The intracellular pharmacology of antiretroviral protease inhibitors

J. Ford*, S. H. Khoo and D. J. Back

Department of Pharmacology and Therapeutics, University of Liverpool, 70 Pembroke Place, Liverpool L69 3GF, UK


    Abstract
 Top
 Abstract
 Introduction
 Bioavailability
 Plasma protein binding
 Physiochemical properties
 Protease inhibitor intracellular...
 Drug transporter proteins
 Summary
 Acknowledgements
 References
 
Therapeutic drug monitoring (TDM) of antiretroviral protease inhibitors (PIs) has been suggested to have the potential to both reduce toxicity and optimize individual therapy. However, the major target of PIs is within cells infected with HIV. Therefore clinical outcome ultimately must be related to intracellular drug concentrations since antiviral activity of PIs is highly correlated with intracellular concentrations in vitro. Intracellular pharmacokinetics provides information regarding drug disposition in a compartment where HIV replication occurs and combined with plasma data may be useful in understanding therapeutic failure in relation to cellular resistance. In order to improve therapeutic efficacy, it is therefore important that the intracellular pharmacokinetics of drugs, such as PIs, is studied in addition to plasma pharmacokinetics. Multidrug resistance transporters may result in a lower cellular concentration of drug via an efflux mechanism, thus contributing to sanctuary site formation. However, conclusive proof that transporters contribute to clinical drug resistance is still lacking, although recent studies have attempted to address this issue. In relation to host and cellular factors, this review considers several issues involved in influencing intracellular drug concentrations and discusses the intracellular levels of PIs recently published from cellular studies.

Keywords: P-glycoprotein , pharmacokinetics , HIV , efflux , influx


    Introduction
 Top
 Abstract
 Introduction
 Bioavailability
 Plasma protein binding
 Physiochemical properties
 Protease inhibitor intracellular...
 Drug transporter proteins
 Summary
 Acknowledgements
 References
 
The critical role of the HIV intracellular protease enzyme in virus maturation has established the HIV protease as an important target for therapeutic intervention, leading to the development of protease inhibitors (PIs) that prevent the post-integration step of HIV replication.1 Inadequate suppression of HIV replication remains a major limitation in the achievement of treatment success.2 The failure of antiretroviral therapy is a complex interplay of many factors, including poor adherence, virological resistance and pharmacological issues, such as protein binding and cellular resistance.3

There is increasing interest in the use of therapeutic drug monitoring (TDM), to aid optimization of antiretroviral therapy; this has the potential to both reduce toxicity and ensure adequate viral suppression. However, the major target of PIs is within cells infected with HIV and clinical outcome ultimately must be related to intracellular drug concentrations. Therefore, it is important that factors influencing viral replication and evolution within lymphocytes be fully understood. In HIV-1-infected peripheral blood mononuclear cells (PBMCs) and cell lines, the antiviral activity of PIs is highly correlated with the intracellular concentration of drug,4,5 which has formed the rationale for in vivo studies on intracellular PIs. Intracellular pharmacokinetics provides information regarding drugs in a compartment where HIV replication occurs and combined with plasma data may give insights into understanding therapeutic failure in relation to cellular resistance.

In vitro PIs accumulate in the following order: nelfinavir > saquinavir > ritonavir > indinavir in an adenocarcinoma cell line,6 nelfinavir > saquinavir > ritonavir = lopinavir > indinavir in buffy coat cell subsets ex vivo7 and saquinavir > amprenavir > indinavir in subcellular HeLa cell fractions.8 Furthermore, there is a hierarchy of intracellular accumulation in HIV-1 infected patients in vivo, saquinavir > ritonavir > indinavir9 and nelfinavir > amprenavir > indinavir.10 The main areas of pharmacology that influence intracellular drug concentrations are oral bioavailability, plasma protein binding (altering the free fraction of drug), physiochemical properties (such as lipophilicity, degree of ionization), cellular protein binding and cellular influx/efflux active transport (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. Some physiochemical properties of the protease inhibitors

