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Comparative study of the persistence of anti-HIV activity of deoxynucleoside HIV reverse transcriptase inhibitors after removal from culture.

Paintsil E, Grill SP, Dutschman GE, Cheng YC - AIDS Res Ther (2009)

Bottom Line: The persistence ranking was derived from assays based on measures of single viral replication-cycle and cumulative inhibition at multiple time-points.Therefore, a better indicator of the pharmacodynamic property of an inhibitor.The persistence of anti-HIV activity assay may complement in vitro potency assays to better predict in vivo performance of nucleoside analogs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA. yccheng@yale.edu

ABSTRACT

Background: Most in vitro assays of drug potency may not adequately predict the performance in vivo. Methods to assess the persistence of antiviral activity of deoxynucleoside analogs, which require intracellular activation to the active metabolites that can persist in cells, will be important for designing dosages, combination regimens, and assessing treatment compliance. Using an HIV-IIIB/TZM-bl indicator cell culture system, we assessed the ability of an inhibitor to protect cells from infection and to delay viral rebound after removal of inhibitor from culture.

Results: The order of protection of cells from HIV-infection was 4'-Ed4T > LFD4C > DDI > D4T > 3TC > AZT > FTC > NVP. The fold-increase in EC50 to delay viral rebound was DDI < 4'-Ed4T < LFD4C < FTC < D4T < 3TC < NVP < AZT. The ranking of persistence of anti-HIV activity of the inhibitors based on the two-component assay was DDI > 4'-Ed4T > LFD4C > FTC = D4T > 3TC > NVP > AZT.

Conclusion: The persistence ranking was derived from assays based on measures of single viral replication-cycle and cumulative inhibition at multiple time-points. Therefore, a better indicator of the pharmacodynamic property of an inhibitor. The persistence of anti-HIV activity assay may complement in vitro potency assays to better predict in vivo performance of nucleoside analogs.

No MeSH data available.


Related in: MedlinePlus

Protection of cells from HIV infection after removal of inhibitor from culture. The protection of cells from HIV infection was determined using a TZM-bl indicator cell line based-single cycle replication assay described in the "Materials and Methods" section and illustrated in Figure 1A. TZM-bl cells were cultured in the presence of various concentrations inhibitor (LFD4C, FTC, DDI, or 3TC) 24 h. The cells were then washed to remove extracellular drug, and infected with HIV-1 IIIB virus at an MOI of 0.1 at 0, 24, and 48 h of drug removal. The percentage inhibition of HIV-1 replication was determined by measuring the luciferase activity. The curves represent: percent inhibition of HIV-1 replication when cells were infected at the time of drug treatment and incubated together for 24 h (black circle); percent inhibition when cells were pre-incubated with drugs for 24 h and the media changed without replacement of drug prior to infection and incubated for 24 h (black triangle); and percent inhibition when cells were pre-incubated with drugs for 24 h and then the media was changed to remove drug at 0 and 24 h without drug replacement prior to infection (black square). Results are the average of at least three independent experiments. Error bars indicate standard deviations.
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Figure 2: Protection of cells from HIV infection after removal of inhibitor from culture. The protection of cells from HIV infection was determined using a TZM-bl indicator cell line based-single cycle replication assay described in the "Materials and Methods" section and illustrated in Figure 1A. TZM-bl cells were cultured in the presence of various concentrations inhibitor (LFD4C, FTC, DDI, or 3TC) 24 h. The cells were then washed to remove extracellular drug, and infected with HIV-1 IIIB virus at an MOI of 0.1 at 0, 24, and 48 h of drug removal. The percentage inhibition of HIV-1 replication was determined by measuring the luciferase activity. The curves represent: percent inhibition of HIV-1 replication when cells were infected at the time of drug treatment and incubated together for 24 h (black circle); percent inhibition when cells were pre-incubated with drugs for 24 h and the media changed without replacement of drug prior to infection and incubated for 24 h (black triangle); and percent inhibition when cells were pre-incubated with drugs for 24 h and then the media was changed to remove drug at 0 and 24 h without drug replacement prior to infection (black square). Results are the average of at least three independent experiments. Error bars indicate standard deviations.

