A Review of the Pharmacokinetics and Pharmacodynamics of Disulfiram and Its Metabolites
Abstract
Background. Transcriptionally silent human immunodeficiency virus blazon i (HIV-ane) DNA persists in resting memory CD4+ T cells despite antiretroviral therapy. In a master cell model, the antialcoholism drug disulfiram has been shown to induce HIV-1 transcription in latently infected resting memory CD4+ T cells at concentrations achieved in vivo.
Methods. Nosotros conducted a unmarried-arm airplane pilot report to evaluate whether 500 mg of disulfiram administered daily for 14 days to HIV-1–infected individuals on stable suppressive antiretroviral therapy would result in reversal of HIV-i latency with a concomitant transient increase in residual viremia or depletion of the latent reservoir in resting retentiveness CD4+ T cells.
Results. Disulfiram was safe and well tolerated. In that location was a loftier level of subject-to-subject area variability in plasma disulfiram levels. The latent reservoir did non change significantly (1.16-fold alter; 95% confidence interval [CI], .seventy- to ane.92-fold; P = .56). During disulfiram administration, rest viremia did non alter significantly compared to baseline (1.53-fold; 95% CI, .88- to 2.69-fold; P = .13), although residuum viremia was estimated to increase by 1.88-fold compared to baseline during the postdosing menses (95% CI, i.03- to three.43-fold; P = .04). In a post hoc analysis, a rapid and transient increment in viremia was noted in a subset of individuals (n = vi) with immediate postdose sampling (HIV-1 RNA increase, 2.96-fold; 95% CI, 1.29- to half dozen.81-fold; P = .01).
Conclusions. Assistants of disulfiram to patients on antiretroviral therapy does not reduce the size of the latent reservoir. A possible dose-related effect on remainder viremia supports future studies assessing the touch on of higher doses on HIV-1 product. Disulfiram affects relevant signaling pathways and can be safely administered, supporting future studies of this drug.
Combination antiretroviral therapy (Art) has dramatically contradistinct the natural history of human being immunodeficiency virus type 1 (HIV-1) infection for most infected individuals with access to treatment [ane]. Fine art reduces plasma HIV-1 RNA to below the limit of clinical detection. It was initially hoped that the virus could be eradicated with two–three years of constructive ART treatment [ii]; nonetheless, a latent grade of HIV-one exists in vivo. Stably integrated, transcriptionally silent viral genomes persist in long-lived resting memory CD4+ T cells [3–7]. The stability of this latent reservoir is the major bulwark to eradication of HIV-1 [iv, 7, eight], requiring patients to remain on Art indefinitely. Given the business concern for adverse effects of ART, also as the financial burden of treatment and need for adherence, strategies to eliminate the latent reservoir have get an urgent research priority.
One eradication strategy that has attracted significant attending involves targeting the latent reservoir through the apply of drugs that reverse latency without inducing global T-prison cell activation [nine, 10]. This strategy is based on the hypothesis that cells in which latency has been reversed volition exist targeted by cytolytic CD8+ T cells or will die by viral cytopathic furnishings [eleven]. Previous attempts to target latently infected cells by inducing global T-cell activation take proven too toxic for use in humans [12–14]. Subsequent inquiry has focused on identifying compounds that will induce HIV-1 factor expression in latently infected resting CD4+ T cells without activating the cell itself [15]. To this finish, several in vitro models take been described that appear to recapitulate the phenotype of HIV-1 latency in resting CD4+ T cells [16–18]. We have described 1 such model that makes employ of Bcl-2–transduced main CD4+ T cells [xvi] and performed a loftier-throughput screen to identify compounds that induce viral gene expression without triggering cellular activation [19]. One hit from this screen was disulfiram, a US Nutrient and Drug Administration (FDA)–approved drug used to treat alcoholism [20]. Disulfiram (bis [diethylthiocarbamoyl] disulphide) inhibits aldehyde dehydrogenase, resulting in an increased concentration of acetaldehyde when alcohol is consumed [21]. This leads to an unpleasant systemic reaction that serves every bit a deterrent to alcohol consumption [22, 23]. Disulfiram has been in clinical use for several decades [24] and has a well-characterized safety profile [25, 26].
The molecular mechanism of in vitro disulfiram-induced HIV-1 latency reactivation is unclear. Disulfiram undergoes a complex metabolism [26] with the downstream metabolite N,Due north-diethylthiolcarbamate sulfoxide (DETC-MeSO) primarily responsible for aldehyde dehydrogenase inhibition and resultant clinical upshot [27]. In contrast, reactivation of latent HIV-1 in vitro occurs only with the parent compound and first metabolite, diethyldithiocarbamic acid (DDTC) [nineteen]. Subsequent metabolites, including diethyldithiocarbamate methyl ester (DDTC-Me), induce no appreciable HIV-one reactivation [xix]. A recent study institute that intracellular depletion of the phosphate and tensin homolog (PTEN) protein by disulfiram led to upregulation of the Akt signaling pathway, resulting in HIV-1 gene transcription in the U1 HIV-1–infected monocyte prison cell line [28]. This potential molecular mechanism of disulfiram activity is under farther investigation.
