a Department of Medical Microbiology and Retroscreen Virology, St Bartholomew's and The Royal London School of Medicine and Dentistry, 64 Turner Street, London, UK; b Departmento de Virologia, Universidade Federal do Rio de Janeiro, 21 941-590 Rio de Janeiro, Brasil; c The Queen Elizabeth Hospital, Department of Medicine, Liver Research Laboratories, Edgbaston, Birmingham, UK
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Abstract |
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Introduction |
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Certain bile salt derivatives have surfactant activity with low cellular toxicity. Within the human intestinal lumen, bile salts can reach a concentration of 350 mg/L without any harmful effect on the individual12 and, in patients suffering from biliary obstruction, blood bile salt concentration can increase 100-fold without serious toxic effects.13 Furthermore, the steroidal ring of bile salts can be modified chemically to yield derivatives with low, moderate or high surfactant activity.
In the present study, four bile salt derivatives with high surfactant activities were examined for their cellular toxicity and then compared with sodium deoxycholate and nonoxynol-9 for anti-HIV-1 virucidal potency.
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Materials and methods |
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Materials.
L-Tyrosine methyl ester hydrochloride (crystalline), chenodeoxycholic acid (99% pure as judged by TLC and GLC), cholylglycine (99% pure as judged by TLC), N,N-dicyclohexylcarbodiimide, N,N' dimethylformamide, triethylamine, 1-hydroxybenzotriazole and Lipidex 5000 were purchased from Sigma Chemical Co., Poole, UK. Chloramine-T was supplied by Hopkin and Williams, Chadwell Heath, UK. Amberlite XAD-2, silica gel 60 F254 TLC plates (Merck 5554) 20 x 20 cm, ethyl acetate (Analar), chloroform (Analar), glacial acetic acid (99% pure) and potassium iodide were obtained from BDH Chemicals Ltd, Poole, UK. Methanol (99% pure) and ethanol (99% pure) were purchased from Fisons Scientific Apparatus, Loughborough, UK. Sep-Pak C18 cartridges were purchased from Waters Associates Ltd, Hertford, UK.
Synthetic methods.
Deoxycholyltyrosine (DCT), chenodeoxycholyltyrosine and cholylglycyltyrosine were synthesized following procedures of Spenney et al.14 (with a slight modification), Tserng et al.15 and Mills et al.16 The di-iodinated derivatives of these compounds were then synthesized. Tyrosine-conjugated bile salt (0.5 mmol) in 5 mL phosphate buffer was added to 1.0 mmol potassium iodide followed by the addition of 0.5 mL of chloramine-T solution (10 mg/mL in 10 mM phosphate buffer pH 7.4) with incubation for 30 min. The rest of the non-radioactive iodination procedure including Amberlite XAD-2 purification of the di-iodinated compound was as described elsewhere.17 Further purification was by silica gel PLC employing ethyl acetate/methanol/acetic acid (70:20:10 by volume). The di-iodo derivatives of deoxycholyltyrosine, chenodeoxycholyltyrosine or cholylglycyltyrosine were isolated from the PLC plates by extraction with methanol and then dried by evaporation in a rotary evaporator at 40°C.
The synthesized compounds, di-iodo-deoxycholyltyrosine (DIDCT), di-iodo-chenodeoxycholyltyrosine (DICDCT), di-iodo-cholylglycyltyrosine (DICGT) and DCT (Figure 1), and sodium deoxycholate as a positive control, were dissolved separately in RPMI 1640 (Sigma), filter sterilized using Millipore 0.22 µm filters, divided into aliquots and stored at 20°C. Nonoxynol-9 was diluted to 1% (v/v) in RPMI 1640 and filter sterilized using a 0.22 µm filter.
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H9 and C8166 cell lines were kindly provided by Dr Harvey Holmes through the Medical Research Council's AIDS Directed Programme, UK. Three different laboratory-adapted strains of HIV-1 were used to evaluate the virucidal activity of the compounds: RF, with an infectivity titre of 106 TCID50/mL for H9 cells; IIIB, with an infectivity titre of 105.4 TCID50/mL for H9 cells; and MN, with an infectivity titre of 107 TCID50/mL for C8166 cells.