Mentions: The schema for the assay for protection of cells from HIV infection is illustrated in Figure 1A; the details of the experiments have been previously published [11]. In brief, TZM-bl cells were plated at 5 × 103 cells per well in a 96-well microtiter plate in 100 μl of Phenol Red Free RPMI 1640 media and allowed to adhere for 15–18 h at 37°C prior to infection or drug treatment. After adherence of the cells, the media was changed and the cells were treated with various concentrations of 3TC, FTC, LFD4C, or DDI. Each drug concentration was replicated five times and the experiment repeated on at least three different occasions. To determine the effective concentration of inhibitor that inhibits 50% of viral growth (EC50), the cells were infected with HIV-1 IIIB virus at an MOI of 0.1 at the time of drug treatment (see Figure 1A, top panel). After 24 h of infection, the relative luciferase activity was determined as described below. The EC50 was calculated as the concentration of inhibitor that produced 50% of the relative luciferase activity of the control wells with HIV-infected cells in the absence of an inhibitor. For the protection of cells from HIV infection, a batch of plates was infected with HIV-1 IIIB virus after 24 h of incubation with drug and the drug removed without replacement (see Figure 1A, middle panel). A second batch of plates was incubated after replacement of media without drug for 24 h then infected with virus after a second change of media to remove any extracellular drug (see Figure 1A, bottom panel). Thus, the second batch of plates had two-24 h media changes without drug replacement prior to HIV infection. The cells in each well were harvested after 24 h of infection and lysed using luciferase assay reagent (Promega, Madison, WI). Firefly luciferase activities were quantified using a dual-luciferase reporter assay system (Promega, Madison, WI), and a microplate luminometer (FARCyte™, Amersham Biosciences Co., Piscataway, NJ). Background luminescence was determined from uninfected cells and subtracted from all experimental wells. Protection of cells from HIV infection was measured as a function of inhibition of viral replication (% of control replication without drug), calculated by dividing the mean number of luciferase units at each concentration of a drug by the mean number from cells containing no drug. The ability of a drug to protect cells from HIV infection is illustrated on plot with percentage inhibition on the y-axis against drug concentration on the x-axis (Figure 2).


Comparative study of the persistence of anti-HIV activity of deoxynucleoside HIV reverse transcriptase inhibitors after removal from culture.

Paintsil E, Grill SP, Dutschman GE, Cheng YC - AIDS Res Ther (2009)

