The availability of potent antiretroviral therapy has reduced morbidity and mortality due to AIDS.1,2 Preferred initial therapies for HIV-1 infection include 2 nucleoside reverse transcriptase inhibitors (NRTIs) plus either an HIV protease inhibitor or a nonnucleoside reverse transcriptase inhibitor (NNRTI).3 The active intracellular anabolites of approved NRTIs block viral replication by competing with endogenous cellular nucleotides for incorporation into proviral DNA and are relatively specific for HIV reverse transcriptase. However, the inhibition of human mitochondrial DNA polymerase-γ by NRTIs4 has been associated with toxicities that include peripheral neuropathy, lipoatrophy, hepatic steatosis, and lactic acidosis.5
Precise incidence rates are difficult to determine but, perhaps, as many as one third of HIV-infected persons may develop symptomatic peripheral neuropathy that is characterized by distal, symmetrical anesthesia, and/or painful dysesthesia.6,7 Although peripheral neuropathy can complicate untreated HIV infection,8 most cases since the availability of antiretroviral therapy have resulted from exposure to NRTIs, particularly the dideoxynucleosides didanosine (ddI) and stavudine (d4T).9,10 There is evidence that NRTI-associated peripheral neuropathy is the result of mitochondrial injury.11,12
AIDS Clinical Trials Group (ACTG) study 384 was a randomized, double-blind treatment strategy trial that randomized subjects using a factorial design to receive either a protease inhibitor (nelfinavir) or an NNRTI (efavirenz) or both, and to receive one of 2 dual NRTI regimens (ddI/d4T or zidovudine [ZDV]/lamivudine [3TC]) as initial therapy for HIV infection. A total of 960 participants enrolled in the United States and Italy between October 1998 and November 1999, and were followed for up to 3 years.13,14 Of the 488 subjects randomized to receive ddI/d4T, 176 (36%) developed peripheral neuropathy during the study compared with 69 (14%) of the 472 receiving ZDV/3TC. The subjects in the ddI/d4T-treated arm developed peripheral neuropathy and dose-modifying toxicities more rapidly than the subjects in the ZDV/3TC-treated arm (P < 0.001).13
Oxidant stress describes the generation of reactive free radicals that damage cells and tissues.15 Mitochondria play a central role in regulation of oxidant stress through the production and scavenging of free radicals. Oxidant stress is increased in human conditions that include Alzheimer disease, Parkinson disease, scleroderma, and hepatorenal syndrome, and exposures such as heavy cigarette smoking.16-21 A central feature of oxidant stress is lipid peroxidation. Measuring the end products of free radical-catalyzed lipid peroxidation in plasma is a noninvasive approach to quantifying this process.15 Prostaglandin F2-like products, termed F2-isoprostanes (F2-IsoPs), are accurate markers of oxidative stress in humans.22,23
Increased and/or dysregulated oxidant stress may play a role in NRTI-associated toxicities, perhaps as a consequence of mitochondrial DNA damage and dysfunction.4 In animal models, ZDV and d4T have been shown to induce mitochondrial damage in skeletal muscle that is attenuated by administration of antioxidants.24,25 Increased plasma F2-IsoP levels have been demonstrated among NRTI-treated patients with symptomatic hyperlactatemia and lipoatrophy.26 In a separate cross-sectional study, the therapeutic control of HIV replication was also associated with increased plasma F2-IsoPs.27 Neither study was designed to address associations with the use of specific antiretroviral drugs. To determine whether NRTI-associated toxicity is associated with evidence of increased systemic oxidant stress, and in an effort to identify a biomarker for predicting the development of peripheral neuropathy, we quantified plasma F2-IsoPs among ACTG 384 participants.
This study included HIV-infected individuals who had enrolled in ACTG study 384 at clinical trial sites in the United States. The eligibility criteria for ACTG 384 included plasma HIV-1 RNA greater than or equal to 500 copies per milliliter and fewer than 7 days of earlier antiretroviral therapy. The NRTIs were open-labeled: ddI (dosage, 400 mg or 250 mg once a day based on weight; enteric-coated tablets were available during the final year of study); d4T (dosage, 40 mg or 30 mg 2 times a day based on weight); and 3TC (dose, 150 mg) and ZDV (dose, 300 mg) administered as a fixed-dose combination 2 times a day. Efavirenz (dosage, 600 mg once a day) and nelfinavir (dosage, 1250 mg 2 times a day) were double-blinded with matching placebos. Clinical assessments were obtained at screening, at entry, at weeks 4, 8, 12, 16, 20, and 24, and every 8 weeks thereafter. ACTG 384 was approved by institutional review boards for each site, and the subjects gave written informed consent. The Vanderbilt Committee for the Protection of Human Subjects and the ACTG approved the use of stored plasma and data for this study.
