646
Two-Drug Combinations of Zidovudine, Didanosine, and Recombinant Interferon-a A Inhibit Replication of Zidovudine-Resistant Human Immunodeficiency Virus Type 1 Synergistically In Vitro Victoria A. Johnson, Debra P. Merrill, Joseph A. Videler, Ting-Chao Chou, Roy E. Byington, Joseph J. Eron, Richard T. D'Aquila, and Martin S. Hirsch
Infectious Disease Unit, Massachusetts General Hospital and Harvard Medical School, Boston; Laboratory of Biochemical Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, New York
The search for effective therapies for AIDS has led to the discovery of many agents that demonstrate activity against human immunodeficiency virus type 1 (HIV-1) replication in vitro [1-8]. Several of these agents show favorable results in patients as well, including zidovudine (AZT, 3'-azido-3'deoxythymidine), didanosine (ddI, 2',3'-dideoxyinosine), and recombinant interferon-a A (rIFN-aA) [9-21]. AZT and ddI are nucleoside analogues that inhibit HIV -1 reverse transcriptase (RT) [4-7]. Although the precise mechanism of inhibition of HI V-I replication by interferon-a is unclear, it may affect virus assembly or release as shown by studies of chronically infected cells [22-25]. Despite the progress made in the development of single-
Received 12 March 1991; revised 31 May 1991. Presented in part: Workshop on Viral Resistance, Washington. DC, October 1990, and the first joint University of Miami and University of California San Diego symposium. HIV Disease: Pathogenesis and Therapy. Grenelefe, Florida. March 1991 (abstract. J Acquir Immune Defic Syndr 1991;4:354). Written informed consent was obtained from all subjects according to protocols approved by the Subcommittee on Human Studies. Division of Research Affairs, Massachusetts General Hospital, Harvard Medical School. Financial support: National Institutes of Health (CA-12464, CA-35020, AI-26056, AI-29193, and National Research Service Award F32-7933 to V.A.J.) and Elsa U. Pardee Foundation. M.S.H. is a member of the BristolMyers AIDS Advisory Board. Reprints or correspondence: Dr. Victoria A. Johnson. Infectious Disease Unit. Gray 5. Massachusetts General Hospital, Boston. MA 02114. The Journal of Infectious Diseases 1991;164:646-55 © 199\ by The University of Chicago. All rights reserved. 0022-1899/91/6404-0003$01.00
agent therapy for HIV -1 infection, monotherapy with either AZT, ddI, or interferon-a has been associated with drug toxicity or failure [17, 26-30]. In addition, AZT-resistant variants of HIV-l have been isolated from patients receiving AZT as extended single-agent therapy [31-37], although the clinical implications of these findings remain uncertain. It is likely that combined therapy will be required for effective long-term treatment of HIV -1 infection, as in the approach to certain other infectious diseases (e.g., tuberculosis) and cancers [38-42]. Anti-HIV-l combination strategies that demonstrate favorable drug interactions (e.g., synergy) may allow the use of individual agents below their toxic concentrations, provide more complete viral suppression, and limit the emergence of drug-resistant HIV-l mutants. We previously demonstrated favorable interactions between agents that interrupt the HIV-1 replication cycle by different or similar mechanisms of action. Synergistic interactions were seen when combining an RT inhibitor (e.g., either AZT or ddI) with an agent that interrupts virus assembly and release (e.g., interferon-a) [43-45]. Additive to synergistic interactions have also been noted between RT inhibitors when combined (e.g., AZT and ddI) [45, 46]. Several clinical trials of combination therapy are in progress or planned in HIV-infected patients to evaluate the two-drug regimens of AZT and interferon-a, ddI and interferon-a, or AZT and ddI [47-50]. To further define the potential role of combination therapy for treatment of AZT-resistant virus variants, we studied AZT-resistant isolates derived from two HIV -I-infected individuals after extended AZT monotherapy. Our goal was to
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Optimal management of human immunodeficiency virus type 1 (HIV-l) infections may require combinations of anti-HIV-I agents, Zidovudine (AZT, 3'-azido-3'-deoxythymidine), didanosine (ddI, 2',3'-dideoxyinosine), and recombinant interferon-a A (rIFN-aA) were evaluated in two-drug regimens against replication of AZT-resistant HIV-1 in vitro. AZT-sensitive and AZTresistant isolate pairs derived from two individuals before and after extended AZT monotherapy were studied. Drug interactions using peripheral blood mononuclear cells infected with HIV-l were evaluated mathematically. Synergistic interactions were seen among AZT, ddI, and rIFNaA in two-drug regimens against AZT-resistant HIV-1 in vitro, even when AZT was included in the treatment regimen. Mixtures of wild-type and mutant reverse transcriptase genes were found in one of the late-AZT therapy isolates, suggesting that the mechanism of synergy of AZT-containing regimens may involve inhibition of AZT -sensitive viruses in the viral pool. These studies suggest that AZT may be useful in drug combination regimens, even when AZT-resistant viruses are isolated in vitro.
110 1991; 164 (October)
AZT-Resistant HIV-I: Combination Therapy
evaluate the ability of two-drug combined regimens to block replication of AZT-resistant HIV-I in vitro using regimens that did or did not include AZT. The regimens studied were AZT alone versus ddI alone versus AZT combined with ddI (i.e., the addition of another anti-Hl Vr l agent that inhibits R T); AZT alone versus rIFN-aA alone versus AZT combined with rIFN-aA (i.e., the addition of an anti-Hl V-I agent acting by a different mechanism); and ddI alone versus rIFN-aA alone versus ddI combined with rIFN-aA (i.e., use of a regimen that did not include AZT).