We describe a airplane pilot trial in which we administered 500 mg of disulfiram daily for 14 days to HIV-one–infected patients who had suppression of viremia on Art to determine whether this compound could reactivate latent HIV-one from resting retention CD4+ T cells. This FDA-approved dose was selected to achieve in vivo concentrations of disulfiram and its metabolites comparable to concentrations that resulted in latency reversal activity in vitro [19]. The safety of college doses is unknown. We hypothesized that disulfiram treatment would be prophylactic and would event in a transient increment in residual viremia due to virus release from latently infected resting CD4+ T cells and a measureable decline in the size of the latent reservoir.
METHODS
Participants
Nosotros conducted an open-label, single-arm, pilot clinical trial at Johns Hopkins Hospital (JHH) and the University of California, San Francisco (UCSF). Inclusion criteria included historic period >18 years, utilize of a Department of Health and Man Services–recommended Fine art regimen continuously for a minimum of xviii months, >90% adherence equally determined by self-report, maintenance of undetectable plasma viral load using standard commercial assays (<l copies RNA/mL) for the previous 12 months, and a CD4+ T-cell count >200 cells/µL for 24 weeks prior to enrollment. Participants had to agree to abstain from alcohol during the 2-calendar week flow of disulfiram administration and the subsequent two weeks.
Exclusion criteria included the presence of an alcohol employ disorder; apply of any drug formulation containing alcohol or medications involved in clinically important drug interactions with disulfiram; serious illness requiring hospitalization in the 3 months prior to enrollment; severe myocardial or coronary artery disease; history of psychosis, peripheral neuropathy, seizure disorder or hypothyroidism; evidence of clinically active hepatitis with aspartate aminotransferase (AST) or alanine transaminase (ALT) serum concentrations >3 times the upper limit of normal; treatment with immunomodulatory drugs in the previous 16 weeks; pregnancy or breastfeeding; and allergy to safe or thiuram derivatives. The protocol was approved by the institutional review boards of both institutions participating in the trial.
Report Design
Potential participants underwent an initial screening visit followed by 2–3 pretreatment visits after enrollment, during which blood was obtained for baseline measurement of residual viremia using a highly sensitive quantitative existent-time reverse transcriptase polymerase chain reaction analysis (the single-copy assay [SCA]) as previously described [29]. Condom laboratory tests including chemistry and liver part profiles and consummate blood counts were obtained weekly during disulfiram administration and at every visit earlier and after the disulfiram assistants period. At day –14, a large peripheral claret sample (180 mL) was obtained to mensurate the frequency of latently infected resting retentivity CD4+ T cells using a previously described quantitative viral outgrowth assay [thirty].
Beginning on 24-hour interval 0, subjects received a direct observed oral dose of 500 mg of disulfiram. Disulfiram was administered daily for 14 days under direct observation on weekdays and by participant assistants on weekends. Participants were evaluated at every visit for potential adverse events using a standardized questionnaire and a detailed face-to-face interview with a study investigator. All antiretroviral medications were connected throughout the trial, with medication adherence determined by self-report. Get-go at twenty-four hours 0, residuum viremia was measured every 2 days (Monday, Wednesday, and Friday) for 3 weeks using the SCA. SCA was performed at every subsequent study visit for an additional 9 weeks. Plasma samples were also used to quantify disulfiram concentrations. A second 180-mL claret sample was obtained at week 12 for a posttreatment measurement of replication-competent HIV-ane in resting memory CD4+ T cells (Effigy i).
Effigy 1.
Timing of trial events. Afterward enrollment, participants underwent several baseline measurements of residual viremia using a highly sensitive quantitative real-time opposite transcriptase polymerase chain reaction assay (the single-re-create assay). Five hundred milligrams of disulfiram was administered daily from twenty-four hours 0 to twenty-four hour period 13. A 180-mL blood sample was obtained at day –fourteen and again on mean solar day 84 to estimate the frequency of latently infected cells in peripheral blood using a limiting dilution co-culture method. Safe laboratory tests including a complete blood count and metabolic panel were drawn weekly before, during, and two weeks later on disulfiram administration, and monthly thereafter. Abbreviation: DSF, disulfiram.
Figure 1.
