Since its first isolation in 1986,1 FIV has been identified worldwide and FIV infection continues to be a major health problem among cats, especially in countries with large populations of free-roaming cats.2–4 In cats, FIV induces an acquired immunodeficiency syndrome similar to that caused by HIV in humans,5,6 and both viruses share many immunopathogenic and genomic features.7,8 Close similarities exist between the RTs of HIV and FIV, and it has been shown that several RT-targeted antiviral compounds active against HIV are also effective in inhibiting FIV replication in vitro.6,9 However, experience with these drugs in vivo in cats is limited, and clinical use of antiviral drugs is still uncommon in veterinary medicine. To date, treatment of FIV-infected cats is largely based on supportive measures and the management of secondary problems.
In the treatment of humans with AIDS, specific targeting of HIV is important, and it is well documented that clinical outcome is improved when plasma viral burden is reduced.5,10,11 Single-agent treatment is no longer recommended for treatment of HIV-infected individuals because mutations of HIV, which are not susceptible to the antiviral agent, can develop. Highly active antiretroviral therapy of HIV-infected patients involves administration of combinations of antiviral drugs from different drug classes, such as 2 NRTIs combined with a protease inhibitor or 2 NRTIs with a non-NRTI.10 Combination treatment with drugs from different drug classes has not been assessed in FIV-infected cats, to our knowledge. This is mainly due to the fact that despite similarities between the HIV and FIV proteases, all but one of the currently available HIV protease inhibitors failed to inhibit the protease of FIV.8,12,13 Similarly, it has been shown that non-NRTIs are highly specific for HIV and are not effective against FIV.12,14 This is in contrast to NRTIs that inhibit HIV as well as other lentiviruses, including FIV.14
Of all antiviral drugs, only the NRTI zidovudine has been assessed thoroughly in cats in terms of in vitro inhibition of nonpathogenic15 and pathogenic16 FIV, inhibition of virion RT purified from in vitro–propagated nonpathogenic15 and pathogenic16 FIV, pharmacokinetics in uninfected cats,17 and clinical response of experimentally18 and naturally FIV-infected cats treated with the drug.19 Zidovudine increases the CD4:CD8 ratio and can improve clinical condition scores in FIV-infected cats with stomatitis19 and neurologic signs.20 Its use, however, can result in adverse effects, such as dose-dependent nonregenerative anemia and neutropenia in cats21,22; moreover, results of an in vitro study in cats23 and reports of humans24,25 and cats26 treated with zidovudine have indicated that mutations conferring resistance against the drug can develop. Therefore, it is important to assess the activities of other antiviral drugs and drug combinations for the treatment of FIV-infected cats.
Because a wide range of antiretroviral drugs have been assessed in humans and some of these (at least drugs from the NRTI class) might be of value in FIV-infected cats, the purpose of the study reported here was to compare 9 NRTIs regarding their cytotoxic effects in feline PBM cells and antiviral efficacy against FIV. The drugs included 3 NRTIs that had not been evaluated in feline cell lines before and 6 well-characterized NRTIs.
Materials and Methods
Test compounds—Of the 9 NRTIs assessed in the study, 3 had not been evaluated in feline cell lines previously; these were amdoxovir,a racivira and dexelvucitabinea (Appendix). The other 6 NRTIs were abacavir,b didanosine,b emtricitabine,a lamivudine,a stavudine,a and zidovudine.a Each test compound was dissolved in dimethyl sulfoxide to create a 40mM stock solution, which was stored at −20°C. This solution was then further diluted with distilled water to provide working solutions at the concentrations required for the respective experiments.
Cats—Housing and husbandry practices were in accordance with federal guidelines. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Georgia.
Isolation of PBM cells from cats—Peripheral blood mononuclear cells were harvested from blood samples collected from 3 castrated male SPF cats.c The cats were used on a rotating schedule. Six to 12 blood samples were collected from each cat (cat 1, 12 samples; cat 2, 7 samples; and cat 3, 6 samples) over a period of 16 months. The samples were collected intermittently. No more than 20 mL of blood was collected from an individual cat more frequently than once monthly.
The collection and isolation of PBM cells were performed as previously described by McCrackin Stevenson and McBroom.16 Briefly, 5 to 20 mL of blood was collected from a jugular vein of each cat into commercial vacuum tubes containing sodium heparin. Anticoagulated blood was diluted with an equal volume of PBS solution and layered over a density gradient followed by centrifugation and PBM cell aspiration. Isolated PBM cells were stimulated for 72 hours in medium (RPMI-1640 cell culture medium containing human recombinant interleukin-2 [29 U/mL],e penicillin [50 U/mL], streptomycin [50 μg/mL], l-glutamine [2mM], HEPES buffer [10mM], β2-mercaptoethanol [5 × 10−5M], sodium pyruvate [1mM], and heat-inactivated 10% fetal bovine serum) containing concanavalin Af (1 μg/mL) and incubated at 37°C in an atmosphere containing 5% CO2. Cells were then transferred to medium without concanavalin A for the remainder of the experiments.