 

    Bioavailability
 Top
 Abstract
 Introduction
 Bioavailability
 Plasma protein binding
 Physiochemical properties
 Protease inhibitor intracellular...
 Drug transporter proteins
 Summary
 Acknowledgements
 References
 
The oral bioavailability of PIs is generally low, due to metabolism by the cytochrome P450 (CYP) system, particularly CYP-3A4 and to a much lesser extent CYP2D6, CYP2C9 and CYP-2C19. In addition, PIs are substrates for multidrug resistance influx and efflux transporters expressed in the gastrointestinal tract and liver, which may also reduce bioavailability. Nonetheless, it is crucial to maintain an adequate concentration of drug in the systemic circulation above the minimum effective concentration, available to target pharmacological sites of action and halt viral replication. Pharmacoenhancement (boosting) with low-dose ritonavir (generally 100 mg), markedly increases the bioavailability of PIs by inhibition of CYP enzymes and efflux transporters.


    Plasma protein binding
 Top
 Abstract
 Introduction
 Bioavailability
 Plasma protein binding
 Physiochemical properties
 Protease inhibitor intracellular...
 Drug transporter proteins
 Summary
 Acknowledgements
 References
 
PIs bind avidly to {alpha}1-acid glycoprotein (AAG)4 thereby reducing the free (unbound) concentration of drug available to traverse cell membranes. However, it is important to note that nelfinavir, which accumulates to the greatest degree in cells, is highly protein bound (>98%), whereas indinavir, which accumulates least, is only 60% bound (Table 1). This highlights the complexity of accumulation since indinavir, a drug with a higher free fraction, accumulates to a low level inside lymphocytes, unlike its counterparts. Thus it appears that protein binding per se is not directly related to the rank order of PI accumulation. However, increasing levels of AAG have been reported to decrease the intracellular accumulation of PIs in vitro, by reducing unbound drug concentrations available to reach target cells.4,11 Whether intracellular drug remains free or is complexed to proteins has not been definitively determined. However, it is highly likely that PIs bind to intracellular proteins due to their high affinity for protein in the plasma compartment. Within cells, a dynamic equilibrium must then exist between drug bound to protein and drug bound to HIV protease, influenced by the affinity of PIs for each protein. PIs have been shown to be located primarily within mitochondrial, nuclear and cytosolic fractions of the cell, and are therefore not just solubilized in the extracellular membrane.9


    Physiochemical properties
 Top
 Abstract
 Introduction
 Bioavailability
 Plasma protein binding
 Physiochemical properties
 Protease inhibitor intracellular...
 Drug transporter proteins
 Summary
 Acknowledgements
 References
 
Passive diffusion/lipophilicity

Passive diffusion through a membrane is determined by the size of the molecule, unbound drug concentrations, degree of ionization and lipophilicity. The partition coefficient of a compound between water and a lipophilic solvent such as n-octanol is one model utilized to describe the potential accumulation of a compound. Nelfinavir is the most lipophilic PI having the highest partition coefficient, and may therefore passively diffuse into the intracellular compartment to a greater extent compared with other PIs. Log P values for the PIs have been reported in the following order: nelfinavir > saquinavir > amprenavir = lopinavir > ritonavir > indinavir (Table 1), providing an indication of the lipophilicity of the compounds.7 Accumulation of PIs in lymphocytes and subsets reflects the lipophilicity rank order, suggesting that passive diffusion is important in the bioaccumulation of each PI.

Ion trapping

Membranes are impermeable to ionized forms of drugs and so the degree of ionization of a molecule affects the concentration that is available for passive diffusion across biological membranes. Since the dissociation constant (pKa) is equal to the pH at which a compound is 50% ionized, then weak bases will mainly be un-ionized in a basic environment and if lipophilic may be absorbed. Therefore nelfinavir, pKa 6.0/11.1, saquinavir pKa 1.1/7.1 and indinavir pKa 6.212 (Table 1), which are weak bases, are more likely to cross lipid membranes of cells or organelles when the pH of the environment is greater than the pKa of PIs. Thus there is the potential for sequestration of basic compounds (pKa > 8) in acidic compartments and the pH gradient between plasma and lymphocytes will influence ion trapping. The pH gradient between plasma and lymphocytes is subject to change depending on the membrane potential.