Methods
duced cytopathic effects (CPE) occurred and stored at -85°C. Titrations of these viral stocks were done in PBMC to determine the TCID so per milliliter. An end-point titration method in 96well microtiter plates was done in sextuplicate as described previously, using 2 X 105 PBMC per well [43]. Peak virus-induced CPE was identified as the end point on day 7 in culture. The TCID so value of each viral stock was calculated by the method of Reed and Muench (cited in [51]). We also evaluated a pair of HI V-I isolates provided by D. Richman (University of California, San Diego) derived from a patient with AIDS (subject AOI2) [31]. These HIV-l isolates, obtained by an initial cocultivation of HIV-I-infected PBMC with MT-2 cells (a continuous T cell line), were designated AOl2B (obtained after 2 months of AZT therapy) and AOl2D (obtained after 26 months of AZT therapy) [31]. Viral stocks were made and titrated in PBMC for subsequent drug susceptibility testing. Compounds. AZT was obtained in powder form from P. A. Furman (Wellcome Research Laboratories, Research Triangle Park, NC). It was dissolved in sterile PBS and stored at a concentration of I mM in aliquots at -20°C until used. ddI was obtained in powder form from C. McLaren (Bristol-Myers Squibb, Wallingford, CT). It was dissolved in sterile PBS and stored at a concentration of 1 mM in aliquots at -20°C until used. The rIFN-aA was obtained from I. Sim (IFN-a-2a, Roferon-A; Hoffmann-La Roche, Nutley, NJ). Aliquots ofrlFN-aA were stored at a concentration of 105 Ll/rnl in sterile PBS at -85°C until used. The actual concentration of interferon was determined as described previously [8]. Viral replication assays. Cell-free culture supernatant fluids were assayed by an HIV-l p24 antigen ELISA (NEN Research Products, Boston) as described previously [52]. RT genotypic analysis. For sequencing analysis of the RT genes of the AZT-resistant isolates, PHA-P-stimulated PBMC were infected in vitro (in the absence of any drug) with the identical viral stock used in combination therapy experiments 2 (using isolate 14a-6/89) and 8 (using isolate AO12D). The earlyAZT therapy isolate 14a-4/87 R T gene was also sequenced. After maximal CPE occurred, cell pellets were lysed in detergent (at 3 X 106 cells/ml) and digested with proteinase K. Standard methods of polymerase chain reaction (PCR) were used, including Taq polymerase and an automated thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) [53]. PCR was done using 1.2 X 105 cell equivalents oflysate per amplification. Routine stringent precautions to prevent PCR product or plasmid clone carryover included having separate personnel do cell lysis and PCR setup at a site remote from that of both viral culture and PCR product and plasmid clone handling. PBMC from HIV -I-seronegative individuals were pelleted and lysed in parallel with experimental samples to control for carryover. PCR primers corresponded to conserved regions [54] of the HIV-l RT gene in the sense strand (bases 2058-2082) and the antisense strand (bases 2650-2628) of HIV-l BHIO-2 (GenBank accession number M 15654). DNA sequencing was done directly from gel-purified amplification products to identify the dominant base at each position [55]. PCR products were separated on 2% agarose gels, and the 590-bp RT gene fragments were excised and electroeluted. Se-
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Cells. Peripheral blood mononuclear cells (PBMC) from a single HIV-l-seronegative donor were obtained by ficollhypaque density-gradient centrifugation of heparinized venous blood. A different donor was used for each experiment. Cells were treated with phytohemagglutinin (PHA-P, 10 Jlg/ml; Difco Laboratories, Detroit), propagated in R-20 medium (RPMi 1640 medium supplemented with 20% heat-inactivated fetal calf serum [Sigma Chemical, St. Louis], 250 units [U]!ml penicillin, 250 Jlg/ml streptomycin, 2 mM L-glutamine, and 10 mM HEPES buffer [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]) supplemented with 10% interleukin-2 (IL-2; Pharmacia Diagnostics, Silver Spring, MD), and incubated at 37°C in 5% CO 2 , Viral stocks. Paired HIV-l isolates were derived from an HIV-L-seropositive individual (subject 14a) before and after 26 months of uninterrupted AZT monotherapy. These isolates were designated 14a-4/87 (early-AZT therapy) and 14a-6/89 (late-AZT therapy). This individual was diagnosed with HIV-related Kaposi's sarcoma in February 1987 and had the following clinical parameters when the April 1987 isolate was obtained: weight, 61. 7 kg; Karnofsky score, 100; and CD4+ cells, 174/ mrrr', The subject received AZT, 200 mg every 4 h, until May 1987, when the dose was reduced to 100 mg every 4 h. Pneumocystis carinii pneumonia was diagnosed in May 1987. When the late-AZT therapy isolate (l4a-6/89) was obtained, the subject had progressive cutaneous Kaposi's sarcoma and the following clinical parameters: weight, 57.6 kg; Karnofsky score, 90; and CD4+ cells, 8/mm 3 . PBMC from this HIV-l-seropositive individual before and after prolonged therapy with AZT were separated from heparinized venous blood using ficoll-hypaque density-gradient centrifugation and were then cocultivated with 4-day PHA-P-stimulated PBMC from normal donors. Cultures were maintained in R-Ill medium (R-20 medium supplemented with 10% IL-2 and polybrene [2 Jlg/ml; Sigma]). Twice weekly, cell-free culture supernatant fluids were stored as aliquots at -85°C. To prepare viral stocks for drug susceptibility testing, these aliquots were used to infect normal donor 4-day PHA-P-stimulated PBMC (lOX 106 cells) in 10 ml ofR-III medium in 25-cm 2 vented tissue culture flasks (Costar, Cambridge, MA) and incubated at 37°C in 5% CO 2 , Cultures were maintained with twiceweekly exchanges of medium and once-weekly additions of 4day PHA-P-stimulated PBMC (5 X 106 cells). Cell-free supernatant fluids were harvested 1-2 days after peak virus-in-
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110 1991; 164 (October)
Table 1. Experimental design to evaluate zidovudine (AZT), didanosine (ddI), and recombinant interferon-a A (rIFN-aA) against human immunodeficiency virus type I (HIV -I) replication in peripheral blood mononuclear cells. Experiment
HIV-l isolates Early-AZT therapy" Late-AZT therapy' HIV-I inoculum" Range of AZT tested (JlM)
14a-4/87 14a-6/89 1000 0.01 0.1 1.0
Range of ddI tested (JlM)
Range of rIFN-aA tested (units/nil) 16 32 NOTE.
