1 Department of Pharmacology and Therapeutics, University of Liverpool, Pharmacology Research Laboratories, 70 Pembroke Place, Liverpool; 2 Department of Sexually Transmitted Diseases, Royal Free and University College, Mortimer Market Centre, London, UK
Received 8 January 2002; returned 31 May 2002; revised 13 June 2002; accepted 25 June 2002
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patients and methods: Drug efflux transporter expression (P-glycoprotein and MRP1) on peripheral blood mononuclear cells isolated from HIV-positive patients was investigated using flow cytometry. In addition, plasma and intracellular ritonavir and saquinavir concentrations were measured by HPLC/mass spectrometry. The ratio of intracellular:plasma drug concentration was used to quantify intracellular drug accumulation.
Results: Patients with lower MRP1 expression (<median) had a significantly higher accumulation of both ritonavir and saquinavir than those with higher MRP1 expression (P = 0.035, CI = 1.70 to 0.06 and P = 0.043, CI = 12.79 to 0.11, respectively). Ritonavir accumulation was significantly greater in patients with lower P-glycoprotein expression (<median) than in patients with higher expression (P = 0.014, CI = 1.56 to 0.14). There was no relationship between saquinavir accumulation in patients and P-glycoprotein expression (P = 0.219, CI = 5.02 to 2.40). Combining expression of P-glycoprotein and MRP1 {expression index, EI = [(P-glycoprotein 1) + (MRP1 1) x 100]} resulted in a statistically significant relationship between transporter expression and intracellular accumulation of both saquinavir (r2 = 0.195, P = 0.035) and ritonavir (r2 = 0.220, P = 0.049).
Conclusion: Increased expression of P-glycoprotein and MRP1 on lymphocytes is associated with lower intracellular accumulation of saquinavir and ritonavir. These two transporters may play a role in the efflux of ritonavir and saquinavir from lymphocytes in vivo.
Keywords: P-glycoprotein, MRP, accumulation, ritonavir, saquinavir
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A number of cellular and tissue compartments exist that may provide sanctuary for HIV.3,4 In these areas the virus may persist and replicate, despite apparent suppression of replication in plasma.5 It is thought that multidrug resistance transporters may play a role in lowering intracellular drug concentrations at these sites via an efflux mechanism.4,6
P-glycoprotein and MRP (multidrug resistance associated protein) are two well characterized transporters of great interest in the field of HIV research, as their substrates include both the protease inhibitors (e.g. ritonavir and saquinavir) and the nucleoside analogues.712 P-glycoprotein and MRP1 are known to be expressed on human lymphocytes,13,14 a major site of HIV replication and antiretroviral drug action.15
There have been many in vitro studies illustrating the mechanisms by which these transporters may impact upon successful therapy of HIV infection.6,1618 However, studies have yet to examine the role of this active transport-mediated efflux on drug levels in a clinical setting.
We wished to investigate whether natural biological differences in the expression of P-glycoprotein and MRP1 on human lymphocytes would impact on intracellular protease inhibitor concentrations in an HIV-infected population.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Saponin was purchased from Sigma Chemical Co. Ltd (Poole, UK). Phosphate-buffered saline (PBS) tablets were purchased from Gibco Life Technologies Ltd (Paisley, UK). Lymphoprep was purchased from Nycomed Pharma AS (Oslo, Norway) and Cellfix from Becton Dickinson (Oxford, UK). Goat anti-mouse IgG2a:RPE and mouse IgG2a negative control were purchased from Serotec Ltd (Oxford, UK). The anti-human P-glycoprotein antibody, UIC2, was obtained from Immunotech (Marseilles, France). The anti-human MRP antibody, MRPm5 (specific for MRP1),19 was obtained from Kamiya Biomedical Company (Seattle, WA, USA). Ammonium formate, acetonitrile and methanol were purchased from Fisher Scientific (Loughborough, UK). Hypurity 5C18 column was purchased from Hypersil (Manchester, UK).