Determination of endpoint for the cytotoxicity assay—To determine the ideal endpoint for cytotoxicity assays, a preliminary experiment was conducted that incorporated uninfected PBM cells from 1 of the 3 SPF cats used in the present study. Ten quadruplicates of concanavalin A–stimulated feline PBM cells were seeded in each well of a 96-well plate (5 × 104 cells/well). Then, 200 μL of medium without concanavalin A was added to each well. The plate was incubated at 37°C in an atmosphere containing 5% CO2. After incubation for 24 hours, 10 μL of suspension was harvested from the first set of quadruplicate wells to determine the cell count per milliliter of cell suspension. The cell counts for replicate wells were averaged to provide a mean value. This step was repeated on 9 consecutive days, and data were used to create a curve representing cell growth kinetics. These data (not shown) suggested that day 5 would be an ideal endpoint for the cytotoxicity assay because day 5 was on the linear portion of the growth curve.
Cytotoxicity assays—All cytotoxicity assays were conducted with PBM cells from only 1 of the 3 SPF cats used in the present study. For a cytotoxicity assay, concanavalin A–stimulated feline PBM cells were seeded in each well of a 96-well plate (5 × 104 cells/well), and 200 μL of medium without concanavalin A was added to each well. Each test compound was added to quadruplicate wells at serial 1:5 and 1:10 dilutions, resulting in final concentrations ranging from 0.001 to 500μM. Sterile distilled water was used as the negative control. After incubation at 37°C in an atmosphere containing 5% CO2 for 5 days, 20 μL of the tetrazolium reagent MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulphophenyl]-2H-tetrazolium)g was added to each well, and the plates were incubated under the same conditions for 4 hours. Viable cells are able to reduce the tetrazolium compound to a formazan product, which was quantified colorimetrically with a 96-well microplate readerh at a wavelength of 490 nm. Cell viability was expressed as a percentage of the negative control value. Mean cell viability was calculated from results of replicate wells. Two experiments were performed for each drug, and an overall mean cell viability was calculated.
Determination of the CCID50 for the antiviral assay—To determine the CCID50, virus stocki of a pathogenic molecular clone of FIV (FIV-pPPR)27 produced in the continuous interleukin-2–dependent feline T-cell line, 104-C1,28,j was titered in two 24-well plates by culturing quadruplicate replicas of 200 μL of 5-fold serial dilutions of virus starting with either a 1:10 dilution or 1:5 dilution (1 plate for each dilution). To each well, 2 × 105 SPF feline PBM cells and 500 μL of medium were added (day 0).k Three quadruplicate replicas of uninfected tissue culture supernatant were set up in a separate plate as a negative control. The plates were incubated at 37°C in an atmosphere containing 5% CO2. On day 3, 500 μL of fresh medium was added to each well. On day 7, 200 μL of supernatant was harvested from each well and stored at −70°C for determination of FIV p24 antigen concentration.
The supernatant samples were tested for FIV p24 antigen by use of an antigen capture ELISA as described by Joshi et al.29 All wells with absorbance readings that exceeded the mean of the absorbance readings of the negative control wells by > 2 SDs were designated positive. The Spearman-Karber equation30 was used to calculate the virus titers for the 2 plates; the mean of these 2 values was the final CCID50.
Antiviral assay—The pathogenic molecular clone FIV-pPPR was used for the antiviral assay as described by McCrackin Stevenson and McBroom.16 Concanavalin A–stimulated SPF feline PBM cells were cultured for 3 to 5 days and subsequently exposed at a density of 5 × 106 cells/mL to 1 mL of virus-positive tissue culture supernatant containing 450 × CCID50/mL (as determined by assessment of the titer of the virus stock). The cells were inoculated in 2 mL of medium for 2 to 4 hours. One 24-well plate was set up for each test compound and contained quadruplicate replicas of 5 drug dilutions (final concentrations from 0.1 to 10μM) and sterile distilled water (negative control) in 2 mL of medium. Virus-exposed PBM cells (2 × 105 cells) were then added to each well, and plates were incubated at 37°C in an atmosphere containing 5% CO2 for 7 days. Three and a half days after the end of inoculation, 1 mL of supernatant was removed from each well without disturbing the cells at the bottom, and fresh medium containing half of the initial amount of drug was added. Seven days after the end of inoculation, supernatants were harvested. This endpoint was selected in accordance with results from a previous study.16 Replication of FIV was determined by use of an FIV p24 antigen capture ELISA as described by Joshi et al.29 Production of p24 antigen was expressed as a percentage of the control value generated by FIV-infected, untreated cells in control wells. Optical density readings of the supernatant from uninfected PBM cells were subtracted neither from the OD readings of the negative control wells nor from the OD readings of the drug-containing wells. The mean OD reading was calculated for results of replicate wells. Two experiments were performed for each drug, and an overall mean value was calculated from the results.
Statistical analysis—Results are reported as mean ± SEM. Differences in toxic effects among the antiviral compounds at the concentration of 500μM and differences in antiviral efficacy among the antiviral compounds at the concentration of 10μM were assessed by use of the Kruskal-Wallis test and Dunn multiple comparisons test. A commercially available software packagel was used for the analyses. A value of P ≤ 0.05 was considered significant.
Results
Cytotoxicity of the NRTIs—All 9 NRTIs had cytotoxic effects on feline PBM cells at concentrations > 10μM; the severity of the effects increased with increasing drug concentration (Figure 1). The cytotoxic effects induced by each drug at a concentration of 500μM were compared (Figure 2). Toxic effects among the 9 drugs varied significantly (P < 0.001) at this concentration; both didanosine and amdoxovir were significantly (P < 0.05) less toxic to feline PBM cells than was abacavir.