    Protease inhibitor intracellular accumulation
 Top
 Abstract
 Introduction
 Bioavailability
 Plasma protein binding
 Physiochemical properties
 Protease inhibitor intracellular...
 Drug transporter proteins
 Summary
 Acknowledgements
 References
 
Trials evaluating intracellular PI accumulation are few in comparison to the number of plasma pharmacokinetic studies that have been carried out. To determine intracellular PI concentrations in vivo, patients are required to donate large volumes of blood and it is vital that stringent assay conditions be adhered to, consequently, intracellular pharmacokinetic studies have been carried out in only a relatively small number of patients. Intracellular concentrations are calculated on the basis of a single PBMC volume of 0.4 pL and total cell density of samples.13 To date, the volume of a single cell is the best surrogate marker to calculate intracellular concentrations, however, this may not reflect the volume of aqueous cellular medium for which free drug is available. The intracellular concentration is therefore calculated from the cell count and the cell volume. It is important to note that the intracellular concentration of drug is actually the total drug concentration associated with the cells. Intracellular accumulation data are usually quantified and expressed as a ratio of the intracellular area under the curve (AUC) over the total plasma AUC throughout the dosage interval. The methods that have been utilized to determine intracellular drug concentrations are broadly similar, however, there are some differences in terms of cell collection and quantification of drug levels.1416 Therefore, measurement of cellular drug concentration must be standardized and reach a consensus recommendation, so as to generate consistent, accurate and reproducible assays in human PBMCs.

The accumulation of PIs in vivo mirrors the hierarchy that has been observed from in vitro studies nelfinavir > saquinavir > amprenavir > M8 > lopinavir ≥ ritonavir > indinavir (Table 2) and accumulation of PIs appears to reflect the rank order of lipophilicity. However, some in vitro and ex vivo studies suggest that passive diffusion is not the only explanation for accumulation6,7 and other factors such as active transport may be important.


View this table:
[in this window]
[in a new window]
 
Table 2. The intracellular accumulation ratio of the PIs nelfinavir, saquinavir, ritonavir, amprenavir, lopinavir and indinavir alone and in combination with ritonavir observed in vivo from several published studies

 
Saquinavir has been the most extensively studied intracellular PI in vivo in both once and twice daily regimens (Table 3). Consistent intracellular accumulation ratios have been obtained for co-administered saquinavir and ritonavir in both once and twice daily regimens (median values 2.7, 3.3, 3.6, 4.0) in contrast to saquinavir administered as a sole PI where the ratio was more variable (median values 7.5, 9.5, 17.6). These studies have demonstrated that boosted saquinavir lowered the accumulation ratio of saquinavir (although absolute intracellular concentrations and AUC values were increased), whereas addition of saquinavir to a ritonavir regimen increased the accumulation ratio of ritonavir from 1.0 to 1.5. Preliminary data with respect to ritonavir (Table 2), where intracellular accumulation was boosted ~five-fold when co-administered with lopinavir, suggest that this process has the potential to be manipulated. The changing PI accumulation ratios when co-administered with ritonavir may reflect competition for active transporters; it at least suggests that accumulation has the potential to be modulated.