During ACTG 384 study, signs, symptoms, or diagnoses were recorded at study entry and at each study visit using a symptom distress self-report questionnaire that included pain and neuropathy. Peripheral neuropathy was a targeted toxicity in ACTG 384 and was graded according to the Division of AIDS (National Institutes of Health, Bethesda, MD) Table for Severity of Adult Adverse Experiences.28 Grade 2 peripheral neuropathy was defined as moderate paresthesia requiring nonnarcotic analgesia, or moderate neurosensory impairment, such as decreased vibratory, pinprick, or hot/cold sensation to the ankle, or decreased position sense or mild impairment that is not asymmetrical. Subjects with peripheral neuropathy at study entry were excluded from this study, and follow-up was censored at the time that a subject changed any study drug (excluding dose adjustments) from those to which they were originally randomized. The cases were subjects who developed peripheral neuropathy of at least grade 2 while receiving ddI/d4T, and who had stored plasma available from baseline and at the time of peripheral neuropathy (or within the previous 8 weeks). The controls were subjects receiving ddI/d4T who did not develop peripheral neuropathy during follow-up. Wherever possible, for each case, a single control subject was selected at random from among the potential control subjects that had stored plasma available and at the same duration of follow-up as when the case's peripheral neuropathy occurred. To assess the effects of different NRTI combinations on oxidant stress, we also included an alternate control group comprised of subjects receiving ZDV/3TC who developed at least grade 2 peripheral neuropathy.
Plasma was from EDTA anticoagulated whole blood. The specimens were centrifuged at 400g for 10 minutes at room temperature and the plasma aliquots were stored at −70°C within 4 to 6 hours. Plasma levels of 15-F2t-isoprostane (8-iso-PGF2α) were quantified using a gas chromatographic/negative ion chemical ionization mass spectrometric approach using stable isotope dilution as described by Morrow and Roberts.29 The assay has an intraday variability of less than 10%. Normal F2-IsoP concentration in plasma of healthy volunteers is considered 35 pg/mL ± 1 SD (6 pg/mL).29
Demographics and baseline laboratory values are presented as proportions and median (interquartile range [IQR]). Comparisons of peripheral neuropathy and F2-IsoP levels were performed using Wilcoxon rank sum test.
A total of 164 ACTG 384 participants were included in this analysis: 75 cases; 71 control subjects; and 18 alternate control subjects. Most were men (84%) and non-Hispanic white (44%); the median age was 38 years (Table 1). Median baseline plasma HIV-1 RNA concentrations and CD4 lymphocyte counts were 5.2 (IQR, 4.5-5.7) log10 copies/mL and 211 (IQR, 42-405) cells/mm3, respectively. At baseline, cases (median age, 38 [IQR, 33-45] years) were older than control subjects (median age, 36 [IQR, 30-42] years; P = 0.03) and had lower CD4 lymphocyte counts (cases' median, 119 [IQR, 32-316] cells/mm3; control subjects' median, 315 [IQR, 80-455] cells/mm3; P = 0.002).
Median F2-IsoP values at baseline were 53 pg/mL (IQR, 40-85) in cases, 57 pg/mL (IQR, 41-77) in control subjects, and 53 pg/mL (IQR, 47-101) in alternate control subjects (Table 1). Median baseline values did not differ significantly between cases and control subjects (P = 0.78), or between cases and alternate control subjects (P = 0.6). F2-Median IsoPs at the time of peripheral neuropathy also did not differ from those at a corresponding time among control subjects (59 [IQR, 43-88] vs. 61 [IQR, 43-79] pg/mL, respectively; P = 0.67), or among alternate control subjects (49 [IQR, 42-72] pg/mL; P = 0.19). Median changes in F2-IsoPs from baseline were 10 pg/mL (IQR, −17 to 26) for cases, 4 pg/mL (IQR, −11 to 17) for control subjects, and 1 pg/mL (IQR, −48 to 10) for alternate control subjects; these changes were not significantly different when comparing case patients and control subjects (P = 0.48) or comparing case patients and alternate control subjects (P = 0.21).
In this case-control study of 164 individuals who received NRTI-containing regimens during a prospective, randomized clinical trial, including 146 subjects who were randomized to receive ddI/d4T-containing regimens, the development of peripheral neuropathy was not associated with increased systemic oxidant stress as assessed by quantification of F2-IsoPs in plasma.