3
4
14a-6/89 1000 0.125 0.25 0.5 1.0 0.625 1.25 2.5 5.0
14a-6/89 1000 0.125 0.25 0.5 1.0 0.625 1.25 2.5 5.0
14a-6/89 1000 0.125 0.25 0.5 1.0 0.625 1.25 2.5 5.0
4 8 16 32
4 8 16 32
4 8 16 32
5
AOl2B AOl2D 1000 0.01 0.1 1.0 0.01 0.1 1.0 10.0 NT NT NT NT
6
7
8
AOl2D 5000 0.125 0.25 0.5 1.0 0.625 1.25 2.5 5.0
AOl2D 100 0.125 0.25 0.5 1.0 0.625 1.25 2.5 5.0
AOl2D 100 0.125 0.25 0.5 1.0 0.625 1.25 2.5 5.0
4 8 16 32
4 8 16 32
4 8 16 32
NT, not tested.
* Subjects received AZT I indicate synergism. additive effects, and antagonism, respectively. CI values were determined from median-effect plot parameters m (slope) and D m (EDso) of each drug and each combination based on classic isobologram equation asdescribed previously [5759]. All calculations were done with computer software using an IBM PC [57]. * HIV -I p24 antigen ELISA done on day 10 in culture; all other ELISA results were on day 7.
technique, which evaluates drug interactions by a dose-oriented geometric method. Full details of these methods and the computer data analysis have been described previously [57-59]. Infection kinetics were dependent on the MOl used. CI values (table 2) are presented only for experimental harvest days with ongoing replication of virus (i.e., not beyond peak infection). Thus, harvest days are depicted only if HIV-I p24 antigen values of the untreated-infected control cultures were equal to or greater than those of the previous harvest day.
Results Effects ofone-drug regimens in PBMC using HIV-l isolates derived from patients before and after prolonged AZT monotherapy. To establish whether our late-AZ'I' therapy isolate demonstrated decreased susceptibility to AZT in vitro when compared to the early-AZT therapy isolate, the first experiment evaluated the single-agent susceptibility of the pair of HIV-1 isolates derived from subject 14a before (l4a-4/87)
and after (14a-6/89) prolonged AZT monotherapy. Because cross-resistance to ddl using AZT-resistant virus has not been demonstrated [16], ddl was used as the treatment control. It was determined previously that AZT ~ 10 u.M and ddl ~ 100 u.M significantly inhibited cell growth and viability by day lOin culture in the absence of virus (data not shown), and therefore these concentrations were not tested in subsequent antiviral studies. As shown in figure I, the late-AZT therapy isolate 14a-6/ 89 displayed marked AZT resistance when compared to the early-AZT therapy isolate 14a-4/87 on day 7 in culture, although both isolates were susceptible to ddl at similar concentrations. The £D 50 of each agent required to block HIV-I p24 antigen production in cultures infected with the earlyAZT therapy isolate was 0.00026 f.lM AZT and 0.67 f.lM ddI. In contrast, the £D 50 of each agent required to block the late-AZT therapy isolate was 0.37 f.lM AZT and 0.92 p-M ddI. The late-AZT therapy isolate from subject 14a was "'"-' I 400-fold less susceptible to AZT than the early-AZT therapy isolate in this experiment. HIV-1 isolates AO 12B and AO 120 were tested similarly in experiment 5. The E0 50 of each of the single agents was 0.00198 f.lM AZT and 0.2 f.lM ddl for the early-AZT therapy isolate AO 12B. In contrast, the E0 50 of each of the single agents required to block the late-AZT therapy isolate AO 12D was 4.8 f.lM AZT and 0.3 f.lM ddI in this experiment (data not shown).