Representative dose-response curves for the cytotoxic effects of the NRTIs emtricitabine (A) and dexelvucitabine (B) on purified PBM cells obtained from a single SPF cat. The PBM cells were seeded in each well of a 96-well plate (5 × 104 cells/well), and 200 μL of medium without concanavalin A was added to each well. Each test compound was added to quadruplicate wells at final concentrations ranging from 0.001 to 500μM. Sterile distilled water was used as the negative control. After incubation at 37°C in an atmosphere containing 5% CO2 for 5 days, 20 μL of a tetrazolium reagent was added to each well, and the plates were incubated under the same conditions for 4 hours. Viable cells were quantified colorimetrically at a wavelength of 490 nm. Cell viability was expressed as a percentage of the negative control value. Mean cell viability was calculated from results of replicate wells; 2 experiments were performed for each drug, and an overall mean ± SEM cell viability was calculated.
Citation: American Journal of Veterinary Research 75, 3; 10.2460/ajvr.75.3.273
Representative dose-response curves for the cytotoxic effects of the NRTIs emtricitabine (A) and dexelvucitabine (B) on purified PBM cells obtained from a single SPF cat. The PBM cells were seeded in each well of a 96-well plate (5 × 104 cells/well), and 200 μL of medium without concanavalin A was added to each well. Each test compound was added to quadruplicate wells at final concentrations ranging from 0.001 to 500μM. Sterile distilled water was used as the negative control. After incubation at 37°C in an atmosphere containing 5% CO2 for 5 days, 20 μL of a tetrazolium reagent was added to each well, and the plates were incubated under the same conditions for 4 hours. Viable cells were quantified colorimetrically at a wavelength of 490 nm. Cell viability was expressed as a percentage of the negative control value. Mean cell viability was calculated from results of replicate wells; 2 experiments were performed for each drug, and an overall mean ± SEM cell viability was calculated.
Citation: American Journal of Veterinary Research 75, 3; 10.2460/ajvr.75.3.273
Representative dose-response curves for the cytotoxic effects of the NRTIs emtricitabine (A) and dexelvucitabine (B) on purified PBM cells obtained from a single SPF cat. The PBM cells were seeded in each well of a 96-well plate (5 × 104 cells/well), and 200 μL of medium without concanavalin A was added to each well. Each test compound was added to quadruplicate wells at final concentrations ranging from 0.001 to 500μM. Sterile distilled water was used as the negative control. After incubation at 37°C in an atmosphere containing 5% CO2 for 5 days, 20 μL of a tetrazolium reagent was added to each well, and the plates were incubated under the same conditions for 4 hours. Viable cells were quantified colorimetrically at a wavelength of 490 nm. Cell viability was expressed as a percentage of the negative control value. Mean cell viability was calculated from results of replicate wells; 2 experiments were performed for each drug, and an overall mean ± SEM cell viability was calculated.
Citation: American Journal of Veterinary Research 75, 3; 10.2460/ajvr.75.3.273
Comparison of the cytotoxic effects of 9 NRTIs (each at a concentration of 500μM) on feline PBM cells from a single SPF cat. The PBM cells were seeded in each well of a 96-wel plate (5 × 104 cells/well), and 200 μL of medium without concanavalin A was added to each well. Each test compound was added to quadruplicate wells at a final concentration of 500μM. Sterile distilled water was used as the negative control. After incubation at 37°C in an atmosphere containing 5% CO2 for 5 days 20 μL of a tetrazolium reagent was added to each well, and the plates were incubated under the same conditions for 4 hours. Viable cells were quantified colorimetrically at a wavelength of 490 nm. Cell viability was expressed as a percentage of the negative control value. The mean ± SEM cell viability for each drug treatment was derived from 2 experiments, each of which was conducted with 4 replicates for each drug. Viability varied significantly (P < 0.001); both didanosine and amdoxovir were significantly (P < 0.05) less toxic than abacavir.
Citation: American Journal of Veterinary Research 75, 3; 10.2460/ajvr.75.3.273
Comparison of the cytotoxic effects of 9 NRTIs (each at a concentration of 500μM) on feline PBM cells from a single SPF cat. The PBM cells were seeded in each well of a 96-wel plate (5 × 104 cells/well), and 200 μL of medium without concanavalin A was added to each well. Each test compound was added to quadruplicate wells at a final concentration of 500μM. Sterile distilled water was used as the negative control. After incubation at 37°C in an atmosphere containing 5% CO2 for 5 days 20 μL of a tetrazolium reagent was added to each well, and the plates were incubated under the same conditions for 4 hours. Viable cells were quantified colorimetrically at a wavelength of 490 nm. Cell viability was expressed as a percentage of the negative control value. The mean ± SEM cell viability for each drug treatment was derived from 2 experiments, each of which was conducted with 4 replicates for each drug. Viability varied significantly (P < 0.001); both didanosine and amdoxovir were significantly (P < 0.05) less toxic than abacavir.
Citation: American Journal of Veterinary Research 75, 3; 10.2460/ajvr.75.3.273
Comparison of the cytotoxic effects of 9 NRTIs (each at a concentration of 500μM) on feline PBM cells from a single SPF cat. The PBM cells were seeded in each well of a 96-wel plate (5 × 104 cells/well), and 200 μL of medium without concanavalin A was added to each well. Each test compound was added to quadruplicate wells at a final concentration of 500μM. Sterile distilled water was used as the negative control. After incubation at 37°C in an atmosphere containing 5% CO2 for 5 days 20 μL of a tetrazolium reagent was added to each well, and the plates were incubated under the same conditions for 4 hours. Viable cells were quantified colorimetrically at a wavelength of 490 nm. Cell viability was expressed as a percentage of the negative control value. The mean ± SEM cell viability for each drug treatment was derived from 2 experiments, each of which was conducted with 4 replicates for each drug. Viability varied significantly (P < 0.001); both didanosine and amdoxovir were significantly (P < 0.05) less toxic than abacavir.