View this table:
[in this window]
[in a new window]
 
Table 3. Intracellular accumulation ratios obtained for saquinavir (SQV) and ritonavir (RTV) in vivo from several published studies

 
There are consistent data emerging from saquinavir pharmacokinetic studies. One study showed 86% of patients were virologically suppressed, despite what were considered suboptimal plasma saquinavir concentrations throughout the study period17 and it is reasonable to suggest that intracellular drug levels may be responsible for the observed virological response. Another report demonstrated a disconnect between very low plasma saquinavir concentrations and high intracellular drug accumulation in a subset of patients with durable virological suppression, despite receiving low doses (600 mg, every 8 h) of unboosted hard gel saquinavir.9 In a different study,18 in patients receiving hard gel saquinavir in combination with ritonavir administered once daily, a similar trend emerged with plasma saquinavir trough concentrations below the minimum effective concentration, whilst intracellular trough concentrations were much higher, and patients remained virologically suppressed. Moreover, saquinavir demonstrated a statistically longer intracellular half-life of 5.9 h in comparison to a plasma half-life of 4.5 h. This finding is consistent with in vitro data5 reporting that saquinavir remained associated with cells more than 2 h after removal of drug from the culture medium. Together, these data indicate that cellular concentrations of saquinavir may be present when plasma drug levels are low, although further studies are warranted to investigate this phenomenon for other PIs and to determine the cellular mechanisms involved.

Another issue regarding intracellular drug concentrations is whether extrapolation from simultaneous plasma concentrations can be carried out and if plasma TDM has the potential to be a surrogate marker for cell-associated concentrations. Conflicting data surrounding this matter have been reported with studies suggesting extrapolation of saquinavir, indinavir and nelfinavir may be possible15,1820 whilst for other PIs lopinavir,15 ritonavir and M816,21 studies suggest extrapolation is difficult. Generally interpretation of intracellular PI concentrations from corresponding plasma levels is complicated with many interrelated factors involved and each individual PI may behave differently. Further clinical trials using large patient numbers are required to address this issue. A recent study has shown that baseline lopinavir mutations giving rise to virological failure were associated with both low intracellular and plasma concentrations. The authors suggested that monitoring the efficacy of lopinavir and ritonavir should include measurement of baseline mutations, intracellular and plasma concentrations.22

Generally, intracellular drug concentrations obtained from different laboratories have generated consistent data, however, some differences in the data are present. In order to improve the performance of assays, and expand intracellular pharmacokinetic data, procedures should be standardized so as to produce accurate and reproducible assays in human peripheral blood mononuclear cells.


    Drug transporter proteins
 Top
 Abstract
 Introduction
 Bioavailability
 Plasma protein binding
 Physiochemical properties
 Protease inhibitor intracellular...
 Drug transporter proteins
 Summary
 Acknowledgements
 References
 
Multidrug transport proteins function as a protective barrier to potential toxic agents lowering the intracellular concentration of a broad range of chemically unrelated substrates, a phenomenon known as multidrug resistance.23 P-glycoprotein (P-gp), encoded by the MDR1 gene, is a 170 kDa phosphorylated and glycosylated plasma membrane protein belonging to the ATP-binding cassette (ABC) superfamily of transport proteins.24 P-gp substrates have diverse structures and include many clinically relevant drugs such as anticancer drugs, immunosuppressive agents, antibiotics and antiretrovirals25 including the PIs.26,27 P-gp is 1280 amino acids long, consisting of two homologous halves of 610 amino acids joined to a linker region of 60 amino acids.28 Each half contains six transmembrane domains followed by a hydrophilic domain that comprises a nucleotide-binding site for ATP29,30 (Figure 1). Although P-gp is the most extensively researched transporter, other ABC transporters namely multidrug resistance associated protein (MRP1) and breast cancer-resistant protein or mitoxantrone resistance protein (BCRP/MXR) have demonstrated multidrug resistance to tumours.3134 P-gp, MRP1 and BCRP transport proteins are three key multidrug resistance transporters with overlapping substrate specificity and display a broad tissue localization (Table 4).32,35 MRP shares approximately 15% amino acid sequence identity with P-gp36 and to date there are nine members of the MRP family35,37 that all transport organic anions or neutral drugs conjugated to glutathione, glucuronide or sulphate.38



View larger version (65K):
[in this window]
[in a new window]
 
Figure 1. Membrane topology of MDR-related ABC transporters P-gp, MRP1 and BCRP.