Timing of trial events. After enrollment, participants underwent several baseline measurements of remainder viremia using a highly sensitive quantitative real-time reverse transcriptase polymerase chain reaction assay (the unmarried-copy assay). V hundred milligrams of disulfiram was administered daily from day 0 to 24-hour interval 13. A 180-mL blood sample was obtained at day –14 and again on twenty-four hours 84 to estimate the frequency of latently infected cells in peripheral blood using a limiting dilution co-culture method. Safety laboratory tests including a consummate blood count and metabolic panel were drawn weekly before, during, and 2 weeks after disulfiram administration, and monthly thereafter. Abbreviation: DSF, disulfiram.
Disulfiram Mass Spectrometry
An ultra-functioning liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) assay was developed to measure out disulfiram concentrations in plasma. The UPLC-MS system consisted of a Dionex Ultimate 3000 UPLC system coupled to a TSQ Vantage Triple Stage Quadrupole mass spectrometer (Thermo Fisher Scientific). A deuterated analogue (d20-disulfiram; deuterated at 20 positions on the molecule) was used every bit the internal standard. The analytes were extracted via protein precipitation and were chromatographically separated on a Phenomenex Kinetex column (phase C18; diameter, 2.1 mm × fifty mm; particle size, 1.7 µm; pore size, 100A°) using mobile phases consisting of (1) water, 0.one% formic acid and (two) acetonitrile, 0.i% formic acrid delivered at a flow charge per unit of 400 µL/minute. The resolved analytes were detected by mass spectrometry in selected reaction monitoring mode under negative electrospray ionization using the following transitions: disulfiram thousand/z 297.2/116.1 and d20-disulfiram m/z 317.2/126.1. The assay was linear from 15 ng/mL to 6400 ng/mL of disulfiram with an r two of 0.996 (SD 0.001). Interday and intraday precision ranged from one.viii% to 6.3% and 1.2% to 5.viii%, respectively, whereas the accuracies were 95.4% to 104%, and 93.half-dozen% to 105%, respectively.
Biostatistical Assay
Maximum likelihood estimation of rates of latently infected resting CD4+ T cells was carried out using the NLMIXED procedure in SAS version 9.2 (SAS Institute, Cary, N Carolina), with Wald 95% confidence intervals (CIs) calculated for log IUPM (infectious units per million) and then back-transformed to the IUPM scale. For cases with no positive co-cultures, the estimated IUPM was zero and an upper 95% confidence jump was calculated as 3/(full number of cells tested) [31]. Nosotros modeled the upshot of the postdisulfiram time point (vs predisulfiram) on log IUPM across all participants, using the primary data from each co-civilisation of whether it was positive or negative and how many cells it independent. This model was fit by maximum likelihood in the NLMIXED procedure in SAS, and included parameters for each participant'due south baseline log IUPM to account for the matched pre–post nature of the information inside individuals. Additional models allowed the postdisulfiram outcome to differ for those who ever had a detectable disulfiram level compared to those who did not. The software provided Wald P values and 95% CIs. We dorsum-transformed estimates and CIs to fold-effects on IUPM. We performed sensitivity analyses by excluding particular wells that had probability <.001 given the estimated IUPM.
We modeled residual viremia measured past SCA using negative binomial regression, with a random intercept to account for within-person correlation, once more using the SAS NL mixed procedure. To forbid very large values from dominating the analyses, we set SCA values >56 to equal 56, which was the 97th percentile of all observed values. We initially fit a model with one parameter for how viremia during disulfiram administration differed from the baseline period and 1 parameter for how information technology differed postdisulfiram compared to baseline. We then fit models that examined a number of possible refinements: assuasive viremia ii hours after the first dose (measured at JHH) to differ from viremia at other times during disulfiram administration; assuasive viremia during and afterwards assistants to differ depending on whether disulfiram was ever detected in any the patient'southward blood specimens; and assuasive viremia during administration to be influenced past the concurrently measured blood level of disulfiram. Nosotros chose the primary model for presentation equally the simplest one for which all farther refinements had a P value >.05.
RESULTS
Study Participants and Safety Outcomes
We enrolled 16 participants (11 at JHH, 5 at UCSF; Table ane). The median CD4+ T-cell count and per centum at the time of enrollment were 609 cells/μL (range, 224–1168 cells/μL; interquartile range [IQR], 366 cells/μL) and 30% (range, 12.6%–42.vii%; IQR, 11%), respectively. The median time of viral suppression (<fifty copies/mL) was 79 months (range, 16–162 months; IQR, 79 months). Art regimens for 8 participants combined ii nucleoside reverse transcriptase inhibitors (NRTIs) with the nonnucleoside reverse transcriptase inhibitor efavirenz, and regimens for the other 6 patients combined 2 NRTIs with a ritonavir-additional protease inhibitor. 2 participants were taking regimens that included agents from >2 antiretroviral drug classes. One dropped out of the report subsequently completing 12 days of disulfiram therapy.