Citation: American Journal of Veterinary Research 75, 3; 10.2460/ajvr.75.3.273
Antiviral efficacy—The ability of the 9 NRTIs to inhibit FIV replication in feline PBM cells was tested at noncytotoxic concentrations ranging from 0.1 to 10μM. The quantities of FIV p24 antigen (expressed as a percentage of OD readings from FIV-infected, untreated cells in control wells) in the supernatant from PBM cells incubated with 10μM of each drug were compared (Figure 3). Results indicated that emtricitabine, didanosine, and lamivudine were the most potent inhibitors and that dexelvucitabine was the weakest inhibitor, although the findings did not differ significantly (Figure 4).
Comparison of the inhibitory effect of 9 NRTIs (each at a concentration of 10μM) on FIV replication in SPF feline PBM cells. The pathogenic molecular clone FIV-pPPR was used for the antiviral assay. Concanavalin A–stimulated feline PBM cells were cultured for 3 to 5 days and subsequently exposed at a density of 5 × 106 cells/mL to 1 mL of virus-positive tissue culture supernatant containing 450 × CCID50/mL (as determined by assessment of the titer of the virus stock). The cells were inoculated in 2 mL of medium for 2 to 4 hours. Virus-exposed PBM cells (2 × 105 cells) were then added to each well of a 24-well plate, and 10μM of 1 of the 9 drugs or sterile distilled water (negative control) was added to separate wells containing 2 mL of medium; plates were incubated at 37°C in an atmosphere containing 5% CO2 for 7 days. Three and a half days after the end of inoculation, 1 mL of supernatant was removed and refreshed with medium containing a concentration of drug or distilled water equal to that present in the well. Seven days after the end of inoculation, supernatants were harvested. Replication of FIV was determined by use of an FIV p24 antigen capture ELISA. Production of p24 antigen was expressed as a percentage of the control value generated by FIV-infected, untreated cells in control wells. Optical density readings of the supernatant from uninfected PBM cells were not subtracted from the OD readings of the negative control wells or from the OD readings of the drug-containing wells. The mean OD reading was calculated for results of replicate wells. Two experiments were performed for each drug, and an overall mean ± SEM value was calculated from the results.
Citation: American Journal of Veterinary Research 75, 3; 10.2460/ajvr.75.3.273
Comparison of the inhibitory effect of 9 NRTIs (each at a concentration of 10μM) on FIV replication in SPF feline PBM cells. The pathogenic molecular clone FIV-pPPR was used for the antiviral assay. Concanavalin A–stimulated feline PBM cells were cultured for 3 to 5 days and subsequently exposed at a density of 5 × 106 cells/mL to 1 mL of virus-positive tissue culture supernatant containing 450 × CCID50/mL (as determined by assessment of the titer of the virus stock). The cells were inoculated in 2 mL of medium for 2 to 4 hours. Virus-exposed PBM cells (2 × 105 cells) were then added to each well of a 24-well plate, and 10μM of 1 of the 9 drugs or sterile distilled water (negative control) was added to separate wells containing 2 mL of medium; plates were incubated at 37°C in an atmosphere containing 5% CO2 for 7 days. Three and a half days after the end of inoculation, 1 mL of supernatant was removed and refreshed with medium containing a concentration of drug or distilled water equal to that present in the well. Seven days after the end of inoculation, supernatants were harvested. Replication of FIV was determined by use of an FIV p24 antigen capture ELISA. Production of p24 antigen was expressed as a percentage of the control value generated by FIV-infected, untreated cells in control wells. Optical density readings of the supernatant from uninfected PBM cells were not subtracted from the OD readings of the negative control wells or from the OD readings of the drug-containing wells. The mean OD reading was calculated for results of replicate wells. Two experiments were performed for each drug, and an overall mean ± SEM value was calculated from the results.
Citation: American Journal of Veterinary Research 75, 3; 10.2460/ajvr.75.3.273
Comparison of the inhibitory effect of 9 NRTIs (each at a concentration of 10μM) on FIV replication in SPF feline PBM cells. The pathogenic molecular clone FIV-pPPR was used for the antiviral assay. Concanavalin A–stimulated feline PBM cells were cultured for 3 to 5 days and subsequently exposed at a density of 5 × 106 cells/mL to 1 mL of virus-positive tissue culture supernatant containing 450 × CCID50/mL (as determined by assessment of the titer of the virus stock). The cells were inoculated in 2 mL of medium for 2 to 4 hours. Virus-exposed PBM cells (2 × 105 cells) were then added to each well of a 24-well plate, and 10μM of 1 of the 9 drugs or sterile distilled water (negative control) was added to separate wells containing 2 mL of medium; plates were incubated at 37°C in an atmosphere containing 5% CO2 for 7 days. Three and a half days after the end of inoculation, 1 mL of supernatant was removed and refreshed with medium containing a concentration of drug or distilled water equal to that present in the well. Seven days after the end of inoculation, supernatants were harvested. Replication of FIV was determined by use of an FIV p24 antigen capture ELISA. Production of p24 antigen was expressed as a percentage of the control value generated by FIV-infected, untreated cells in control wells. Optical density readings of the supernatant from uninfected PBM cells were not subtracted from the OD readings of the negative control wells or from the OD readings of the drug-containing wells. The mean OD reading was calculated for results of replicate wells. Two experiments were performed for each drug, and an overall mean ± SEM value was calculated from the results.