 

View this table:
[in this window]
[in a new window]
 
Table 4. Tissue distribution of influx and efflux transport proteins (adapted from Refs 72,73,77,80)

 
MRP1 is 190 kDa, 1531 amino acids long with two potential nucleotide binding sites and many membrane spanning helices (Figure 1). BCRP is 70 kDa and a unique half transporter (Figure 1) and recent data show that this protein forms an active homodimer becoming 140 kDa to carry out its transport function.32,39 BCRP, a member of the ABC White subfamily, has a wide tissue distribution that has similarities to the tissue location of P-gp and MRP1.40 Transporters with wide tissue distribution and broad substrate specificity may limit drug penetration into HIV cellular targets and pharmacological sanctuaries.41,42 Furthermore, since such transporters are expressed on lymphocytes,4345 active drug efflux may reduce cellular accumulation in vivo (Figure 2). Transporters can potentially influence orally administered drugs at two main sites, at the bioavailability level of the intestine/liver or at the cellular lymphocyte level. Currently, it is unclear whether regulation of transporter expression is tissue specific, for example, whether a high level of P-gp expression throughout the intestine corresponds to a high level of P-gp expression on lymphocytes. However, one study has demonstrated induction of CYP3A4 by efavirenz in the liver despite no induction in the intestine, which suggests that various organs have independent expression of the cytochrome enzyme.46 Since reports have established CYP3A4 and P-gp to act in synergy and to have a similar tissue expression,47,48 a similar finding may also occur for P-gp. To provide more evidence surrounding this issue, studies have revealed that P-gp expression is progressively increased from proximal to distal regions of the human intestine.49



View larger version (43K):
[in this window]
[in a new window]
 
Figure 2. Potential influx and efflux transport proteins expressed on a lymphocyte that may impact accumulation.

 
Studies using cell lines as models in vitro have proved useful in investigating the PIs as substrates for transporters such as P-gp50 and MRP1.51 Furthermore, in vitro studies have indicated that cells overexpressing P-gp (CEMVBL) and MRP1 (CEME1000) had a reduced accumulation of PIs in relation to the parent CEM cell line.6 An ex vivo study7 illustrated a differential cell subset accumulation of each PI, such that CD8 cells, which express more P-gp than CD4 cells, had lower accumulation of PI. In vitro studies are useful in demonstrating the potential impact that transporters may have in vivo, however, the clinical relevance of such findings is unclear. The potential role of P-gp in modulating cell susceptibility to HIV infection has been reported. A 70-fold decrease in viral production in cells overexpressing P-gp and a 50-fold increase in cells overexpressing MRP1 compared with control were observed.52 However, reports have questioned the use of cell lines in this type of model with non-physiological levels of transporter and different levels of CD4 and CXCR4 which may affect viral cell entry.53 Another study demonstrated that differences in physiological levels of MDR1 expression and MDR1 haplotypes did not modify HIV-1 infection in vitro or influence permissiveness to infection.54

P-gp plays a major role in cancer therapy resistance, preventing sufficient intracellular accumulation of several anticancer agents due to expression in various types of cancer. Therefore it is thought that resistance can be overcome by inhibition of P-gp. Many first- and second-generation inhibitors have displayed poor efficacy. However, third-generation inhibitors such as XR9576 have shown higher potency, selectivity and demonstrated inhibition of P-gp in a solid tumour model.55 Relatively few studies have compared surface expression levels of transporters with the intracellular accumulation of PIs in vivo. Some in vivo studies have shown P-gp expression on the surface of lymphocytes to be related to accumulation of ritonavir56 and lopinavir and ritonavir21 whilst others have shown no relationship with nelfinavir or M816 or once daily saquinavir and ritonavir18 accumulation. Another study showed an inverse relationship between overexpression of the P-gp MDR1 gene and lower intracellular concentration of PI.10 Therefore there appears to be a disconnect between P-gp transporter data in vitro and in vivo and one must be careful when interpreting in vitro data. Similar disconnects between in vitro and clinical data have been observed previously when in vitro studies showed nelfinavir to up-regulate P-gp expression in vitro at 10 µM5760 but patients on PI- and non-PI-based regimens exhibited no difference in expression of P-gp.60,61