Tabular array one.
Participant Baseline Characteristics
| Participant No. | Age | Sex | Race/Ethnicity | Duration of Viral Suppressiona | ART Regimen | Screening CD4 Count (%)b |
|---|---|---|---|---|---|---|
| 7150 | 24 | Thousand | AA | xvi | FTC/TDF/EFV | 524 (27) |
| 7151 | 55 | M | West | 87 | FTC/TDF/DRV/r | 275 (31) |
| 7152 | 43 | Thou | Westward | 45 | FTC/TDF/EFV | 644 (39) |
| 7153 | 37 | M | AA | 138 | 3TC/ABC/EFV | 1157 (39) |
| 7154 | 46 | M | W | 150 | FTC/TDF/NVP/FPV/r | 613 (28) |
| 7155 | 52 | Thou | W | 101 | FTC/TDF/EFV | 886 (43) |
| 7156 | 57 | 1000 | W | 162 | FTC/TDF/DRV/r | 1168 (35) |
| 7157 | 38 | One thousand | AA | 53 | 3TC/ABC/EFV | 604 (28) |
| 7158 | 46 | M | Westward | 79 | 3TC/ABC/ATV/r | 953 (42) |
| 7160 | 44 | M | H | 24 | 3TC/ABC/ATV/r | 224 (xiii) |
| 7161 | 48 | F | AA | 37 | ABC/3TC/DRV/r | 871 (28) |
| 2006 | threescore | M | West | 178 | 3TC/TDF/ATV/r | 561 (29) |
| 2135 | 53 | Grand | W | 78 | FTC/TDF/EFV | 749 (42) |
| 2428 | 48 | Thou | West | 13 | FTC/TDF/EFV | 511 (21) |
| 2432 | 57 | M | W | 26 | FTC/TDF/EFV | 503 (34) |
| 3037 | 52 | M | AA | 58 | 3TC/ABC/TDF/ZDV/ETV/DRV/r | 504 (23) |
| Participant No. | Age | Sex | Race/Ethnicity | Duration of Viral Suppressiona | Art Regimen | Screening CD4 Count (%)b |
|---|---|---|---|---|---|---|
| 7150 | 24 | M | AA | sixteen | FTC/TDF/EFV | 524 (27) |
| 7151 | 55 | M | Due west | 87 | FTC/TDF/DRV/r | 275 (31) |
| 7152 | 43 | M | W | 45 | FTC/TDF/EFV | 644 (39) |
| 7153 | 37 | M | AA | 138 | 3TC/ABC/EFV | 1157 (39) |
| 7154 | 46 | M | W | 150 | FTC/TDF/NVP/FPV/r | 613 (28) |
| 7155 | 52 | M | Due west | 101 | FTC/TDF/EFV | 886 (43) |
| 7156 | 57 | Thou | West | 162 | FTC/TDF/DRV/r | 1168 (35) |
| 7157 | 38 | M | AA | 53 | 3TC/ABC/EFV | 604 (28) |
| 7158 | 46 | G | W | 79 | 3TC/ABC/ATV/r | 953 (42) |
| 7160 | 44 | Yard | H | 24 | 3TC/ABC/ATV/r | 224 (13) |
| 7161 | 48 | F | AA | 37 | ABC/3TC/DRV/r | 871 (28) |
| 2006 | sixty | One thousand | W | 178 | 3TC/TDF/ATV/r | 561 (29) |
| 2135 | 53 | M | W | 78 | FTC/TDF/EFV | 749 (42) |
| 2428 | 48 | M | W | 13 | FTC/TDF/EFV | 511 (21) |
| 2432 | 57 | Thousand | Due west | 26 | FTC/TDF/EFV | 503 (34) |
| 3037 | 52 | Chiliad | AA | 58 | 3TC/ABC/TDF/ZDV/ETV/DRV/r | 504 (23) |
Abbreviations: 3TC, lamivudine; AA, African American; ABC, abacavir; ART, antiretroviral therapy; ATV/r, atazanavir boosted with ritonavir; DRV/r, darunavir boosted with ritonavir; EFV, efavirenz; ETV, etravirine; FPV/r, fosamprenavir boosted with ritonavir; FTC, emtricitabine; H, Hispanic; NVP, nevirapine; TDF, tenofovir; West, non-Hispanic white; ZDV, zidovudine.
a Sequent months of documented viral load (plasma HIV-i RNA) suppression below limit of clinical detection on ART.
b Absolute CD4+ T-cell count measured in cells/μL.
Table ane.