Citation: American Journal of Veterinary Research 75, 3; 10.2460/ajvr.75.3.273
Representative dose-response curves for effects of the NRTIs emtricitabine (A) and dexelvucitabine (B) on FIV replication in SPF feline PBM cells. The pathogenic molecular clone FIV-pPPR was used for the antiviral assay. Concanavalin A–stimulated feline PBM cells were cultured for 3 to 5 days and subsequently exposed at a density of 5 × 106 cells/mL to 1 mL of virus-positive tissue culture supernatant containing 450 × CCID50/mL. The cells were inoculated in 2 mL of medium for 2 to 4 hours. Virus-exposed PBM cells (2 × 105 cells) were then added to each well of a 24-well plate, and 5 dilutions (final concentrations from 0.1 to 10μM) of 1 of the 2 drugs or sterile distilled water (negative control) were added to separate wells (quadruplicate replicates) containing 2 mL of medium; plates were incubated at 37°C in an atmosphere containing 5% CO2 for 7 days. Three and a half days after the end of inoculation, 1 mL of supernatant was removed and refreshed with medium containing a concentration of drug or distilled water equal to that present in the well. Seven days after the end of inoculation, supernatants were harvested. Replication of FIV was determined by use of an FIV p24 antigen capture ELISA. Production of p24 antigen was expressed as a percentage of the control value generated by FIV-infected, untreated cells in control wells. Optical density readings of the supernatant from uninfected PBM cells were not subtracted from the OD readings of the negative control wells or from the OD readings of the drug-containing wells. The mean OD reading was calculated for results of replicate wells. Two experiments were performed for each drug, and an overall mean ± SEM value was calculated.
Citation: American Journal of Veterinary Research 75, 3; 10.2460/ajvr.75.3.273
Representative dose-response curves for effects of the NRTIs emtricitabine (A) and dexelvucitabine (B) on FIV replication in SPF feline PBM cells. The pathogenic molecular clone FIV-pPPR was used for the antiviral assay. Concanavalin A–stimulated feline PBM cells were cultured for 3 to 5 days and subsequently exposed at a density of 5 × 106 cells/mL to 1 mL of virus-positive tissue culture supernatant containing 450 × CCID50/mL. The cells were inoculated in 2 mL of medium for 2 to 4 hours. Virus-exposed PBM cells (2 × 105 cells) were then added to each well of a 24-well plate, and 5 dilutions (final concentrations from 0.1 to 10μM) of 1 of the 2 drugs or sterile distilled water (negative control) were added to separate wells (quadruplicate replicates) containing 2 mL of medium; plates were incubated at 37°C in an atmosphere containing 5% CO2 for 7 days. Three and a half days after the end of inoculation, 1 mL of supernatant was removed and refreshed with medium containing a concentration of drug or distilled water equal to that present in the well. Seven days after the end of inoculation, supernatants were harvested. Replication of FIV was determined by use of an FIV p24 antigen capture ELISA. Production of p24 antigen was expressed as a percentage of the control value generated by FIV-infected, untreated cells in control wells. Optical density readings of the supernatant from uninfected PBM cells were not subtracted from the OD readings of the negative control wells or from the OD readings of the drug-containing wells. The mean OD reading was calculated for results of replicate wells. Two experiments were performed for each drug, and an overall mean ± SEM value was calculated.
Citation: American Journal of Veterinary Research 75, 3; 10.2460/ajvr.75.3.273
Representative dose-response curves for effects of the NRTIs emtricitabine (A) and dexelvucitabine (B) on FIV replication in SPF feline PBM cells. The pathogenic molecular clone FIV-pPPR was used for the antiviral assay. Concanavalin A–stimulated feline PBM cells were cultured for 3 to 5 days and subsequently exposed at a density of 5 × 106 cells/mL to 1 mL of virus-positive tissue culture supernatant containing 450 × CCID50/mL. The cells were inoculated in 2 mL of medium for 2 to 4 hours. Virus-exposed PBM cells (2 × 105 cells) were then added to each well of a 24-well plate, and 5 dilutions (final concentrations from 0.1 to 10μM) of 1 of the 2 drugs or sterile distilled water (negative control) were added to separate wells (quadruplicate replicates) containing 2 mL of medium; plates were incubated at 37°C in an atmosphere containing 5% CO2 for 7 days. Three and a half days after the end of inoculation, 1 mL of supernatant was removed and refreshed with medium containing a concentration of drug or distilled water equal to that present in the well. Seven days after the end of inoculation, supernatants were harvested. Replication of FIV was determined by use of an FIV p24 antigen capture ELISA. Production of p24 antigen was expressed as a percentage of the control value generated by FIV-infected, untreated cells in control wells. Optical density readings of the supernatant from uninfected PBM cells were not subtracted from the OD readings of the negative control wells or from the OD readings of the drug-containing wells. The mean OD reading was calculated for results of replicate wells. Two experiments were performed for each drug, and an overall mean ± SEM value was calculated.