Reports have indicated that patients with MRP1 expression lower than the median displayed significantly higher saquinavir and ritonavir accumulation,56 whereas lopinavir accumulation was not related to expression of MRP1.21 The PIs are substrates for MRP2, another transporter that may influence intracellular drug levels.62 However, this transporter is not expressed on lymphocytes63 and so will not affect lymphocyte accumulation of PIs directly. However, BCRP may play an important role in the transport of some antiretrovirals, since it appears to be expressed on lymphocytes45 to a similar extent as P-gp, and confers cellular resistance to zidovudine, a nucleoside reverse transcriptase inhibitor.45 Although few studies have investigated BCRP transport of substrates, a recent article suggests that the PIs are not substrates for BCRP.64 Relationships between PI accumulation and BCRP expression remain to be determined in vitro and in vivo.

Polymorphisms in the genes encoding transporter proteins, receptors, and drug-metabolizing enzymes have been associated with inter-individual variation. Genetic variations in the MDR1 gene have been extensively studied and generated inconsistent and variable data.65 Single nucleotide polymorphisms (SNPs) have been identified in the MDR1 gene and a relationship has been observed between a C3435T SNP in exon 26 and P-gp expression in the intestine66 and on PBMCs.67 Some studies have demonstrated individuals with the TT genotype to have high plasma digoxin levels68 whereas other studies have shown TT individuals to have low plasma fexofenadine69 or efavirenz and nelfinavir concentrations.67 One recent study demonstrated an association between individuals with the homozygous variant genotype (TT) and higher plasma and intracellular nelfinavir exposure.70 However, the SNP is in a non-coding ‘wobble’ position. This has generated many questions as to how this SNP actually correlates with reduced expression and function of P-gp. Hypotheses69,71 propose that the polymorphism in exon 26 is linked to other SNPs such as G2677T located in exon 21, since haplotype combinations of SNPs are likely to be superior to single SNPs. It is possible that C3435T is linked to a SNP that affects MDR1 gene expression in a regulatory region such as a promoter or enhancer region or is possibly linked to another polymorphic gene such as steroid xenobiotic receptor (SXR) or CYP P450 enzymes. Clearly, to understand polymorphic studies, assays for measuring protein and mRNA need to be standardized.

The lack of consistency in reported data indicate that combinations of multiple efflux transporters are more likely to be responsible. In addition, since PIs have different affinities for different transporters such as P-gp,51 it may be that a particular PI is influenced by one transporter more than another. Moreover, many different methods are available to measure expression, function or gene expression of transporters. Generally, extrapolation of in vitro to clinical data is difficult when many interrelating factors are involved in vivo.

Organic anion and cation transporters are two other major classes of transport proteins, however few data exist about their expression on lymphocytes. Organic anion transporters (OAT) have been cloned and belong to one of the following gene families: OAT, OATP or OAT-K72 whilst organic cation transporters (OCT) belong to either the OCT or OCTN family73 (Table 4). Few studies have investigated the role of organic anion and cation transport proteins in the transport of antiretrovirals. However, the PIs saquinavir, ritonavir, indinavir and nelfinavir have been shown to inhibit OCT1 in vitro, which has the potential to cause drug interactions.74 A more recent study identified that OATP-A transports saquinavir in vitro75 and the clinical relevance of this finding remains to be determined. SNPs have also been identified in OATP-C, causing a functional alteration in transport, since several variants exhibited reduced uptake of OATP-C substrates.76 Three SNPs have been identified in the human carnitine transporter gene (OCTN-2) leading to subsequent carnitine deficiency.77 In addition, variants of OAT1 have been identified as a result of alternative splicing with some splice variants (OAT1-3 and OAT1-4) reported to be potentially non-functional.73