Patients
A total of 32 HIV-infected patients attending clinics in the Mortimer Market Centre, London, Royal Liverpool University Hospital and North Manchester General Hospital were recruited. Ethical approval for the study was obtained from relevant local ethics committees. All patients gave informed written consent. All patients at the time of study had an undetectable plasma viral load (<50 copies/mL) on combination antiretroviral therapy including a protease inhibitor. Patients undergoing treatment with drugs known to be substrates/inhibitors of P-glycoprotein or MRP (other than a protease inhibitor, such as non-steroidal anti-inflammatory drugs, anti-neoplastic agents, verapamil) were excluded from the trial. Table 1 shows patient characteristics. Venous blood samples (25 mL) were collected into lithium heparin tubes over a full dosing interval (0, 2, 4, 8, 12 h for 12 hourly dosing, and 0, 1, 2, 4, 8 h for 8 hourly dosing).
|
Plasma was separated from 5 mL of blood. Peripheral blood mononuclear cells were isolated by density cushion centrifugation, adhering stringently to a protocol developed previously.20 Samples were washed in ice-cold PBS and centrifuged (700g, 6 min, 4°C). After two more washes cells were quantified using a haemocytometer before extraction in 60% methanol. Retention of PBS at 4°C and the rapidity of these wash steps are crucial for minimization of drug loss from the cells; <10% of intracellular drug effluxes out of the cells under these conditions. Following overnight extraction, samples were centrifuged (700g, 6 min, 4°C) and the supernatant fraction transferred to a glass tube prior to evaporation to dryness.
Internal standard was added to both heat-inactivated plasma (40 min, 58°C) and dried cell extracts (resuspended in 200 µL of distilled water) prior to further extraction using diethyl ether. The aqueous layer was frozen and the organic layer was transferred to a clean tube and evaporated to dryness. Samples were reconstituted in the mobile phase [10 mM ammonium formate buffer/acetonitrile (30:70, v/v)] prior to injection on the column. Protease inhibitors were eluted on a Hypurity Elite 5C18 column (5 µm particle size; 250 x 4.6 mm) with the mobile phase maintained at 1.2 mL/min. Quantification of ions resulting from fragmentation of the parent compound (protease inhibitor) was analysed using a mass spectrometer (electrospray ionization) and Xcalibur software. Saquinavir (retention time 4.2 min) and ritonavir (retention time 3.8 min) were analysed by fragmentation of the parent compounds and quantification of the resulting fragments (monitoring of ions m/z ritonavir 721.4/426.1, 296.0; saquinavir 671.4/570.3, 433.2). The internal standard (retention time 8.0 min) was monitored at m/z 674.4/573.3, 388.2.
The lower limits of detection for saquinavir and ritonavir on the column are less than 5 and 10 pg, respectively. These correspond to plasma concentrations of 375 and 750 pg/mL or peripheral blood mononuclear cell (10 x 106 cells) concentrations of 10 and 20 ng/mL, respectively. The inter-assay coefficients of variation (CV) for saquinavir were 9.7, 3.9 and 7.1% at concentrations of 100 ng/mL, 5 µg/mL and 15 µg/mL, respectively. The intra-assay CV were 2.0, 3.5 and 6.1% at the same concentrations. The inter-assay CV for ritonavir were 8.5, 7.0 and 4.9% at concentrations of 250 ng/mL, 2 µg/mL and 8 µg/mL, respectively. The intra-assay CV were 5.5, 5.8 and 5.7% at the same concentrations.
Detection of transporter expression in human lymphocytes
Isolated human peripheral blood mononuclear cells (3.57.0 x 106 cells) were fixed (Cellfix, 1.5 mL; 25 min, 25°C), then washed (1 mL PBS, 4°C; centrifugation 700g, 6 min, 4°C) and resuspended in PBS to a concentration of 2 x 106 cells/mL. Aliquots (200 µL) of cell suspension were transferred to 5 mL plastic sample tubes.