Citation: American Journal of Veterinary Research 75, 3; 10.2460/ajvr.75.3.273
Discussion
The main aim of the study reported here was to evaluate 3 NRTIs (amdoxovir, dexelvucitabine, and racivir) in terms of their cytotoxic effects on primary feline PBM cells and efficacy against a pathogenic molecular clone of FIV, with the goal of identifying potential novel treatment options for cats naturally infected with FIV. To this end, the efficacies of these NRTIs were compared with the efficacies of other NRTIs that are either currently used in the treatment of FIV-infected cats in the field (zidovudine) or potential therapeutic options from the NRTI class for which previously published data existed (ie, abacavir, didanosine, emtricitabine, lamivudine, and stavudine). The results indicated that although amdoxovir, dexelvucitabine, and racivir appeared to have acceptable cytotoxicity profiles in feline PBM cells, compared with those of other NRTIs used for the treatment of HIV infection in humans, their efficacies were less (albeit not significantly) than those of didanosine, emtricitabine, lamivudine, stavudine, and zidovudine. On the basis of the data obtained in the present study, amdoxovir, dexelvucitabine, and racivir appear to be treatment options for future studies investigating their potential use in FIV-infected cats. However, regarding the reduction of the viral burden of FIV-infected cats, there was no evidence to suggest that amdoxovir, dexelvucitabine, or racivir is superior to existing NRTIs.
The cytotoxic effects of some of the drugs investigated in the present study, namely lamivudine,12,21,31 zidovudine,12,21,31–33 abacavir,12 didanosine,9,31 and stavudine,31,34 have been previously evaluated in feline cell lines. In those previous studies, the drugs had cytotoxic effects comparable to those detected in the present study; all those drugs were fairly nontoxic in cell culture at clinically relevant plasma concentrations with noticeable cytotoxic effects only at doses > 10μM. Similar results for zidovudine and lamivudine in feline PBM cells were observed by Arai et al.21 Abacavir was the most toxic drug in the present study and was significantly more toxic than didanosine and amdoxovir. Also in a study of the effects of abacavir, zidovudine, and lamivudine in CRFK cells by Bisset et al,12 abacavir was the most toxic NRTI. The concentration of drug required to inhibit PBM cell proliferation by 50% was 22.9μM for abacavir, compared with 216.8μM for zidovudine and 170.5μM for lamivudine.12 In a study by Smyth et al31 that assessed cytotoxic effects of several compounds (including didanosine, lamivudine, stavudine, and zidovudine) in feline lymphocytes, didanosine had the lowest toxicity, which also corresponds to the findings in the present study.
In the present study, dose-response curves of each drug revealed that cytotoxic effects were only observed at concentrations > 10μM. At concentrations of 100μM, the investigated drugs had only a mild to moderate toxic effect on cell viability (Figure 1). However, a plasma concentration of zidovudine of 100μM has been associated with acute, transient hemolysis after a single IV infusion of 25 mg/kg in cats; thus, this dose and plasma concentration are too high for clinical use.17 Cytotoxicity of each drug was tested at concentrations up to 500μM in the present study. This is a very high concentration, which far exceeds the circulating concentrations that will be attained in cats when drugs are given at dosages that are typically administered to cats (as demonstrated for zidovudine,22 which is usually given at a dosage of 5 to 10 mg/kg, PO or SC, q 12 h,20 and results in a serum concentration22 of 20 to 30μM). In addition, at high doses of drug, the concentration of dimethyl sulfoxide, the solvent for all NRTIs in the present and previous studies,12,31 was at its highest, and dimethyl sulfoxide is known to cause mild cytotoxic changes in feline PBM cells at similar concentrations.31 Even at these high concentrations of drug (500μM) and dimethyl sulfoxide, cell viability was not completely suppressed by any of the test compounds in the present study.
Prior to the present study, the NRTIs amdoxovir, dexelvucitabine, and racivir had not been assessed for their cytotoxic effects in feline cells, to our knowledge. The study results indicated that cytotoxicity of these drugs did not differ significantly from that of the other test compounds, except for amdoxovir, which was significantly less toxic than abacavir. Low cytotoxicity in vitro, however, does not necessarily exclude toxicity in vivo. For example, didanosine is a widely used antiretroviral drug with low cytotoxicity against human cells in culture (and in feline PBM cells as demonstrated in the present study), yet it can cause acute pancreatitis and peripheral neuropathy when used at higher doses in HIV-infected patients.35,36 It has also been shown that didanosine treatment can cause sensory neuropathy (as detected by sophisticated testing methods) in experimentally FIV-infected cats,5 although the clinical relevance of this finding in cats naturally infected with FIV is not clear. Toxic effects on mitochondria in certain tissues have been associated with many NRTIs, and this mechanism of mitochondrial changes appears to be involved in the development of NRTI-related adverse effects, although other pathophysiologic mechanisms are likely to contribute as well.5,37 Amdoxovir also has little cytotoxicity against human cell lines, which corresponds with the finding for this drug in feline PBM cells in the present study.
From a pharmacokinetic study22 of zidovudine in cats, it is known that when the drug is administered at routinely used dosages of 5 to 10 mg/kg every 12 hours, the highest serum concentration attained is 20 to 30μM. Administration of zidovudine at a concentration of 30μM does not induce noteworthy cytotoxic changes in feline lymphocytes,32 as confirmed by the results of the present study in feline PBM cells. The other drugs were not significantly more toxic than zidovudine. Therefore, as far as cytotoxic properties are concerned, it can be assumed that all compounds evaluated in the present study could be used in vivo at dosages comparable to that for zidovudine, although pharmacological data from cats are not available for most of the drugs.