Effects oftwo-drug regimens in PBMC using HIV-l isolates derived from patients after prolonged AZT monotherapy. The ability of two-drug combination regimens to inhibit HIV-l replication by AZT-resistant virus in vitro was next evaluated in experiments 2-4 using the late-AZT therapy isolate 14a-6/89. The regimens tested did or did not include AZT (AZT + ddI, AZT + rIFN-aA, or ddl + rIFN-aA). As shown in figure 2, more complete viral suppression was achieved by each of the two-drug regimens tested when compared with each of the agents used singly in experiment 2 on day 7 in culture. This occurred even if the two-drug treatment regimen included AZT, despite the flat dose-effect curve seen testing AZT as a single agent over the concentration range used. Synergistic interactions were seen between either AZT and ddl, AZT and rIFN-aA, or ddl and rIFN-aA, as determined mathematically, with CI values < I (table 2). Similar results were obtained in experiment 3 for all of these twodrug regimens. In experiment 4, when AZT and rIFN-aA both achieved 1). However, synergistic interactions were still evident between AZT + ddl and ddl + rIFN-aA. To extend these findings to another AZT-resistant isolate, we evaluated combined therapy against the replication of
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2
Combination
649
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lID 1991;164 (October)
120.----------------,---------------,
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Late -AZT Therapy Isolate
-e
-a:i
t )) c: 80
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i=:
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40
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Infected .01.10 1.0 Control AZT (IlM)
.01.10 1.0 10 ddl (IlM)
Infected .01 .10 1.0 Control AZT (IlM)
late-AZ'T therapy isolate AO120 in experiments 6-8. In experiment 6. synergistic interactions were seen between either AZT and ddI or ddI and rIFN-aA, as determined mathematically, with CI values < I. As in experiment 4, when AZT and rIFN-aA tested as single agents both achieved I. To achieve single-agent dose-effect curves that are necessary for evaluation of combined drug interactions. the input MOl was reduced in subsequent experiments 7 and 8 to 100 TCIO so/ I0 6 cells. In experiments 7 and 8, synergistic interactions were seen between either AZT and ddl, AZT and rIFN-aA, or ddl and rlFN-aA. Representative data from experiment 8 on day 10 in culture are shown in figure 3. There was no cytotoxicity of these agents, either alone or in the two-drug combination regimens, over the concentration ranges tested. RT genotypic analysis. Mutations at codons 67, 70, 215, and 219 in the HIV-I RT gene that cause three or four specific amino acid substitutions have been shown to confer AZT resistance [32]. We evaluated the RT genotypes of the isolates from subject 14a (early-AZ'T therapy isolate 14a-4/ 87 and late-AZT therapy isolate 14a-6/89). Only wild-type HIV -I RT sequences were seen at all four of these codons in the early AZT-therapy isolate. Four specific amino acid substitutions dominated in the late-AZT therapy isolate (figure 4A), although evidence for viral genetic heterogeneity was seen (figure 4B). Mixtures of the bases encoding the wildtype lysine and the mutant arginine at codon 70, and the wild-type aspartate and the mutant asparagine at codon 67, were seen in overlapping sequencing reactions done on PCR product templates from two separate amplifications of the same infected PBMC lysate (figure 4B). These PBMC were infected with the identical 14a-6/89 viral stock used in combination therapy experiment 2. RT genes amplified from a PBMC lysate infected with a different 14a-6/89 viral stock
.01.10 1.0 10 ddl (IlM)
not used in a combination therapy experiment were also sequenced; only the dominant genotype encoding the mutant amino acids at codons 67 and 70 was seen. Only mutant sequences were found at codons 215 and 219 in all of the infected cell Iysates. Using the late AZT-therapy isolate AO120, only mutant sequences were seen at all four codons in PBMC Iysates infected with the same viral stock used in combination therapy experiment 8.
Discussion Our results demonstrate synergistic interactions among AZT, ddI, and IFN-aA in vitro in two-drug regimens against the replication of AZT-resistant HIV-I, even when AZT is included in the treatment regimen . These findings were seen with two AZT -resistant HIV-I isolates derived from different clinical sources. The concentrations of the agents tested (i.e., 0.125-1.0 J.LM AZT, 0.625-5.0 J.LM ddI, and 4-32 U/ml rlFN-aA) are easily achievable in vivo [60, 61). The absolute concentrations of each agent required to inhibit HI V-I replication, either alone or in two-drug regimens, varied in each experiment depending on the input viral inoculum, the ability of the clinical isolate to infect PBMC from a particular donor, the kinetics of infection, and the duration of the experiment. For these reasons, the EO so values generated for single-agent susceptibility testing in experiments I and 5 are not easily compared. However, it is clear that the late-AZT therapy isolates from subjects 14a and AO12 had a > 1000fold increase in EO so for AZT relative to the early-AZT therapy isolates. Due to the variability expected in biologic assays, no attempt was made to compare degrees of synergism or the efficacy of two-drug regimens across multiple experiments . Synergistic interactions were seen between agents that interrupt the HIV-I replication cycle by different or similar mechanisms of action, provided that single-agent dose-effect
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Figure I. Inhibition of human immunodeficiency virus type I (HrV-I) p24 antigen production in acutely infected peripheral blood mononuclear cells (experiment I) treated with zidovudine (AZT) or didanosine (ddl) as single agents. Data are shown for day 7 in culture. The HIV-I isolates used were earlyAZT therapy isolate 14a-4/87 and lateAZT therapy isolate 14a-6/89 from subject 14a. The untreated infected control is also shown. Similar results were obtained when the data were adjusted for viable cell numbers.
::::::-
AZT-Resistant HIV-I : Combination Therapy
JID 1991;164 (October)
1 5 0 . - - - - - - - - - - - - - - - - - - - -- ----,
A AZTandddI
G
Infected Control
~
Singl. Agents 2-!lrug Regimen
_
AZ:r ddI AZ:r ArT ddI AZ:r AZ:r ddI AZ:r AZ:r ddI AZ:r 1.0 5.0 + .5 2.5 + 25 125 + .125. m + ~M ~M ddI ~M ~M ddI ~M ~M ddI ~M ~M ddI 150.--------- - - -- - - - - - - - - - - - - - , Infected Control
8 AZTand rIFN-. -A
~M
~M U/ rrj IFN
150.-----
-
-
-
-
-
-
-
-
AZ:r IFN AZ:r .5 18 +
U/ rrj IFN ~ M U/ rrj IFN - - -- - - - -
AZ:r I FN AZ:r 1.0 32 + ~M
-
U/rrj IFN - - --,
C ddI and rIFN-.-A
100...------ - - - - - - - - - - -- ------, A AZT andddI
Infected Control
AZ:r ddI AZ:r .125 .825 + ~M ~M ddI
~ Infected Control ~
Singl. Agents
_
2-llrugRegimen
AZ:r ddI AZ:r 25 125 +
AZ:r ddI ArT .5 2.5 +
ArT ddI ArT 1.0 5.0 +
~M
~M
~M
~M
ddI
~M
ddI
~M
ddI
100...-- - - - - - - - - - - - - - - - - - - - - -, 8 AZTand rIFN-.-A
Infected Control
AZ:r IFN AZ:r .125 4 +
AZ:r IFN AZ:r 25 8 +
~M U/rrj IFN
~M U/rrj I FN
AZ:r IFN AZ:r .5 18 +
..