Three cell samples were incubated with the UIC2 antibody (2.4 mg/L) and three with the isotype control antibody IgG2a (4.8 mg/L) for 30 min on ice (unstained cell samples were used as a negative control). All cell samples were washed twice with PBS (1 mL, 4°C) followed by centrifugation (700g, 6 min, 4°C) and incubated with (R)-phycoerythrin-bound IgG2a secondary antibody (2.0 mg/L) for 30 min on ice, in the dark. The cells were washed twice with PBS (1 mL, 4°C; centrifugation 700g, 6 min, 4°C) and fixed (Cellfix, 0.5 mL). All samples were analysed via flow cytometry.
MRP1 expression was determined as above but with additional steps for permeabilization of the cells. This was necessary as the antibody MRPm5 is directed towards an internal epitope of MRP1. Isolated peripheral blood mononuclear cells were fixed and washed as described previously before resuspension in PBS containing saponin (500 mg/L) to a concentration of 2 x 106 cells/mL. Samples were left for 20 min to allow permeabilization of the cells, then 200 µL of cell suspension was transferred to the individual 5 mL tubes. Cells were incubated with MRPm5 (1.2 mg/L) or isotype control IgG2a (4.8 mg/L) for 30 min on ice (unstained cells used as a negative control). Cells were then treated as described previously but all washes used PBS containing saponin.
Flow cytometry
Flow cytometry was conducted on a Coulter Epics XL-MCL flow cytometer. For each sample 5000 events were collected. Forward scatter and side scatter signals were detected on a linear scale and fluorescence was detected on a logarithmic scale. Lymphocytes were electronically gated. (R)-Phycoerythrin-positive fluorescence (P-glycoprotein expression) was followed in channel 2 (FL2) and the amount of fluorescence plotted as a histogram of FL2 staining. Data acquisition was performed using the computer program WINMDI version 2.6 to determine median FL2 fluorescence analysis values. Data were expressed as a fold increase in fluorescence (FL2), calculated by dividing the median fluorescence intensity seen with UIC2 or MRPm5 by that seen with the IgG2a isotype control for P-glycoprotein and MRP, respectively. In addition, an expression index (EI) was utilized in order to assess the combined effects of both transporters on drug efflux from cells. This was calculated as:
EI = [(P-glycoprotein expression 1) + (MRP1 expression 1)] x 100.
The relationship between EI and drug accumulation was investigated using linear regression analysis.
Statistical analysis
Intracellular concentrations of ritonavir and saquinavir were calculated on the basis of a single peripheral blood mononuclear cell volume of 0.4 pL (determined by flow cytometry and Furman et al.21) and total cell count. The intracellular concentrations calculated were total drug associated with the cells.
Area under the curve (AUC) values for plasma and intracellular ritonavir and saquinavir were evaluated by non-compartmental modelling by using the linear trapezoid rule (TOPFIT computer software; Gustav Fischer Verlag, Stuttgart, Germany). Patients received different dosage regimens (Table 1), which made direct comparison of transporter data with either intracellular or plasma data alone unfeasible. Intracellular accumulation data were therefore quantified and presented as a ratio of intracellular AUC to the plasma AUC over a whole dosing interval. Statistical analysis of drug accumulation in peripheral blood mononuclear cells with low and high P-glycoprotein and MRP1 expression (stratified about the median) was performed using a MannWhitney U-test.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The median intracellular:plasma concentration ratio of saquinavir was 2.74 (range 0.3222.74) and ritonavir was 1.25 (range 0.204.19).
Stratification of the expression of P-glycoprotein and MRP1, into two groups around the median level of transporter expression, was performed for patients receiving either ritonavir (n = 23) or saquinavir (n = 18) (Figure 1). Patients with greater than median expression of MRP1 had significantly lower intracellular accumulation of both protease inhibitors studied (P = 0.035, CI = 0.17 to 0.06 for ritonavir, P = 0.043, CI = 12.79 to 0.11 for saquinavir). Patients with a P-glycoprotein expression greater than the median showed no significant difference in saquinavir accumulation (P = 0.219, CI = 5.02 to 2.40) but had lower ritonavir accumulation (P = 0.014, CI = 1.56 to 0.14).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vitro studies have reported decreased intracellular accumulation of ritonavir and saquinavir in cell lines overexpressing either P-glycoprotein or MRP1.6 It is unclear whether the amount of measured drug is predominantly complexed to intracellular proteins or unbound and thus able to exert its antiviral effect. Further studies are required in order to delineate this. Of equal importance, studies demonstrating that these in vitro observations are also maintained in vivo are lacking. There are no published data correlating intracellular accumulation of a drug with transporter expression in HIV-positive patients.