Human and feline PBM cells are widely used for cell culture studies involving HIV or FIV10,16,21,32,38 because these cell populations contain CD4-positive lymphocytes, which are the primary target of these lentiviruses. Hence, feline PBM cells were used in the present study and FIV-pPPR, a pathogenic molecular clone of FIV, was used for the infection of those cells. It has been shown that in PBM cells, FIV-pPPR behaves similarly to FIV-Maxam, a natural FIV isolate, and that results are therefore applicable to natural FIV infection.16 However, a first-pass virus derived from a molecular clone is a more homogenous viral population, compared with a natural isolate. McCrackin Stevenson and McBroom16 showed that FIV-Maxam was more susceptible to lamivudine, compared with findings for FIV-pPPR, and concluded that results of studies of the susceptibility of FIV-pPPR to NRTIs might overestimate the resistance of FIV populations found in naturally infected cats to these drugs. This might in part explain why susceptibility of the virus to the evaluated NRTIs in the present study was lower than previously described.
Because the dose-response curves indicated that there were no observable cytotoxic effects on feline PBM cells for any of the 9 drugs at a concentration of 10μM, concentration that is commonly achieved in plasma in cats administered zidovudine at the recommended dosage20,22 of 5 to 10 mg/kg, PO or SC, every 12 hours, the 10μM concentration was set as the highest dose to be investigated in the part of the present study designed to assess the antiviral efficacy of the test compounds. All drugs induced a concentration-dependent reduction of FIV replication; however, none of the drugs achieved 50% reduction of virus replication at the highest concentration (10μM) investigated. No significant difference in antiviral efficacy among the test compounds was detected; therefore, all drugs can be considered comparable in their in vitro antiviral efficacy against FIV. Of the drugs investigated in the present study, lamivudine,12,16,21,31 zidovudine,9,12,16,21,31–33,39 abacavir,12 didanosine,9,23,31 stavudine,9,31,34 and emtricitabine16,23 have been assessed previously for their anti-FIV efficacy in different feline cell lines. To our knowledge, the results of the present study have indicated the anti-FIV efficacy of amdoxovir, dexelvucitabine, and racivir for the first time.
Among the previous studies of lamivudine, zidovudine, abacavir, didanosine, stavudine, and emtricitabine, antiviral efficacy against FIV was demonstrated despite the use of different cell culture systems. Vahlenkamp et al33 detected an 80-fold difference in the antiviral efficacy of zidovudine when the drug was used in different cell lines (CRFK cells vs thymocytes), and van der Meer et al38 found a 6-fold difference in the inhibitory potency of zidovudine in thymocytes versus a dendritical cell-thymocyte coculture system. In another study,32 a difference in the EC50 (ie, the concentration of drug required to inhibit FIV p24 expression by 50%) for zidovudine between peripheral blood leukocytes and CRFK cell cultures was observed. Thus, the cell culture systems used markedly influence the EC50 values achieved. When results of different studies are compared, the cell culture system used has to be taken into consideration, and a comparison of a newly investigated drug with drugs of known in vitro efficacy (eg, zidovudine) is more useful than just comparison of EC50 values.
The cell system used in the present study involved primary feline PBM cells. Results of the previous studies that compared PBM cells with other feline cell lines, such as CRFK cells, generally indicated that the test compounds had greater inhibitory potency in PBM cells, compared with findings in CRFK cells. The fact that none of the compounds evaluated in the present study achieved a 50% reduction in virus replication at a concentration of 10μM was surprising because in other studies,9,12,16,33,34 much lower concentrations were required to induce 50% virus inhibition. In the present study, OD readings generated by supernatants of uninfected PBM cells were not determined. These background OD readings were therefore not subtracted from the readings of the plate wells containing infected cells. This might have led to an underestimation of the percentage reduction in p24 antigen concentration achieved in plate wells treated with the test compounds and might explain, at least in part, why reduction of viral replication by 50% was not achieved. Differences in FIV strains might also partly explain this finding. However, McCrackin Stevenson and McBroom16 used both the same cell system and virus and found EC50 values for zidovudine, lamivudine, and emtricitabine that were much lower than 10μM. Gobert et al9 made a similar observation; the EC50 values for zidovudine against 2 FIV strains detected in a previous study15 in their laboratory were higher than the values determined in the later study.9 They concluded that these differences might be related to variations in the batches of fetal bovine serum used in the experiments. However, independent of the system used, the outcome of the present study was that the antiviral efficacies of all drugs investigated were comparable.
The fact that reduction of viral load by 50% was not attained in the present study does not preclude the clinical usefulness of the investigated compounds. In a study by Arai et al,21 combination treatment of zidovudine and lamivudine administered to chronically FIV-infected cats at a high dosage of 20 mg of each drug/kg, PO, every 12 hours, did not result in a significant decrease in FIV load. However, it is well known that in cats that are naturally infected with FIV, zidovudine administration at much lower dosages results in beneficial effects, such as improvement of stomatitis and clinical condition scores,19,22,40 reduction in severity of neurologic signs,20 and improvement of CD4:CD8 ratios.19,22 Ideally, a drug that is considered for in vivo testing should be effective and have very low toxicity. However, the limiting factor as to whether an NRTI other than zidovudine should be considered for in vivo testing might be the drug's associated cytotoxicity rather than its ability to maximally suppress viral replication.
A limitation of the present study was that EC50 values could not be reported for the test compounds because the highest drug dose investigated did not achieve a 50% reduction of virus replication. However, comparison of the test compounds at the highest dose investigated was nevertheless considered useful because it allowed comparison of newly investigated drugs with drugs that had been previously tested in feline cell cultures.