~M U/ rrj IFN
~M U/ m1 IFN
AZ:r IFN AZ:r 1.0 32 +
100.--- - - - - - - - - - - - - - - - - - - - -, C ddI and rIFN-.-A
~
~
100
iii
Two-Drug Combinations of Zidovudine, Didanosine, and Recombinant Interferon-a A Inhibit Replication of Zidovudine-Resistant Human Immunodeficiency Virus Type 1 Synergistically In Vitro Victoria A. Johnson, Debra P. Merrill, Joseph A. Videler, Ting-Chao Chou, Roy E. Byington, Joseph J. Eron, Richard T. D'Aquila, and Martin S. Hirsch
Infectious Disease Unit, Massachusetts General Hospital and Harvard Medical School, Boston; Laboratory of Biochemical Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, New York
The search for effective therapies for AIDS has led to the discovery of many agents that demonstrate activity against human immunodeficiency virus type 1 (HIV-1) replication in vitro [1-8]. Several of these agents show favorable results in patients as well, including zidovudine (AZT, 3'-azido-3'deoxythymidine), didanosine (ddI, 2',3'-dideoxyinosine), and recombinant interferon-a A (rIFN-aA) [9-21]. AZT and ddI are nucleoside analogues that inhibit HIV -1 reverse transcriptase (RT) [4-7]. Although the precise mechanism of inhibition of HI V-I replication by interferon-a is unclear, it may affect virus assembly or release as shown by studies of chronically infected cells [22-25]. Despite the progress made in the development of single-
Received 12 March 1991; revised 31 May 1991. Presented in part: Workshop on Viral Resistance, Washington. DC, October 1990, and the first joint University of Miami and University of California San Diego symposium. HIV Disease: Pathogenesis and Therapy. Grenelefe, Florida. March 1991 (abstract. J Acquir Immune Defic Syndr 1991;4:354). Written informed consent was obtained from all subjects according to protocols approved by the Subcommittee on Human Studies. Division of Research Affairs, Massachusetts General Hospital, Harvard Medical School. Financial support: National Institutes of Health (CA-12464, CA-35020, AI-26056, AI-29193, and National Research Service Award F32-7933 to V.A.J.) and Elsa U. Pardee Foundation. M.S.H. is a member of the BristolMyers AIDS Advisory Board. Reprints or correspondence: Dr. Victoria A. Johnson. Infectious Disease Unit. Gray 5. Massachusetts General Hospital, Boston. MA 02114. The Journal of Infectious Diseases 1991;164:646-55 © 199\ by The University of Chicago. All rights reserved. 0022-1899/91/6404-0003$01.00
agent therapy for HIV -1 infection, monotherapy with either AZT, ddI, or interferon-a has been associated with drug toxicity or failure [17, 26-30]. In addition, AZT-resistant variants of HIV-l have been isolated from patients receiving AZT as extended single-agent therapy [31-37], although the clinical implications of these findings remain uncertain. It is likely that combined therapy will be required for effective long-term treatment of HIV -1 infection, as in the approach to certain other infectious diseases (e.g., tuberculosis) and cancers [38-42]. Anti-HIV-l combination strategies that demonstrate favorable drug interactions (e.g., synergy) may allow the use of individual agents below their toxic concentrations, provide more complete viral suppression, and limit the emergence of drug-resistant HIV-l mutants. We previously demonstrated favorable interactions between agents that interrupt the HIV-1 replication cycle by different or similar mechanisms of action. Synergistic interactions were seen when combining an RT inhibitor (e.g., either AZT or ddI) with an agent that interrupts virus assembly and release (e.g., interferon-a) [43-45]. Additive to synergistic interactions have also been noted between RT inhibitors when combined (e.g., AZT and ddI) [45, 46]. Several clinical trials of combination therapy are in progress or planned in HIV-infected patients to evaluate the two-drug regimens of AZT and interferon-a, ddI and interferon-a, or AZT and ddI [47-50]. To further define the potential role of combination therapy for treatment of AZT-resistant virus variants, we studied AZT-resistant isolates derived from two HIV -I-infected individuals after extended AZT monotherapy. Our goal was to
Downloaded from http://jid.oxfordjournals.org/ at Cambridge University on August 17, 2015
Optimal management of human immunodeficiency virus type 1 (HIV-l) infections may require combinations of anti-HIV-I agents, Zidovudine (AZT, 3'-azido-3'-deoxythymidine), didanosine (ddI, 2',3'-dideoxyinosine), and recombinant interferon-a A (rIFN-aA) were evaluated in two-drug regimens against replication of AZT-resistant HIV-1 in vitro. AZT-sensitive and AZTresistant isolate pairs derived from two individuals before and after extended AZT monotherapy were studied. Drug interactions using peripheral blood mononuclear cells infected with HIV-l were evaluated mathematically. Synergistic interactions were seen among AZT, ddI, and rIFNaA in two-drug regimens against AZT-resistant HIV-1 in vitro, even when AZT was included in the treatment regimen. Mixtures of wild-type and mutant reverse transcriptase genes were found in one of the late-AZT therapy isolates, suggesting that the mechanism of synergy of AZT-containing regimens may involve inhibition of AZT -sensitive viruses in the viral pool. These studies suggest that AZT may be useful in drug combination regimens, even when AZT-resistant viruses are isolated in vitro.