In this study we have demonstrated an inverse relationship between the intracellular accumulation of saquinavir or ritonavir and transporter expression in vivo. Decreased ritonavir accumulation was associated with a high expression of each transporter on lymphocytes. Decreased intracellular accumulation of saquinavir was observed with high MRP1 expression.
The combined contribution of P-glycoprotein and MRP1 to drug efflux of ritonavir and saquinavir was assessed using the EI. There are limitations to this, not least that it represents an oversimplification, since many efflux transporters have been characterized on lymphocytes and the contribution of any individual transporter to the overall efflux of drug will depend upon a number of factors including levels of expression, in vivo functional abilities and drug specificities. Nevertheless, a statistically significant relationship was observed between the EI and intracellular drug accumulation in patients receiving either saquinavir or ritonavir. Although expression of these transporters may not necessarily correlate with functional efflux, these results are the first to suggest that accumulation of the protease inhibitors in vivo may be influenced by the efflux transporters P-glycoprotein and MRP1.
In conclusion, the relationship observed between P-glycoprotein and MRP1 expression and intracellular accumulation of ritonavir and saquinavir suggests that these drug efflux transporters may have a clinically important role in limiting drug exposure within cells. These in vivo findings are in agreement with previous in vitro studies.6 Whether or not some of the biological variability in drug response may be explained by genetic polymorphisms of the MDR-1 gene,22 or other factors influencing transporter expression, e.g. pregnane X receptor, requires further investigation. The effect of agents that may induce transporter expression, including HIV drugs themselves, also requires study.
Finally, our observations suggest that specific inhibitors of these transporters could potentially modify intracellular drug accumulation. This represents one possible novel pharmacological strategy to enhance drug penetration into sanctuary sites.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Khoo, S. H., Gibbons, S. E. & Back, D. J. (2001). Therapeutic drug monitoring as a tool in treating HIV infection. AIDS 15, S17181.[ISI][Medline]
3 . Siliciano, R. F. (1999). Latency and reservoirs for HIV-1. AIDS 13, S4958.[ISI][Medline]
4 . Hoetelmans, R. M. (1998). Sanctuary sites in HIV-1 infection. Antiviral Therapy 3, 137.
5 . Chun, T. W., Carruth, L., Finzi, D., Shen, X., DiGiuseppe, J. A., Taylor, H. et al. (1997). Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 387, 1838.[ISI][Medline]
6 . Jones, K., Hoggard, P. G., Sales, S. D., Khoo, S., Davey, R. & Back, D. J. (2001). Differences in the intracellular accumulation of HIV protease inhibitors in vitro and the effect of active transport. AIDS 15, 67581.[ISI][Medline]
7 . Profit, L., Eagling, V. A. & Back, D. J. (1999). Modulation of P-glycoprotein function in human lymphocytes and Caco-2 cell monolayers by HIV-1 protease inhibitors. AIDS 13, 16237.[ISI][Medline]
8
.
Kim, A. E., Dintaman, J. M., Waddell, D. S. & Silverman, J. A. (1998). Saquinavir, an HIV protease inhibitor, is transported by P-glycoprotein. Journal of Pharmacology and Experimental Therapeutics 286, 143945.
9
.
Srinivas, R. V., Middlemas, D., Flynn, P. & Fridland, A. (1998). Human immunodeficiency virus protease inhibitors serve as substrates for multidrug transporter proteins MDR1 and MRP1 but retain antiviral efficacy in cell lines expressing these transporters. Antimicrobial Agents and Chemotherapy 42, 315762.
