a PHLS Antiviral Susceptibility Reference Unit, Division of Immunity and Infection, University of Birmingham; b Department of Sexual Medicine, Birmingham Heartlands Hospital, Birmingham; c Department of Pharmacology & Therapeutics, University of Liverpool, UK
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Abstract |
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Introduction |
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The central nervous system and genital tract represent two such sanctuary sites. Although the antiviral responses are usually concordant in the blood and genital tract, discordant viral load suppression in these compartments and differences in the rate of emergence of drug resistance-associated viral mutations have been documented.2 Differential penetration of antiviral drugs into semen may be one factor influencing viral suppression and evolution in the male genital tract. In this study we have compared the concentrations of three protease inhibitors in the semen and blood of HIV-1-infected men.
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Materials and methods |
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HIV-1-infected men taking indinavir or a combination of ritonavir plus saquinavir, in combination with one or two nucleoside or non-nucleoside reverse transcriptase inhibitors, were studied. All had been taking their medication for at least 4 weeks before samples were obtained. All provided written informed consent and the study was approved by the local ethics committee. All patients were tested for evidence of other sexually transmitted infections at the time of semen production.
Viral load determination
Viral loads (VL) in blood plasma (BP) and semen plasma (SP) were determined by nucleic acid sequence-based amplification (NASBA; Nuclisens, Organon Teknica, Durham, NC, USA) as described previously,3 with a lower limit of detection of 400 copies/mL for BP and 800 copies/mL for SP.
Sample collection times were carefully documented in relation to the time of drug ingestion. When samples of semen and blood were obtained within 1 h of each other and with no intervening drug administration, a SP:BP concentration ratio was calculated. The sample pairs were divided into groups according to their time of collection (02, 26 or 68 h post-drug ingestion for indinavir analysis and 03, 39 or 912 h for ritonavir and saquinavir calculations). These intervals enabled the initial groups to include peak BP concentrations during the absorption phase and the final groups to capture the lowest BP concentrations).
Drug concentration analysis
Drug concentrations were analysed by sensitive highperformance liquid chromatography assays as described previously.4,5 The lower limits of quantification (LLQ) of the saquinavir, ritonavir and indinavir assays were 20, 25 and 20 ng/mL, respectively. Samples that fell below the LLQ were assigned a value equal to the LLQ for statistical analysis.
Drug concentrations were compared with EC95 values for the respective drugs (EC95 = concentration of drug required to inhibit viral replication by 95% in an in vitro cell culture assay). In this study the reported EC95 values6 were adjusted for the plasma protein binding of the drugs as described by Molla et al.7 This produced EC95 values of 42.9 ng/mL (68 nM) for indinavir, 515 ng/mL (2100 nM) for ritonavir and 278 ng/mL (416 nM) for saquinavir.
Differences in BP and SP drug concentrations in sample pairs and differences in SP:BP ratios generated at different time points within the same individuals were compared using the Wilcoxon signed rank test.
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Results |
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Indinavir
Median BP indinavir concentrations at 02, 26 and 68 h were 4473 ng/mL (range 13806020 ng/mL, n = 7), 937 ng/mL (range 3603070 ng/mL, n = 7) and 360 ng/mL (range 2012090 ng/mL, n = 7), respectively (Figure 1a). The paired SP indinavir concentrations at 02, 26 and 68 h were 2527 ng/mL (range 3193775 ng/mL), 1259 ng/mL (range 2163567 ng/mL) and 558 ng/mL (range 272 3178 ng/mL), respectively. Only during the 02 h interval were SP and BP concentrations significantly different from each other (P < 0.01). All BP and SP indinavir concentrations exceeded the EC95 for indinavir of 42 ng/mL.
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Median BP ritonavir concentrations at 03, 39 and 912 h were 16 310 ng/mL (range 349333 610 ng/mL, n = 11), 9815 ng/mL (range 239114 000 ng/mL, n = 4) and 6020 ng/mL (range 89412 510 ng/mL, n = 9), respectively (Figure 1b). The paired SP ritonavir concentrations were 319 ng/mL (range 60790 ng/mL), 406 ng/mL (range <25 790 ng/mL) and 295 ng/mL (range <25870 ng/mL), respectively. SP concentrations were lower than BP concentrations in all time intervals post-drug ingestion (P < 0.003, P = 0.06, P < 0.007, respectively). All but one patient had detectable SP ritonavir concentrations at all time periods; however, the median SP ritonavir concentrations were below the EC95 of ritonavir (515 ng/mL) throughout the dosing interval. In contrast, BP ritonavir concentrations were above the EC95 at all time points.
Median SP:BP ritonavir ratios at 03, 39 and 912 h were 0.02 (range 0.010.05), <0.04 (range <0.010.06) and <0.04 (range <0.020.11) (data not shown).
Saquinavir
Median BP saquinavir concentrations at 03, 39 and 912 h were 1904 ng/mL (range 7426260 ng/mL, n = 7), 1547 ng/mL (range 10813236 ng/mL, n = 4) and 661 ng/mL (range 5021376 ng/mL, n = 7), respectively (Figure 1c). The paired SP saquinavir concentrations were 40 ng/mL (range <2084 ng/mL), <20 ng/mL (<2083 ng/mL) and <20 ng/mL (range <2034 ng/mL), respectively. SP saquinavir concentrations were below BP saquinavir concentrations at all time intervals (P < 0.01, P = 0.07, P < 0.02, respectively). All BP saquinavir concentrations were above the EC95 for saquinavir (278 ng/mL) at all time points post-drug ingestion. In contrast, 13/18 SP saquinavir concentration were below the limit of detection of the assay and, of the seven detectable concentrations, all were below the EC95 of saquinavir.