Toxicities resulting from prolonged exposure to antiretroviral drugs can compromise the responses to multidrug antiretroviral regimens. In particular, the NRTI class has been implicated as a cause of several insidious and sometimes irreversible chronic toxicities, including peripheral neuropathy, that are thought to result from mitochondrial damage. No intervention has been shown to prevent NRTI-associated peripheral neuropathy, and there is no specific therapy for NRTI-associated peripheral neuropathy other than avoidance of further exposure to potentially neurotoxic drugs. In particular, combined use of ddI/d4T should be avoided. In an effort to identify a biomarker that might be clinically useful for identifying individuals at greatest risk for developing peripheral neuropathy, we investigated F2-IsoPs, products of cellular oxidative damage that may be increased in the setting of mitochondrial dysfunction.30
This is the first study to explore F2-IsoPs as a potential marker of oxidant injury during NRTI-associated peripheral neuropathy. We have previously reported that F2-IsoPs are increased among HIV-infected individuals with HIV-1 RNA levels less than 400 copies per milliliter while receiving various antiretroviral regimens, compared with individuals receiving no antiretrovirals.27 In another survey, McComsey and Morrow26 described increased F2-IsoP levels in individuals with symptomatic hyperlactatemia, lactic acidosis, or lipoatrophy while receiving NRTI-containing regimens, compared with similarly treated persons with asymptomatic hyperlactatemia and control subjects. We hypothesized that the increases in F2-IsoPs would be greater in subjects who developed peripheral neuropathy than in asymptomatic individuals with similar NRTI exposure, and would be greatest among persons exposed to ddI and d4T, which have greater affinity for mitochondrial DNA polymerase-γ31 and mitochondrial toxicity.32
As has been previously reported,7,33 older age at the time of drug treatment and lower baseline CD4 lymphocyte count identified individuals with the increased risk of developing symptomatic peripheral neuropathy. This could reflect underlying asymptomatic peripheral neuropathy in some subjects that progressed after NRTI therapy. Subjects that developed grade 2 or higher-graded symptomatic peripheral neuropathy while receiving ddI/d4T did not have higher F2-IsoP levels at the time of neuropathy or greater changes in F2-IsoP levels than did the control subjects receiving ddI/d4T or subjects who developed peripheral neuropathy while receiving ZDV/3TC. One explanation for the lack of association between plasma F2-IsoP and peripheral neuropathy in this study is because neuronal injury, which may involve oxidant stress, is not of sufficient magnitude to alter circulating lipid peroxidation profiles. Oxidative damage during peripheral neuropathy may be localized to tissue and may not be discernible with a plasma-based assay, in contrast with systemic toxicities (eg, lipoatrophy or lactic acidosis). Additionally, the dynamics of oxidant stress in the setting of treatment initiation and decreasing viral replication and cellular activation are likely very complex and may have limited the capacity to detect NRTI-related changes in oxidant stress in this population.
This study had several limitations. Cigarette smoking is known to increase systemic oxidant stress and plasma F2-IsoP concentrations.21 Because smoking status was not ascertained during the ACTG 384 study, we could not control for this variable. In addition, although peripheral neuropathy was characterized uniformly as a targeted toxicity during the ACTG 384 study, ascertainment was primarily based on clinical signs and symptoms, preventing us from assessing the relationships between oxidant stress and asymptomatic or subclinical peripheral neuropathy that may be identified with more detailed neurological assessments. Plasma from ACTG 384 participants was collected and cryopreserved at numerous clinical research sites. For some specimens, inconsistent processing may have spuriously elevated F2-IsoPs because of ex vivo lipid peroxidation.
In summary, symptomatic NRTI-associated peripheral neuropathy was not associated with increased plasma F2-IsoPs among ACTG 384 participants. Additional studies in this area should focus on well-characterized phenotypes while controlling for potential confounders.
The authors gratefully acknowledge the participants of ACTG study 384. We also acknowledge M. Hirsh, T. Merrigan, V. De Grutolla, R. D'Aquila, and other members of the ACTG 384 team, including M. Fischl (Miami University), M. Dube (Indiana University), C. Pettinelli, A. Martinez (National Institutes of Health), R. Delapenha (Howard University), M. Nokta (University of Texas, Galveston), V. Johnson (University of Alabama, Birmingham), G. Morse (State University of New York, Buffalo), B. Putnam (University of Colorado), M. Klebert (Washington University), M. Testa (Harvard School of Public Health), A. Chiesi, C. Tomino (Istituto Superiore de Sanita), S. Deeks (University of California, San Francisco), T. Nevin (Social & Scientific Systems), J. Levin, V. French, O. Fennell (Adult AIDS Clinical Trials Group Community Constituency Group), M. Stevens, R. Grosso, B. Dusak, S. Hodder (Bristol-Myers Squibb), J. Tolson, C. Brothers (GlaxoSmithKline), R. Leavitt (Merck), D. Manion, N. Ruiz, K. Morrissey (DuPont Pharmaceuticals), M. Becker, B. Quart (Agouron), C. Jennings (Northwestern University), L. Gedeon, S. Dascomb, M. Cooper, M. Murphy, K. Blakelock (Frontier Science and Technology Foundation), and A. Doolan (Massachusetts General Hospital).
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