In the present study, examination of dose-response curves for cytotoxic effects and antiviral efficacy of emtricitabine and dexelvucitabine revealed that the cell viability (as a percentage of the negative control value) at the lower drug concentrations was just > 100%. This finding was likely attributable to experimental variabilities, which might have led to slightly higher OD readings in individual plate wells of the assay. Similarly, in the antiviral assay, variabilities among individual plate wells might have contributed to the calculated FIV p24 antigen concentrations being slightly > 100% at low dexelvucitabine concentrations.
Zidovudine treatment in cats has well-known adverse effects, such as development of nonregenerative anemia and neutropenia,19,21,22 which can necessitate cessation or interruption of treatment. Drug-resistant viral mutants have been detected in HIV-infected patients treated with zidovudine.23–25 Mutations leading to drug resistance have also been reported for FIV in vitro studies15,16,23 and in naturally infected cats treated with zidovudine for ≥ 5 years.26 Therefore, the in vivo investigation of other NRTIs that have demonstrated efficacy against FIV in vitro similar to that of known compounds can result in identification of useful antiviral drugs, which might provide veterinarians with an alternative treatment option for FIV-infected cats.
Although it is difficult to make recommendations about clinical treatment only on the basis of in vitro data, the findings of the present study have suggested that further investigation of didanosine in the treatment of cats naturally infected with FIV is warranted. In a study31 assessing cytotoxic effects of 18 antiviral agents on feline lymphocytes, didanosine had the least toxicity, corresponding to the findings in the present study. In addition, the only NRTI tested in the present study that had greater in vitro efficacy than didanosine was emtricitabine. The combined profile of low cytotoxicity and relative efficacy, compared with characteristics of other NRTIs used in the assays performed in the present study, suggests that didanosine might be an interesting candidate drug for further in vivo testing either as a sole agent or in combination with zidovudine. In fact, monotherapy with didanosine (33 mg/kg, PO, q 24 h from 6 to 12 weeks after infection) in neonatal kittens infected with FIV resulted in improvements in multiple variables, compared with findings in untreated kittens, including reduction in plasma viral load, significant improvement in the animals’ neurobehavioral performance, and attenuation of neuroinflammation.41 There is also support for treatment of HIV-infected humans with a combination of zidovudine and didanosine, which resulted in an overall reduction in mortality rate of 32%, compared with results following zidovudine monotherapy.42 Logical next candidates for in vivo testing of potential novel treatments for FIV-infected cats would include the 3 newly evaluated NRTIs, amdoxovir, dexelvucitabine, and racivir.
ABBREVIATIONS
| CCID50 | Viral dose required to infect 50% of cells in culture |
| CRFK | Crandell-Rees feline kidney |
| EC50 | Concentration of a compound at which 50% of its maximal effect is observed |
| NRTI | Nucleoside reverse transcriptase inhibitor |
| OD | Optical density |
| PBM | Peripheral blood mononuclear |
| RT | Reverse transcriptase |
| SPF | Specific pathogen–free |
Synthesized in Dr. R. F. Schinazi's laboratory, Emory University, Atlanta, Ga.
Obtained through the NIH AIDS Reagent Program, Division of AIDS, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md.
Liberty Research Inc, Waverly, NY.
Ficoll-Hypaque, Pharmacia, Uppsala, Sweden.
Provided by Dr. Niels C. Pedersen, Center for Companion Animal Health, School of Veterinary Medicine, University of California-Davis, Davis, Calif.
Concanavalin A, Sigma-Aldrich, St Louis, Mo.
CellTiter 96 AQueous One Solution cell proliferation assay, Promega, Madison, Wis.
VMax, kinetic microplate reader software, SoftMax, Molecular Devices, Sunnyvale, Calif.
Provided by Dr. John H. Elder, Department of Molecular Biology, The Scripps Research Institute, La Jolla, Calif.
Generated by Dr. C. Grant, Custom Monoclonals, West Sacramento, Calif.
Modification of a protocol provided by Dr. Lawrence E. Mathes, Department of Veterinary Biosciences, The Center for Retrovirus Research, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio.
GraphPad Prism, version 5.00 for Windows, GraphPad Software, San Diego, Calif.
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Appendix
Nucleoside RT inhibitors evaluated for antiviral efficacy against FIV in feline PBM cells.
| NRTI | Chemical name |
|---|---|
| Abacavir | {(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]cyclopent-2-enyl}methanol |
| Amdoxovir | [(2R,4R)-4-(2,6-diaminopurin-9-yl)-1,3-dioxolan-2-yl]methanol |
| Dexelvucitabine | β-d-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine |
| Didanosine | 2′,3′-dideoxyinosine |
| Emtricitabine | (−)-2′,3′-deoxy-5-fluoro-3′-thiacytidine |
| Lamivudine | (−)-2′,3′-dideoxy-3′-thiacytidine |
| Racivir | (±)-P-2′,3′-dideoxy-5-fluoro-3′-thiacytidine |
| Stavudine | 2′,3′-didehydo-2′,3′-dideoxythymidine |
| Zidovudine | 3′-azido-3′-deoxythymidine |
Amdoxovir, dexelvucitanine, and racivir had not been previously assessed in feline cell lines, to the authors’ knowledge. Abacavir and didanosine were obtained through the NIH AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Amdoxovir, dexelvucitabine, emtricitabine, lamivudine, racivir, stavudine, and zidovudine were synthesized in the laboratory of one of the authors (RFS).