Participant Baseline Characteristics
| Participant No. | Historic period | Sexual practice | Race/Ethnicity | Duration of Viral Suppressiona | ART Regimen | Screening CD4 Count (%)b |
|---|---|---|---|---|---|---|
| 7150 | 24 | M | AA | 16 | FTC/TDF/EFV | 524 (27) |
| 7151 | 55 | G | West | 87 | FTC/TDF/DRV/r | 275 (31) |
| 7152 | 43 | M | W | 45 | FTC/TDF/EFV | 644 (39) |
| 7153 | 37 | M | AA | 138 | 3TC/ABC/EFV | 1157 (39) |
| 7154 | 46 | One thousand | W | 150 | FTC/TDF/NVP/FPV/r | 613 (28) |
| 7155 | 52 | Thou | W | 101 | FTC/TDF/EFV | 886 (43) |
| 7156 | 57 | Grand | W | 162 | FTC/TDF/DRV/r | 1168 (35) |
| 7157 | 38 | Thousand | AA | 53 | 3TC/ABC/EFV | 604 (28) |
| 7158 | 46 | Thousand | W | 79 | 3TC/ABC/ATV/r | 953 (42) |
| 7160 | 44 | M | H | 24 | 3TC/ABC/ATV/r | 224 (thirteen) |
| 7161 | 48 | F | AA | 37 | ABC/3TC/DRV/r | 871 (28) |
| 2006 | lx | M | W | 178 | 3TC/TDF/ATV/r | 561 (29) |
| 2135 | 53 | One thousand | West | 78 | FTC/TDF/EFV | 749 (42) |
| 2428 | 48 | M | W | thirteen | FTC/TDF/EFV | 511 (21) |
| 2432 | 57 | M | W | 26 | FTC/TDF/EFV | 503 (34) |
| 3037 | 52 | M | AA | 58 | 3TC/ABC/TDF/ZDV/ETV/DRV/r | 504 (23) |
| Participant No. | Age | Sex | Race/Ethnicity | Duration of Viral Suppressiona | Fine art Regimen | Screening CD4 Count (%)b |
|---|---|---|---|---|---|---|
| 7150 | 24 | K | AA | 16 | FTC/TDF/EFV | 524 (27) |
| 7151 | 55 | M | Westward | 87 | FTC/TDF/DRV/r | 275 (31) |
| 7152 | 43 | M | W | 45 | FTC/TDF/EFV | 644 (39) |
| 7153 | 37 | M | AA | 138 | 3TC/ABC/EFV | 1157 (39) |
| 7154 | 46 | Thou | W | 150 | FTC/TDF/NVP/FPV/r | 613 (28) |
| 7155 | 52 | K | W | 101 | FTC/TDF/EFV | 886 (43) |
| 7156 | 57 | Yard | West | 162 | FTC/TDF/DRV/r | 1168 (35) |
| 7157 | 38 | M | AA | 53 | 3TC/ABC/EFV | 604 (28) |
| 7158 | 46 | Thousand | W | 79 | 3TC/ABC/ATV/r | 953 (42) |
| 7160 | 44 | M | H | 24 | 3TC/ABC/ATV/r | 224 (13) |
| 7161 | 48 | F | AA | 37 | ABC/3TC/DRV/r | 871 (28) |
| 2006 | threescore | M | W | 178 | 3TC/TDF/ATV/r | 561 (29) |
| 2135 | 53 | M | W | 78 | FTC/TDF/EFV | 749 (42) |
| 2428 | 48 | M | W | xiii | FTC/TDF/EFV | 511 (21) |
| 2432 | 57 | Yard | West | 26 | FTC/TDF/EFV | 503 (34) |
| 3037 | 52 | M | AA | 58 | 3TC/ABC/TDF/ZDV/ETV/DRV/r | 504 (23) |
Abbreviations: 3TC, lamivudine; AA, African American; ABC, abacavir; Fine art, antiretroviral therapy; ATV/r, atazanavir boosted with ritonavir; DRV/r, darunavir boosted with ritonavir; EFV, efavirenz; ETV, etravirine; FPV/r, fosamprenavir additional with ritonavir; FTC, emtricitabine; H, Hispanic; NVP, nevirapine; TDF, tenofovir; W, non-Hispanic white; ZDV, zidovudine.
a Sequent months of documented viral load (plasma HIV-1 RNA) suppression below limit of clinical detection on Fine art.
b Absolute CD4+ T-cell count measured in cells/μL.
Disulfiram was safe and well tolerated in all participants. Observed adverse events during the study were consistent with grades I and II toxicity. One participant had a unmarried detectable viral load measured by a standard commercial assay (620 copies/mL) at a postdisulfiram time point that returned to an undetectable level (<50 copies/mL) at next written report visit and remained undetectable for the trial duration. All other participants maintained undetectable viral loads every bit measured past commercial viral load assays throughout the trial. No substantial changes in CD4+ T-jail cell count or percent were observed in any participant for the elapsing of the trial.