Evaluation of cellular toxicity
H9 and C8166 cells were prepared at a density of 2 x 105/mL in growth medium [RPMI 1640 medium with 2 mM l-glutamine, 50 IU/mL penicillin, 50 mg/L streptomycin, 25 mM HEPES buffer and 10% fetal calf serum (Sigma, Poole, UK)]. A volume of 180 µL of cell suspension was dispensed into wells of a flat-bottomed 96-well plate. Two-fold dilutions of each virucidal compound were made in growth medium and then 20 µL of each dilution was added to the first six wells and ten-fold dilutions were made along the plate. The last row of the plate was left as a drug-free control for cell viability.18
Plates were incubated at 37°C in 5% CO2 and cell viability was checked using the Trypan Blue exclusion method after 24 h and 3, 5 and 7 days (using two columns of the plate at each time point). The percentage of viable cells in each well was calculated and the mean for each concentration at each time point was calculated and compared with cell-free drug controls.
Virucidal test
Virus/virucidal mix (200 µL of high-titre virus and 200 µL of each test compound) and virus/growth medium mix (200 µL of high-titre virus and 200 µL of growth medium) were prepared and incubated for 15 min at 37°C in 5% CO2. For 96-well tissue culture plates, 180 µL of H9 (when using RF and IIIB strains) or C8166 (when using the MN strain) cells at a density of 2 x 105 cells/mL were added to each well. Twenty microlitres of virus/virucidal mix was added to the first six wells of the first row and virus/growth medium mix (used as positive control for virus-induced syncytium formation) added to the next six wells of the first row. Ten-fold dilutions were made starting from the first row downwards and keeping the last row as cell control. On days 37 after infection, all wells were examined under the microscope and scored for the presence of virus-induced syncytia18 according to the following scoring system: 3, >60 syncytia; 2, between ten and 60 syncytia; 1, between two and ten syncytia, 0.5, one or two syncytia; 0, no syncytia observed in the entire field. The wells containing virus and drug were scored and compared with the wells containing virus but no drug.
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Results |
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Discussion |
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In contrast to the bile salt derivatives, nonoxynol-9 did damage cells, and so it might increase the risk of HIV infection rather than preventing it. Its use against the sexual transmission of HIV is thus unacceptable.
Bile salts in the unconjugated form are relatively potent physiological detergents.20 Side-chain conjugation with amino acid residues, which increases the hydrophilicity of a bile acid molecule, is associated with a predictable decrease in detergent potency.21 Iodine, which tends to increase the electron density of the side chain, also decreases detergent properties. In the present study we found that, of the tyrosine-conjugated bile salt analogues, DCT, which has the lowest critical micellar concentration,22 was the most effective compound against HIV-1, suggesting that it was the most surface active of the tyrosine bile acid conjugates tested (Figure 1). This anti-HIV-1 effect of DCT could be explained, in part, by a preferential uptake of DCT by surface receptors of the HIV-1 envelope but not by viable cells. This selective uptake would then concentrate DCT (a 7
,12
-dihydroxy bile salt analogue) in the phospholipid-enriched bilayer of HIV lipid membranes. The reasoning is consistent with the relatively low hydrophilicity of DCT in comparison with the 3
,7
,12
trihydroxy or the 3
,7
-dihdroxy tyrosine bile acid analogues (unpublished data).
During viral budding, HIV incorporates host-derived cellular components such as the HLA class I and II molecules that are important for in vitro HIV infectivity.23,24 The difference between the effect of bile salt derivatives on the viral envelope and on the cellular plasma membrane might result from the different proportions of such host-derived molecules in the viral envelope and the cellular plasma membrane. This needs to be investigated further.
Bile salts with high surfactant activity may be useful clinically as virucides against HIV-1. Their ability to inactivate relatively high titres of HIV-1 might enable them to be used as biological disinfectants as well as virucidal compounds.
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Acknowledgments |
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Notes |
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References |
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Received 20 July 1999; returned 13 October 1999; revised 25 November 1999; accepted 22 December 1999