110 1991; 164 (October)
AZT-Resistant HIV-I: Combination Therapy
evaluate the ability of two-drug combined regimens to block replication of AZT-resistant HIV-I in vitro using regimens that did or did not include AZT. The regimens studied were AZT alone versus ddI alone versus AZT combined with ddI (i.e., the addition of another anti-Hl Vr l agent that inhibits R T); AZT alone versus rIFN-aA alone versus AZT combined with rIFN-aA (i.e., the addition of an anti-Hl V-I agent acting by a different mechanism); and ddI alone versus rIFN-aA alone versus ddI combined with rIFN-aA (i.e., use of a regimen that did not include AZT).
Methods
duced cytopathic effects (CPE) occurred and stored at -85°C. Titrations of these viral stocks were done in PBMC to determine the TCID so per milliliter. An end-point titration method in 96well microtiter plates was done in sextuplicate as described previously, using 2 X 105 PBMC per well [43]. Peak virus-induced CPE was identified as the end point on day 7 in culture. The TCID so value of each viral stock was calculated by the method of Reed and Muench (cited in [51]). We also evaluated a pair of HI V-I isolates provided by D. Richman (University of California, San Diego) derived from a patient with AIDS (subject AOI2) [31]. These HIV-l isolates, obtained by an initial cocultivation of HIV-I-infected PBMC with MT-2 cells (a continuous T cell line), were designated AOl2B (obtained after 2 months of AZT therapy) and AOl2D (obtained after 26 months of AZT therapy) [31]. Viral stocks were made and titrated in PBMC for subsequent drug susceptibility testing. Compounds. AZT was obtained in powder form from P. A. Furman (Wellcome Research Laboratories, Research Triangle Park, NC). It was dissolved in sterile PBS and stored at a concentration of I mM in aliquots at -20°C until used. ddI was obtained in powder form from C. McLaren (Bristol-Myers Squibb, Wallingford, CT). It was dissolved in sterile PBS and stored at a concentration of 1 mM in aliquots at -20°C until used. The rIFN-aA was obtained from I. Sim (IFN-a-2a, Roferon-A; Hoffmann-La Roche, Nutley, NJ). Aliquots ofrlFN-aA were stored at a concentration of 105 Ll/rnl in sterile PBS at -85°C until used. The actual concentration of interferon was determined as described previously [8]. Viral replication assays. Cell-free culture supernatant fluids were assayed by an HIV-l p24 antigen ELISA (NEN Research Products, Boston) as described previously [52]. RT genotypic analysis. For sequencing analysis of the RT genes of the AZT-resistant isolates, PHA-P-stimulated PBMC were infected in vitro (in the absence of any drug) with the identical viral stock used in combination therapy experiments 2 (using isolate 14a-6/89) and 8 (using isolate AO12D). The earlyAZT therapy isolate 14a-4/87 R T gene was also sequenced. After maximal CPE occurred, cell pellets were lysed in detergent (at 3 X 106 cells/ml) and digested with proteinase K. Standard methods of polymerase chain reaction (PCR) were used, including Taq polymerase and an automated thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) [53]. PCR was done using 1.2 X 105 cell equivalents oflysate per amplification. Routine stringent precautions to prevent PCR product or plasmid clone carryover included having separate personnel do cell lysis and PCR setup at a site remote from that of both viral culture and PCR product and plasmid clone handling. PBMC from HIV -I-seronegative individuals were pelleted and lysed in parallel with experimental samples to control for carryover. PCR primers corresponded to conserved regions [54] of the HIV-l RT gene in the sense strand (bases 2058-2082) and the antisense strand (bases 2650-2628) of HIV-l BHIO-2 (GenBank accession number M 15654). DNA sequencing was done directly from gel-purified amplification products to identify the dominant base at each position [55]. PCR products were separated on 2% agarose gels, and the 590-bp RT gene fragments were excised and electroeluted. Se-
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Cells. Peripheral blood mononuclear cells (PBMC) from a single HIV-l-seronegative donor were obtained by ficollhypaque density-gradient centrifugation of heparinized venous blood. A different donor was used for each experiment. Cells were treated with phytohemagglutinin (PHA-P, 10 Jlg/ml; Difco Laboratories, Detroit), propagated in R-20 medium (RPMi 1640 medium supplemented with 20% heat-inactivated fetal calf serum [Sigma Chemical, St. Louis], 250 units [U]!ml penicillin, 250 Jlg/ml streptomycin, 2 mM L-glutamine, and 10 mM HEPES buffer [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]) supplemented with 10% interleukin-2 (IL-2; Pharmacia Diagnostics, Silver Spring, MD), and incubated at 37°C in 5% CO 2 , Viral stocks. Paired HIV-l isolates were derived from an HIV-L-seropositive individual (subject 14a) before and after 26 months of uninterrupted AZT monotherapy. These isolates were designated 14a-4/87 (early-AZT therapy) and 14a-6/89 (late-AZT therapy). This individual was diagnosed with HIV-related Kaposi's sarcoma in February 1987 and had the following clinical parameters when the April 1987 isolate was obtained: weight, 61. 