Outcome of Disulfiram Administration on the Frequency of Latently Infected Cells
The size of the latent reservoir from each participant was measured by limiting dilution co-civilisation analysis [30] two weeks before and x weeks after disulfiram assistants (Figure 2). There was little change in the geometric mean frequency of latently infected cells ten weeks afterward disulfiram assistants compared to baseline (postdisulfiram fold-issue = 1.xvi; 95% CI, .70–one.92; P = .56). A bulk of participants had a latent reservoir size within the previously described dynamic range of this assay [8].
Figure 2.
Result of disulfiram (DSF) on latent reservoir size. No substantial change in the frequency of latently infected cells was observed 10 weeks after disulfiram administration compared to baseline every bit measured by limiting dilution co-culture analysis (post-DSF fold-issue = 1.16; 95% confidence interval, .seventy–1.92; P = .56). *Dashed lines/open circles stand for 3 co-culture assays in which no infected cells were identified (these co-cultures had 12.5 million to 27.5 million cells assayed). Abbreviation: IUPM, infectious units per one thousand thousand.
Figure ii.
Effect of disulfiram (DSF) on latent reservoir size. No substantial change in the frequency of latently infected cells was observed 10 weeks afterwards disulfiram assistants compared to baseline equally measured by limiting dilution co-culture assay (mail-DSF fold-effect = i.16; 95% confidence interval, .seventy–1.92; P = .56). *Dashed lines/open circles represent 3 co-culture assays in which no infected cells were identified (these co-cultures had 12.five million to 27.5 million cells assayed). Abbreviation: IUPM, infectious units per million.
Effect of Disulfiram Administration and Disulfiram Plasma Concentration on Rest Viremia
Residuum viremia was measured by SCA in plasma samples obtained at enrollment, days –14, –seven, 0, 2, 4, 7, nine, 11, 14, xvi, and 18, and at weeks 3, iv, eight, and 12. Private plasma virus and disulfiram concentrations through calendar week 4 are shown in Figure 3. Our initial model estimated that residual viremia averaged ane.53-fold college during disulfiram administration than during the predisulfiram baseline flow (95% CI, .88- to 2.69-fold; P = .13) and averaged 1.93-fold higher postdisulfiram than baseline (95% CI, one.04- to 3.57-fold; P = .039).
Effigy 3.
Individual single-re-create assay and disulfiram (DSF) plasma concentration results. The gray bar (betwixt days 0 and 13) represents the period of directly observed DSF administration. Half of Johns Hopkins Infirmary participants (numbers 7151–7161) had an increase in viremia within several hours of the offset DSF dose, but viremia during DSF administration after that time averaged only slightly higher than baseline and was not statistically significant. Six of 15 participants had detectable DSF plasma concentrations during the dosing interval. The lower limit of detection of DSF plasma concentrations using mass spectrometry was 15 ng/mL.
Figure three.
Individual single-re-create analysis and disulfiram (DSF) plasma concentration results. The gray bar (between days 0 and 13) represents the period of directly observed DSF administration. Half of Johns Hopkins Hospital participants (numbers 7151–7161) had an increase in viremia within several hours of the get-go DSF dose, but viremia during DSF administration after that fourth dimension averaged only slightly college than baseline and was non statistically significant. Half-dozen of 15 participants had detectable DSF plasma concentrations during the dosing interval. The lower limit of detection of DSF plasma concentrations using mass spectrometry was 15 ng/mL.
Participants at JHH (n = 10) received disulfiram 2 hours prior to plasma sampling at each visit. We observed an increase in residual viremia later the beginning dose of disulfiram (mean solar day 0) in several participants. Residual viremia on day 0 was estimated to average 3.81-fold college than the predisulfiram baseline (95% CI, 1.43- to 10.eighteen-fold; P = .01). Average residual viremia for the remainder of the disulfiram dosing interval did not differ significantly from baseline (1.thirty-fold increase; 95% CI, .74- to 2.27-fold; P = .33). Table 2 shows our model, which takes into account these early increases in viremia.
Tabular array 2.
Unmarried-Copy Assay Kinetics
| SCA Time Point | Fold Changea | % Change | 95% CI | P Value |
|---|---|---|---|---|
| DSF beginning dose (twenty-four hours 0)b | 3.81 | 281.thirty | 42.viii–917.seven | .01 |
| DSF dosing interval (days 1–13)c | 1.30 | 29.90 | −25.7 to 127.4 | .33 |
| Post-DSF interval (days 14–84)d | 1.88 | 87.90 | 2.8–243.2 | .04 |
| SCA Time Point | Fold Changea | % Change | 95% CI | P Value |
|---|---|---|---|---|
| DSF start dose (twenty-four hour period 0)b | 3.81 | 281.30 | 42.8–917.7 | .01 |
| DSF dosing interval (days 1–xiii)c | 1.30 | 29.90 | −25.7 to 127.iv | .33 |
| Mail-DSF interval (days fourteen–84)d | 1.88 | 87.ninety | 2.8–243.2 | .04 |
Abbreviations: CI, conviction interval; DSF, disulfiram; SCA, single-re-create assay.