Protection of cells from HIV infection after removal of inhibitor from culture. The protection of cells from HIV infection was determined using a TZM-bl indicator cell line based-single cycle replication assay described in the "Materials and Methods" section and illustrated in Figure 1A. TZM-bl cells were cultured in the presence of various concentrations inhibitor (LFD4C, FTC, DDI, or 3TC) 24 h. The cells were then washed to remove extracellular drug, and infected with HIV-1 IIIB virus at an MOI of 0.1 at 0, 24, and 48 h of drug removal. The percentage inhibition of HIV-1 replication was determined by measuring the luciferase activity. The curves represent: percent inhibition of HIV-1 replication when cells were infected at the time of drug treatment and incubated together for 24 h (black circle); percent inhibition when cells were pre-incubated with drugs for 24 h and the media changed without replacement of drug prior to infection and incubated for 24 h (black triangle); and percent inhibition when cells were pre-incubated with drugs for 24 h and then the media was changed to remove drug at 0 and 24 h without drug replacement prior to infection (black square). Results are the average of at least three independent experiments. Error bars indicate standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Protection of cells from HIV infection after removal of inhibitor from culture. The protection of cells from HIV infection was determined using a TZM-bl indicator cell line based-single cycle replication assay described in the "Materials and Methods" section and illustrated in Figure 1A. TZM-bl cells were cultured in the presence of various concentrations inhibitor (LFD4C, FTC, DDI, or 3TC) 24 h. The cells were then washed to remove extracellular drug, and infected with HIV-1 IIIB virus at an MOI of 0.1 at 0, 24, and 48 h of drug removal. The percentage inhibition of HIV-1 replication was determined by measuring the luciferase activity. The curves represent: percent inhibition of HIV-1 replication when cells were infected at the time of drug treatment and incubated together for 24 h (black circle); percent inhibition when cells were pre-incubated with drugs for 24 h and the media changed without replacement of drug prior to infection and incubated for 24 h (black triangle); and percent inhibition when cells were pre-incubated with drugs for 24 h and then the media was changed to remove drug at 0 and 24 h without drug replacement prior to infection (black square). Results are the average of at least three independent experiments. Error bars indicate standard deviations.
Mentions: The schema for the assay for protection of cells from HIV infection is illustrated in Figure 1A; the details of the experiments have been previously published [11]. In brief, TZM-bl cells were plated at 5 × 103 cells per well in a 96-well microtiter plate in 100 μl of Phenol Red Free RPMI 1640 media and allowed to adhere for 15–18 h at 37°C prior to infection or drug treatment. After adherence of the cells, the media was changed and the cells were treated with various concentrations of 3TC, FTC, LFD4C, or DDI. Each drug concentration was replicated five times and the experiment repeated on at least three different occasions. To determine the effective concentration of inhibitor that inhibits 50% of viral growth (EC50), the cells were infected with HIV-1 IIIB virus at an MOI of 0.1 at the time of drug treatment (see Figure 1A, top panel). After 24 h of infection, the relative luciferase activity was determined as described below. The EC50 was calculated as the concentration of inhibitor that produced 50% of the relative luciferase activity of the control wells with HIV-infected cells in the absence of an inhibitor. For the protection of cells from HIV infection, a batch of plates was infected with HIV-1 IIIB virus after 24 h of incubation with drug and the drug removed without replacement (see Figure 1A, middle panel). A second batch of plates was incubated after replacement of media without drug for 24 h then infected with virus after a second change of media to remove any extracellular drug (see Figure 1A, bottom panel). Thus, the second batch of plates had two-24 h media changes without drug replacement prior to HIV infection. The cells in each well were harvested after 24 h of infection and lysed using luciferase assay reagent (Promega, Madison, WI). Firefly luciferase activities were quantified using a dual-luciferase reporter assay system (Promega, Madison, WI), and a microplate luminometer (FARCyte™, Amersham Biosciences Co., Piscataway, NJ). Background luminescence was determined from uninfected cells and subtracted from all experimental wells. Protection of cells from HIV infection was measured as a function of inhibition of viral replication (% of control replication without drug), calculated by dividing the mean number of luciferase units at each concentration of a drug by the mean number from cells containing no drug. The ability of a drug to protect cells from HIV infection is illustrated on plot with percentage inhibition on the y-axis against drug concentration on the x-axis (Figure 2).

Bottom Line: The persistence ranking was derived from assays based on measures of single viral replication-cycle and cumulative inhibition at multiple time-points.Therefore, a better indicator of the pharmacodynamic property of an inhibitor.The persistence of anti-HIV activity assay may complement in vitro potency assays to better predict in vivo performance of nucleoside analogs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA. yccheng@yale.edu

ABSTRACT

Background: Most in vitro assays of drug potency may not adequately predict the performance in vivo. Methods to assess the persistence of antiviral activity of deoxynucleoside analogs, which require intracellular activation to the active metabolites that can persist in cells, will be important for designing dosages, combination regimens, and assessing treatment compliance. Using an HIV-IIIB/TZM-bl indicator cell culture system, we assessed the ability of an inhibitor to protect cells from infection and to delay viral rebound after removal of inhibitor from culture.

Results: The order of protection of cells from HIV-infection was 4'-Ed4T > LFD4C > DDI > D4T > 3TC > AZT > FTC > NVP. The fold-increase in EC50 to delay viral rebound was DDI < 4'-Ed4T < LFD4C < FTC < D4T < 3TC < NVP < AZT. The ranking of persistence of anti-HIV activity of the inhibitors based on the two-component assay was DDI > 4'-Ed4T > LFD4C > FTC = D4T > 3TC > NVP > AZT.

Conclusion: The persistence ranking was derived from assays based on measures of single viral replication-cycle and cumulative inhibition at multiple time-points. Therefore, a better indicator of the pharmacodynamic property of an inhibitor. The persistence of anti-HIV activity assay may complement in vitro potency assays to better predict in vivo performance of nucleoside analogs.

No MeSH data available.


Related in: MedlinePlus