7 kg; Karnofsky score, 100; and CD4+ cells, 174/ mrrr', The subject received AZT, 200 mg every 4 h, until May 1987, when the dose was reduced to 100 mg every 4 h. Pneumocystis carinii pneumonia was diagnosed in May 1987. When the late-AZT therapy isolate (l4a-6/89) was obtained, the subject had progressive cutaneous Kaposi's sarcoma and the following clinical parameters: weight, 57.6 kg; Karnofsky score, 90; and CD4+ cells, 8/mm 3 . PBMC from this HIV-l-seropositive individual before and after prolonged therapy with AZT were separated from heparinized venous blood using ficoll-hypaque density-gradient centrifugation and were then cocultivated with 4-day PHA-P-stimulated PBMC from normal donors. Cultures were maintained in R-Ill medium (R-20 medium supplemented with 10% IL-2 and polybrene [2 Jlg/ml; Sigma]). Twice weekly, cell-free culture supernatant fluids were stored as aliquots at -85°C. To prepare viral stocks for drug susceptibility testing, these aliquots were used to infect normal donor 4-day PHA-P-stimulated PBMC (lOX 106 cells) in 10 ml ofR-III medium in 25-cm 2 vented tissue culture flasks (Costar, Cambridge, MA) and incubated at 37°C in 5% CO 2 , Cultures were maintained with twiceweekly exchanges of medium and once-weekly additions of 4day PHA-P-stimulated PBMC (5 X 106 cells). Cell-free supernatant fluids were harvested 1-2 days after peak virus-in-
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Table 1. Experimental design to evaluate zidovudine (AZT), didanosine (ddI), and recombinant interferon-a A (rIFN-aA) against human immunodeficiency virus type I (HIV -I) replication in peripheral blood mononuclear cells. Experiment
HIV-l isolates Early-AZT therapy" Late-AZT therapy' HIV-I inoculum" Range of AZT tested (JlM)
14a-4/87 14a-6/89 1000 0.01 0.1 1.0
Range of ddI tested (JlM)
Range of rIFN-aA tested (units/nil) 16 32 NOTE.
3
4
14a-6/89 1000 0.125 0.25 0.5 1.0 0.625 1.25 2.5 5.0
14a-6/89 1000 0.125 0.25 0.5 1.0 0.625 1.25 2.5 5.0
14a-6/89 1000 0.125 0.25 0.5 1.0 0.625 1.25 2.5 5.0
4 8 16 32
4 8 16 32
4 8 16 32
5
AOl2B AOl2D 1000 0.01 0.1 1.0 0.01 0.1 1.0 10.0 NT NT NT NT
6
7
8
AOl2D 5000 0.125 0.25 0.5 1.0 0.625 1.25 2.5 5.0
AOl2D 100 0.125 0.25 0.5 1.0 0.625 1.25 2.5 5.0
AOl2D 100 0.125 0.25 0.5 1.0 0.625 1.25 2.5 5.0
4 8 16 32
4 8 16 32
4 8 16 32
NT, not tested.
* Subjects received AZT I indicate synergism. additive effects, and antagonism, respectively. CI values were determined from median-effect plot parameters m (slope) and D m (EDso) of each drug and each combination based on classic isobologram equation asdescribed previously [5759]. All calculations were done with computer software using an IBM PC [57]. * HIV -I p24 antigen ELISA done on day 10 in culture; all other ELISA results were on day 7.
technique, which evaluates drug interactions by a dose-oriented geometric method. Full details of these methods and the computer data analysis have been described previously [57-59]. Infection kinetics were dependent on the MOl used. CI values (table 2) are presented only for experimental harvest days with ongoing replication of virus (i.e., not beyond peak infection). Thus, harvest days are depicted only if HIV-I p24 antigen values of the untreated-infected control cultures were equal to or greater than those of the previous harvest day.
Results Effects ofone-drug regimens in PBMC using HIV-l isolates derived from patients before and after prolonged AZT monotherapy. To establish whether our late-AZ'I' therapy isolate demonstrated decreased susceptibility to AZT in vitro when compared to the early-AZT therapy isolate, the first experiment evaluated the single-agent susceptibility of the pair of HIV-1 isolates derived from subject 14a before (l4a-4/87)
and after (14a-6/89) prolonged AZT monotherapy. Because cross-resistance to ddl using AZT-resistant virus has not been demonstrated [16], ddl was used as the treatment control. It was determined previously that AZT ~ 10 u.M and ddl ~ 100 u.M significantly inhibited cell growth and viability by day lOin culture in the absence of virus (data not shown), and therefore these concentrations were not tested in subsequent antiviral studies. As shown in figure I, the late-AZT therapy isolate 14a-6/ 89 displayed marked AZT resistance when compared to the early-AZT therapy isolate 14a-4/87 on day 7 in culture, although both isolates were susceptible to ddl at similar concentrations. The £D 50 of each agent required to block HIV-I p24 antigen production in cultures infected with the earlyAZT therapy isolate was 0.00026 f.lM AZT and 0.67 f.lM ddI. In contrast, the £D 50 of each agent required to block the late-AZT therapy isolate was 0.37 f.lM AZT and 0.92 p-M ddI. The late-AZT therapy isolate from subject 14a was "'"-' I 400-fold less susceptible to AZT than the early-AZT therapy isolate in this experiment. HIV-1 isolates AO 12B and AO 120 were tested similarly in experiment 5. The E0 50 of each of the single agents was 0.00198 f.lM AZT and 0.2 f.lM ddl for the early-AZT therapy isolate AO 12B. In contrast, the E0 50 of each of the single agents required to block the late-AZT therapy isolate AO 12D was 4.8 f.lM AZT and 0.3 f.lM ddI in this experiment (data not shown).