a Fold-modify and percentage alter compare average SCA values with the pre-DSF baseline SCA estimate of two.2 copies/mL (95% CI, .nine–v.4).
b Day 0 SCA comparison performed with Johns Hopkins Hospital (JHH) SCA values just (n = 10) considering University of California, San Francisco subjects did non undergo plasma sampling immediately after day 0 DSF dose.
c JHH SCA values just (n = x).
d Data for all subjects included (n = xv).
Table ii.
Single-Copy Assay Kinetics
| SCA Fourth dimension Point | Fold Modifya | % Alter | 95% CI | P Value |
|---|---|---|---|---|
| DSF first dose (day 0)b | 3.81 | 281.thirty | 42.8–917.7 | .01 |
| DSF dosing interval (days 1–thirteen)c | 1.thirty | 29.90 | −25.seven to 127.four | .33 |
| Post-DSF interval (days 14–84)d | 1.88 | 87.ninety | 2.8–243.two | .04 |
| SCA Time Point | Fold Changea | % Alter | 95% CI | P Value |
|---|---|---|---|---|
| DSF first dose (solar day 0)b | iii.81 | 281.30 | 42.viii–917.7 | .01 |
| DSF dosing interval (days 1–13)c | 1.30 | 29.90 | −25.7 to 127.iv | .33 |
| Post-DSF interval (days xiv–84)d | 1.88 | 87.90 | 2.8–243.2 | .04 |
Abbreviations: CI, confidence interval; DSF, disulfiram; SCA, unmarried-re-create assay.
a Fold-change and pct change compare boilerplate SCA values with the pre-DSF baseline SCA approximate of two.two copies/mL (95% CI, .ix–v.4).
b 24-hour interval 0 SCA comparison performed with Johns Hopkins Hospital (JHH) SCA values simply (due north = ten) because Academy of California, San Francisco subjects did not undergo plasma sampling immediately afterward day 0 DSF dose.
c JHH SCA values but (n = 10).
d Information for all subjects included (n = 15).
Subject-to-Subject Variability in Disulfiram Exposure
We measured plasma levels of disulfiram by mass spectrometry. There was substantial and unexplained variability in disulfiram concentrations that did not appear to exist predicted by treatment regimen (Figure two). Six of 15 subjects had detectable plasma disulfiram concentrations at some point during the two-calendar week dosing interval (subjects 7153, 7154, 7155, 7156, 2006, 3037 shown in Figure three; lower limit of detection 15 ng/mL). There was a nonsignificant trend suggesting higher boilerplate viremia in these six subjects compared to those in whom disulfiram concentrations were non detected (estimated departure, 0.47-fold; 95% CI, .twenty- to 1.ten-fold, P = .077). Comparing postdisulfiram boilerplate viremia to the predisulfiram baseline showed an estimated 2.96-fold increase over baseline amongst these 6 subjects (95% CI, 1.29- to 6.81-fold; P = .01) compared to those without detectable disulfiram (ane.39-fold; 95% CI, .69- to 2.79-fold; P = .33; divergence detected vs not detected, two.thirteen-fold; 95% CI, .85- to five.4-fold; P = .10).
Give-and-take
We conducted a pilot clinical trial in which nosotros administered the FDA-canonical drug disulfiram for 14 days to HIV-1–infected individuals on Fine art to evaluate the safe and efficacy of this intervention equally a ways to adjy the HIV-1 latent reservoir. Disulfiram was well tolerated by all participants. The size of the latent reservoir, measured by a well-validated in vitro viral outgrowth analysis, [30] did not decrease later the intervention. We observed merely a small and non statistically significant average change in residuum viremia during disulfiram treatment compared to baseline. In a post hoc analysis limited to ten subjects with frequent sampling, we observed an unexpected rapid and transient increment in plasma viremia. Disulfiram exposure varied essentially among subjects.
There is much interest in understanding the kinetics of the subsequently stages of viral replication, including proviral gene transcription, translation, viral budding, and release in resting CD4+ T cells. A recently published study in which a single dose of the histone deacetylase inhibitor (HDACi) vorinostat was administered to 8 HIV-1–infected patients with viral suppression estimated a mean 4.eight-fold increase in jail cell-associated HIV-i RNA inside 4–seven hours of drug assistants [32]. Similar data have been presented by Lewin and colleagues with vorinostat [33]. Levin and Tolstrup recently reported rapid increases in plasma viremia after exposure to the HDACi panobinostat in an ongoing phase 1/2 clinical trial (NCT01680094) [34]. In an in vitro study assessing the affect of diverse antilatency drugs on the kinetics of HIV-1 RNA production, the touch on of disulfiram was more than rapid and transient than vorinostat [35], an observation consequent with the data presented hither.
