18, 19, 21). The in vivo pharmacological pressure of a PI-containing regimen may theoretically be able to influence the in vitro susceptibility to other PIs; in fact our multidrug-resistant isolates presented with a common background of marked resistance to PIs. This phenomenon was seen in our isolates, in which a cross-resistant genotypic pattern of drug cross-resistance was present at the baseline for IDV, RTV, and NFV but not for PNU-140690. Of note, it has been reported that treatment with a second PI selected for the emergence of HIV-1 strains with reduced sensitivity to more than one PI (9). The results indicated that initial therapy with one PI might induce genotypic pressure, with the emergence of further mutations in the protease-coding region following the exposure to other compounds within the same class.
The reports by Winters et al. (21) and Dulioust et al. (9) underlined the fact that the emergence of cross-resistance to PIs in vivo is a dynamic process that cannot be adequately predicted by standard phenotypic and genotypic assays (J. H. Condra, D. J. Holder, D. J. Graham, M. Shivaprakash, D. T. Laird, W. A. Schleif, J. A. Chodakewitz, and E. A. Emini, Abstr. Int. Workshop HIV Drug Resistance, Treatment Strategies and Eradication, abstr. 47, p. 48-49, 1997). Nevertheless, it is widely recommended that the antivirologic efficacy of a combination regimen be tested before its eventual replacement and, more appropriately, prior to the introduction of a new therapeutic agent, e.g., PNU-140690.
We performed sequence analysis of the multidrug-resistant isolates recovered from plasma samples during PI therapy and before the in vitro PNU-140690 susceptibility tests and found various patterns of resistance. These amino acid substitutions may be selected by PNU-140690 administration (week 12 data from a phase II trial; Y. Wang, personal communication). The substitutions and their prevalences evidenced in our samples (I15V, 1 of 10 isolates; E35D, 6 of 10 isolates; N37D, 3 of 10 isolates; R41K, 2 of 10 isolates; D60E, 3 of 10 isolates; and A71T, 1 of 10 isolates) did not correlate with the phenotypic susceptibility to PNU-140690. Among the patients who participated in the phase II protocol mentioned above, only D60E A71T, and V77G were not seen at the baseline but were seen at week 12. The mutations reported by Wang and us have not been studied by in situ mutagenesis, and their role in determining PNU-140690 resistance is not yet clear. PNU-140690 retained activity against our clinical isolates that harbored those mutations. As for other PIs (4, 12, 14), resistance to PNU-140690 might require multiple amino acid subsitutions and specific codon changes that could reduce its proteolytic activity. These genotypic characteristics have not yet emerged from the currently available in vivo resistance data.
Despite the variety of PNU-140690 IC90s, PNU-140690 concentrations achieved a constant reduction of viral replication under the culture condition used for the PI-MDR isolates examined in this study. In contrast, the antiviral effects of IDV, RTV, and NFV, when tested, had been suppressed, allowing viral replication, despite the presence of high drug concentrations capable of limiting infection with PI-sensitive isolates and reference HIV-1 strains.
In conclusion, from the results of our study, the possibility has emerged that PI-MDR isolates can be treated with PNU-140690, most likely in combination with other RT inhibitors and possibly with other PIs, especially after taking into consideration the therapeutic approach of combining PNU-140690 with RTV as treatment against RTV-resistant isolates (2), as recently reported. The efficacy of a potent PI like PNU-140690 as part of combination regimens against HIV-1 warrants further in vitro studies and in vivo confirmation in clinical trials.
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ACKNOWLEDGMENTS |
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We thank the HIV-1-infected patients who volunteered to participate in this study, Bianca Maria Ghisi for manuscript preparation, and Elizabeth L. Kaplan for editorial assistance and continuous support. We also acknowledge the support and the helpful discussion with Wanda De Cian and Massimiliano Mucci of Pharmacia & Upjohn (Milan, Italy).
This work was supported by an Istituto Superiore di Sanitá (Rome, Italy) IV Progetto Terapia Antiretrovirale/AIDS 1997 grant (grant 980.1.16) and by a research grant from Pharmacia & Upjohn.
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FOOTNOTES |
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* Corresponding author. Mailing address: Istituto di Malattie Infettive e Tropicali, Universitá di Milano, Ospedale Luigi Sacco, Via GB Grassi 74, 20157 Milano, Italy. Phone: 39.02.35799676. Fax: 39.02.3560805. E-mail: rusconi{at}mailserver.unimi.it.
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Abstract
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Materials and Methods
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Discussion
References
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Antimicrobial Agents and Chemotherapy, May 2000, p. 1328-1332, Vol. 44, No. 5
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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