Effects oftwo-drug regimens in PBMC using HIV-l isolates derived from patients after prolonged AZT monotherapy. The ability of two-drug combination regimens to inhibit HIV-l replication by AZT-resistant virus in vitro was next evaluated in experiments 2-4 using the late-AZT therapy isolate 14a-6/89. The regimens tested did or did not include AZT (AZT + ddI, AZT + rIFN-aA, or ddl + rIFN-aA). As shown in figure 2, more complete viral suppression was achieved by each of the two-drug regimens tested when compared with each of the agents used singly in experiment 2 on day 7 in culture. This occurred even if the two-drug treatment regimen included AZT, despite the flat dose-effect curve seen testing AZT as a single agent over the concentration range used. Synergistic interactions were seen between either AZT and ddl, AZT and rIFN-aA, or ddl and rIFN-aA, as determined mathematically, with CI values < I (table 2). Similar results were obtained in experiment 3 for all of these twodrug regimens. In experiment 4, when AZT and rIFN-aA both achieved 1). However, synergistic interactions were still evident between AZT + ddl and ddl + rIFN-aA. To extend these findings to another AZT-resistant isolate, we evaluated combined therapy against the replication of
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2
Combination
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lID 1991;164 (October)
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late-AZ'T therapy isolate AO120 in experiments 6-8. In experiment 6. synergistic interactions were seen between either AZT and ddI or ddI and rIFN-aA, as determined mathematically, with CI values < I. As in experiment 4, when AZT and rIFN-aA tested as single agents both achieved I. To achieve single-agent dose-effect curves that are necessary for evaluation of combined drug interactions. the input MOl was reduced in subsequent experiments 7 and 8 to 100 TCIO so/ I0 6 cells. In experiments 7 and 8, synergistic interactions were seen between either AZT and ddl, AZT and rIFN-aA, or ddl and rlFN-aA. Representative data from experiment 8 on day 10 in culture are shown in figure 3. There was no cytotoxicity of these agents, either alone or in the two-drug combination regimens, over the concentration ranges tested. RT genotypic analysis. Mutations at codons 67, 70, 215, and 219 in the HIV-I RT gene that cause three or four specific amino acid substitutions have been shown to confer AZT resistance [32]. We evaluated the RT genotypes of the isolates from subject 14a (early-AZ'T therapy isolate 14a-4/ 87 and late-AZT therapy isolate 14a-6/89). Only wild-type HIV -I RT sequences were seen at all four of these codons in the early AZT-therapy isolate. Four specific amino acid substitutions dominated in the late-AZT therapy isolate (figure 4A), although evidence for viral genetic heterogeneity was seen (figure 4B). Mixtures of the bases encoding the wildtype lysine and the mutant arginine at codon 70, and the wild-type aspartate and the mutant asparagine at codon 67, were seen in overlapping sequencing reactions done on PCR product templates from two separate amplifications of the same infected PBMC lysate (figure 4B). These PBMC were infected with the identical 14a-6/89 viral stock used in combination therapy experiment 2. RT genes amplified from a PBMC lysate infected with a different 14a-6/89 viral stock
.01.10 1.0 10 ddl (IlM)
not used in a combination therapy experiment were also sequenced; only the dominant genotype encoding the mutant amino acids at codons 67 and 70 was seen. Only mutant sequences were found at codons 215 and 219 in all of the infected cell Iysates. Using the late AZT-therapy isolate AO120, only mutant sequences were seen at all four codons in PBMC Iysates infected with the same viral stock used in combination therapy experiment 8.
Discussion Our results demonstrate synergistic interactions among AZT, ddI, and IFN-aA in vitro in two-drug regimens against the replication of AZT-resistant HIV-I, even when AZT is included in the treatment regimen . These findings were seen with two AZT -resistant HIV-I isolates derived from different clinical sources. The concentrations of the agents tested (i.e., 0.125-1.0 J.LM AZT, 0.625-5.0 J.LM ddI, and 4-32 U/ml rlFN-aA) are easily achievable in vivo [60, 61). The absolute concentrations of each agent required to inhibit HI V-I replication, either alone or in two-drug regimens, varied in each experiment depending on the input viral inoculum, the ability of the clinical isolate to infect PBMC from a particular donor, the kinetics of infection, and the duration of the experiment. For these reasons, the EO so values generated for single-agent susceptibility testing in experiments I and 5 are not easily compared. However, it is clear that the late-AZT therapy isolates from subjects 14a and AO12 had a > 1000fold increase in EO so for AZT relative to the early-AZT therapy isolates. Due to the variability expected in biologic assays, no attempt was made to compare degrees of synergism or the efficacy of two-drug regimens across multiple experiments . Synergistic interactions were seen between agents that interrupt the HIV-I replication cycle by different or similar mechanisms of action, provided that single-agent dose-effect
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Figure I. Inhibition of human immunodeficiency virus type I (HrV-I) p24 antigen production in acutely infected peripheral blood mononuclear cells (experiment I) treated with zidovudine (AZT) or didanosine (ddl) as single agents. Data are shown for day 7 in culture. The HIV-I isolates used were earlyAZT therapy isolate 14a-4/87 and lateAZT therapy isolate 14a-6/89 from subject 14a. The untreated infected control is also shown. Similar results were obtained when the data were adjusted for viable cell numbers.
::::::-
AZT-Resistant HIV-I : Combination Therapy
JID 1991;164 (October)
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~
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8 AZTand rIFN-. -A
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-
U/rrj IFN - - --,
C ddI and rIFN-.-A
100...------ - - - - - - - - - - -- ------, A AZT andddI
Infected Control
AZ:r ddI AZ:r .125 .825 + ~M ~M ddI
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2-llrugRegimen
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AZ:r ddI ArT .5 2.5 +
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..
~M U/ rrj IFN
~M U/ m1 IFN
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100.--- - - - - - - - - - - - - - - - - - - - -, C ddI and rIFN-.-A
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iii