Disulfiram appeared to have no result on the size of the latent reservoir as measured by quantitative in vitro viral outgrowth. The mechanism of action of disulfiram in inducing proviral transcription is non currently understood. Disulfiram may induce HIV-1 transcription by activating the Akt signaling pathway, as has been recently described in cell line models of HIV-i latency [28]. Alternatively, it is possible that fifty-fifty potent and sustained reversal of latency may not affect the reservoir in a durable manner. 1 important tenet of the "daze and impale" HIV-1 eradication strategy that makes utilize of latency-reversing agents targeting the reservoir is that virus-producing cells volition be cleared by the allowed system or volition be eliminated by viral cytopathic furnishings following viral reactivation. Nevertheless, it appears that reversing latency without T-cell activation may not be sufficient to kill latently infected CD4+ T cells [36]. The written report presented hither suggests that "daze and impale" strategies with drugs such as disulfiram will likely require another step to prime the immune arrangement to articulate virus-producing resting memory CD4+ T cells.
The pharmacokinetics and pharmacodynamics of disulfiram appear to exist highly variable among subjects. Up to fifty% will not take a disulfiram-ethanol reaction with a 250-mg dose [37]. For some individuals, doses of 500 mg are insufficient to instigate this reaction [37]. A formal report of the elimination kinetics of disulfiram establish marked intersubject variability in plasma levels of disulfiram and its metabolites [25]. A separate written report identified a 600-fold variation in disulfiram plasma concentrations among subjects [38]. The mechanism for this variability remains unknown. Using highly sensitive mass spectrometry, we likewise institute substantial subject-to-subject field variability in drug exposure, and could detect plasma disulfiram concentrations in just half dozen of 15 participants. These measurements accept into account only the parent compound; mass spectrometry assays for downstream metabolites may illuminate intersubject disulfiram pharmacokinetics and are in evolution for future studies. These participants had a significant "post-drug" increment in low-level viremia that was sustained over the 2 months following the disulfiram dosing interval, and besides demonstrated a nonsignificant trend toward decrease in the size of the latent reservoir, suggesting that higher exposures to the drug in vivo may have more pronounced and prolonged effects on HIV-1 production.
In summary, this trial attempted to safely interpret in vitro discoveries affecting the latent reservoir into initial in vivo analysis. Disulfiram was safe and well tolerated, but did not announced to significantly perturb the latent reservoir. The apparent exposure-response effect observed in this study highlights significant intersubject variability in disulfiram pharmacokinetics and suggest that higher doses of disulfiram might exist more constructive. It is also possible that combining disulfiram with other latency-reversing agents will have a more pronounced effect on the reservoir, and the favorable prophylactic profile of disulfiram provides support for such combination approaches.
Notes
Acknowledgments. We are indebted to the trial participants for their backbone, dedication, and altruism. We are grateful to Morrie Faiman for contributions regarding disulfiram pharmacology; Joseph Wong for assistance with viral quantification methods; Janice Clements for input on nonhuman primate models of HIV-1 latency; and Joel Gallant, Joe Cofrancesco, and Emily Richie for patient referrals.
Disclaimer. The contents of this piece of work are solely the responsibility of the authors and do not necessarily represent the official view of the Johns Hopkins Institute for Clinical and Translational Research (ICTR), National Eye for Advancing Translational Sciences (NCATS), or National Institutes of Health (NIH). None of these funding sources played whatever role in report pattern, collection, analysis or interpretation of data, writing of the manuscript, or decisions regarding manuscript submission.
Financial back up. This work was supported by an ARCHE Collaborative Research Grant from the Foundation for AIDS Inquiry, with additional back up from Martin Delaney CARE and DARE Collaboratories (NIH grant numbers AI096113 and 1U19AI096109), the National Institute of Allergy and Infectious Diseases (K24 AI069994), the UCSF/Gladstone Center for AIDS Research (P30 AI027763), the UCSF Clinical and Translational Science Constitute (UL1 RR024131), the Johns Hopkins Center for AIDS Research, the NIH (grant 43222 to R. F. S.), the Howard Hughes Medical Institute (to R. F. S.) and the Center for AIDS Prevention Studies (P30 MH62246). This work was as well supported by the Johns Hopkins ICTR, which is funded in office by grant number UL1 TR 000424-06 from NCATS, a component of the NIH, and NIH Roadmap for Medical Research.
Potential conflicts of interest. All authors: No potential conflicts of involvement.
All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
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Writer notes
a A. M. Due south. and A. A. contributed equally to this work.
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