10 . Signoretti, C., Romagnoli, G., Turriziani, O., Antonelli, G., Dianzani, F. & Cianfriglia, M. (1997). Induction of the multidrug-transporter P-glycoprotein by 3'-azido-3'-deoxythymidine (AZT) treatment in tumor cell lines. Journal of Experimental Clinical Cancer Research 16, 2932.[ISI][Medline]
11 . Antonelli, G., Turriziani, O., Cianfriglia, M., Riva, E., Dong, G., Fattorossi, A. et al. (1992). Resistance of HIV-1 to AZT might also involve the cellular expression of multidrug resistance P-glycoprotein. AIDS Research and Human Retroviruses 8, 183944.[ISI][Medline]
12 . Schuetz, J. D., Connelly, M. C., Sun, D., Paibir, S. G., Flynn, P. M., Srinivas, R. V. et al. (1999). MRP4: a previously unidentified factor in resistance to nucleoside-based antiviral drugs. Nature Medicine 5, 104851.[ISI][Medline]
13 . Neyfakh, A. A., Serpinskaya, A. S., Chervonsky, A.V., Apasov, S. G. & Kazarov, A. R. (1989). Multidrug-resistance phenotype of a subpopulation of T-lymphocytes without drug selection. Experimental Cell Research 185, 496505.[ISI][Medline]
14 . Abbaszadegan, M. R., Futscher, B. W., Klimecki, W. T., List, A. & Dalton, W. S. (1994). Analysis of multidrug resistance-associated protein (MRP) messenger RNA in normal and malignant hematopoietic cells. Cancer Research 54, 46769.[Abstract]
15 . Fauci, A. S. (1988). The human immunodeficiency virus: infectivity and mechanisms of pathogenesis. Science 239, 61722.[ISI][Medline]
16 . Lee, C. G., Ramachandra, M., Jeang, K. T., Martin, M. A., Pastan, I. & Gottesman, M. M. (2000). Effect of ABC transporters on HIV-1 infection: inhibition of virus production by the MDR1 transporter. Federation of American Societies for Experimental Biology Journal 14, 51622.
17 . Gollapudi, S. & Gupta, S. (1990). Human immunodeficiency virus I-induced expression of P-glycoprotein. Biochemical and Biophysical Research Communications 171, 10027.[ISI][Medline]
18 . Lee, C. G., Gottesman, M. M., Cardarelli, C. O., Ramachandra, M., Jeang, K. T., Ambudkar, S. V. et al. (1998). HIV-1 protease inhibitors are substrates for the MDR1 multidrug transporter. Biochemistry 37, 3594601.[ISI][Medline]
19
.
Scheffer, G. L., Kool, M., Heijn, M., de Haas, M., Pijnenborg, A. C., Wijnholds, J. et al. (2000). Specific detection of multidrug resistance proteins MRP1, MRP2, MRP3, MRP5, and MDR3 P-glycoprotein with a panel of monoclonal antibodies. Cancer Research 60, 526977.
20 . Khoo, S. H., Hoggard, P. G., Newton, P., Meaden, E. R., Smith, A., Williams, I. et al. (2001). Intracellular accumulation of the protease inhibitors saquinavir (SQV) and indinavir (IDV)the effect of co-administration with ritonavir (RTV). In Program and Abstracts of the Eighth European Conference on Clinical Aspects and Treatment of HIV Infection, Athens, Greece, 2001. Abstract 159, p. 117. European AIDS Clinical Society.
21 . Furman, P. A., Fyfe, J. A., St Clair, M. H., Weinhold, K., Rideout, J. L., Freeman, G. A. et al. (1986). Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase. Proceedings of the National Academy of Sciences, USA 83, 83337.[Abstract]
22
.
Hoffmeyer, S., Burk, O., von Richter, O., Arnold, H. P., Brockmoller, J., Johne, A. et al. (2000). Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo. Proceedings of the National Academy of Sciences, USA 97, 34738.