SP:BP saquinavir ratios at 03, 39 and 912 h were <0.02 (range <0.0030.11), <0.04 (range 0.010.07) and <0.04 (range <0.02<0.06), respectively (data not shown).
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Discussion |
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When trying to establish reasons for these differences, the physicochemical properties of this class of drugs must be considered. All three drugs are of similar size, are lipid soluble and are weak bases, so that ion trapping within the prostate gland (pH 6.6) may contribute to lower rates of elimination,8 although the degree of active drug transport into and out of the genital tract is unknown. The most plausible reason for the differences in semen penetration is the different protein binding of the drugs. Indinavir is 60% bound to plasma proteins but ritonavir and saquinavir are each >98% protein bound. This hypothesis is supported by the results with amprenavir, which is c. 90% protein bound and achieves semen concentrations of c. 20% of plasma concentrations.8
Since all the patients enrolled in this study were receiving nucleoside analogues in addition to protease inhibitors (PIs), it is difficult to draw conclusions regarding the efficacy of different PI combinations on suppression of virus in semen. Our observations support previous findings that indinavir concentrations in semen exceed proteincorrected EC95 values9 and thus would be expected to be effective. The SP concentrations of saquinavir and ritonavir were, however, usually low. It is interesting that addition of low-dose ritonavir significantly increased seminal plasma indinavir concentrations in another study; the proposed mechanism suggested to be via inhibition of P glycoprotein.9 However, in our study, despite all patients taking saquinavir in combination with ritonavir, saquinavir concentrations remained low and, because no patients were taking saquinavir alone, we were unable to determine whether ritonavir increased seminal saquinavir concentrations to any significant extent. The semen concentrations were generally below the calculated EC95s. This needs to be interpreted with some caution, however, since it is not known to what degree these drugs become protein bound within the semen and what effect this will have on the free drug available for either antiviral activity or active drug transport. Furthermore, although four patients receiving ritonavir and saquinavir had elevated SP VL, they all had detectable virus in the BP (where concentrations were above the EC95, and three had been receiving therapy for <4 weeks, which may be insufficient time to fully suppress the VL). The fourth patient has been studied in more detail, and virus in both compartments demonstrated the presence of PI resistance mutations.10
In summary, we have demonstrated major differences between PIs in their capacity to penetrate the male genital tract. The impact of such differential penetration into this compartment on virus suppression and evolution of resistant virus requires further study.
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Acknowledgements |
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Notes |
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References |
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2 . Eron, J. J., Vernazza, P. L., Johnston, D. M., Seillier-Moiseiwitsch, F., Alcorn, T. M., Fiscus, S. A. et al. (1998). Resistance of HIV-1 to antiretroviral agents in blood and seminal plasma: implications for transmission. AIDS 12, F1819.[ISI][Medline]
3 . Dyer, J. R., Gilliam, B. L., Eron, J. J., Jr, Grosso, L., Cohen, M. S. & Fiscus, S. A. (1996). Quantitation of human immunodeficiency virus type. 1 RNA in cell free seminal plasma: comparison of NASBA with Amplicor reverse transcriptionPCR amplification and correlation with quantitative culture. Journal of Virological Methods 60, 16170.[ISI][Medline]
4 . Merry, C., Barry, G., Mulcahy, F., Ryan, M., Heavey, J., Tija, J. et al. (1998). Saquinavir pharmacokinetics alone and in combination with ritonavir in HIV-infected patients. AIDS 11, F2933.[ISI]
5 . Merry, C., Barry, M. G., Ryan, M., Tija, J. F., Hennessy, M., Eagling, V. A. et al. (1999). Interaction of sildenafil and indinavir when co-administered to HIV-positive patients. AIDS 13, F1017.[ISI][Medline]
6 . Condra, J. H., Petropoulous, C. J., Ziermann, R., Schleif, W. A., Shivaprakash, M. & Emini, E. A. (2000). Drug resistance and predicted virological responses to human immunodeficiency virus type. 1 protease inhibitor therapy. Journal of Infectious Diseases 182, 75865.[ISI][Medline]
7 . Molla, A., Vasavanonda, S., Kumar, G., Sham, H. L., Johnson, M., Grabowski, B. et al. (1998). Human serum attenuates the activity of protease inhibitors toward wild-type and mutant human immunodeficiency virus. Virology 250, 25562.[ISI][Medline]
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Taylor, S. & Pereira, A. (2001). Antiretroviral drug concentrations in semen of HIV-1 infected men. Sexually Transmitted Infections 77, 411.
9 . Van Praag, R. M., Weverling, G. J., Portegies, P., Jurriaans, S., Zhou, X., Turner-Foisy, M. L. et al. (2000). Enhanced penetration of indinavir in cerebrospinal fluid and semen after the addition of low dose ritonavir. AIDS 14, 118794.[ISI][Medline]
10 . Choudhury, B., Cane, P. A., Workman, J., Taylor, S. & Pillay, D. (2000). Impact of poor drug compliance and interupted therapy on HIV-1 evolution in blood and semen. Antiviral Therapy 5, 147.
Received 1 December 2000; returned 10 May 2001; revised 28 May 2001; accepted 13 July 2001