    Summary
 Top
 Abstract
 Introduction
 Bioavailability
 Plasma protein binding
 Physiochemical properties
 Protease inhibitor intracellular...
 Drug transporter proteins
 Summary
 Acknowledgements
 References
 
New information on the intracellular accumulation of PIs is emerging, although studies are still required to clarify the issues discussed in this review and to determine whether intracellular accumulation of PIs is clinically relevant. Comparative studies of PIs suggest that many factors govern antiviral potency, and not simply the hierarchy of intracellular accumulation. Nonetheless, preliminary data indicate that intracellular accumulation can be modulated, and potentially accounts for a disconnect between plasma drug concentration and virological response. Clearly, the interaction of multiple influx and efflux transport may be important in accumulation, however, inter-individual variation in the expression of transporters, metabolizing enzymes and nuclear receptors, makes interpretation of data complicated. Hopefully, a greater understanding of intracellular pharmacology will improve long-term therapy by reducing cellular resistance.


    Acknowledgements
 Top
 Abstract
 Introduction
 Bioavailability
 Plasma protein binding
 Physiochemical properties
 Protease inhibitor intracellular...
 Drug transporter proteins
 Summary
 Acknowledgements
 References
 
Much of the work carried out was supported by an Academic Institute Grant from the British Society for Antimicrobial Chemotherapy (BSAC). Jennifer Ford was supported by a PhD studentship from AVERT.


    Footnotes
 
* Corresponding author. Tel: +44-151-794-5565; Fax: +44-151-794-5656; Email: jford{at}liv.ac.uk


    References
 Top
 Abstract
 Introduction
 Bioavailability
 Plasma protein binding
 Physiochemical properties
 Protease inhibitor intracellular...
 Drug transporter proteins
 Summary
 Acknowledgements
 References
 
1 . Robins, T. & Plattner, J. (1993). HIV protease inhibitors: their anti-HIV activity and potential role in treatment. Journal of Acquired Immune Deficiency Syndromes 6, 162–70.[Medline]

2 . Kuritzkes, D. R. (2003). Management of patients with virologic and metabolic failure. AIDS Reader 13, S17–S22.[Medline]

3 . Cinatl, J., Jr, Cinatl, J., Rabenau, H. et al. (1994). Failure of antiretroviral therapy: role of viral and cellular factors. Intervirology 37, 307–14.[ISI][Medline]

4 . Bilello, J. A. & Drusano, G. L. (1996). Relevance of plasma protein binding to antiviral activity and clinical efficacy of inhibitors of human immunodeficiency virus protease. Journal of Infectious Diseases 173, 1524–6.[ISI][Medline]

5 . Nascimbeni, M., Lamotte, C., Peytavin, G. et al. (1999). Kinetics of antiviral activity and intracellular pharmacokinetics of human immunodeficiency virus type 1 protease inhibitors in tissue culture. Antimicrobial Agents and Chemotherapy 43, 2629–34.[Abstract/Free Full Text]

6 . Jones, K., Hoggard, P. G., Sales, S. D. et al. (2001). Differences in the intracellular accumulation of HIV protease inhibitors in vitro and the effect of active transport. AIDS 15, 675–81.[CrossRef][ISI][Medline]

7 . Ford, J., Hoggard, P. G., Khoo, S. et al. (2003). Intracellular accumulation of protease inhibitors in subpopulations of lymphocytes with P-glycoprotein expression. British Pharmacology Society, Winter Meeting.

8 . Lamotte, C., Peytavin, G., Clavel, F. et al. (2003). Subcellular distribution of HIV protease inhibitors (PI) in cell culture. In Program and Abstracts of the Forty-third Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2003. Abstract A-1801. American Society for Microbiology, Washington, DC, USA.

9 . Khoo, S. H., Hoggard, P. G., Williams, I. et al. (2002). Intracellular accumulation of human immunodeficiency virus protease inhibitors. Antimicrobial Agents and Chemotherapy 46, 3228–35.[Abstract/Free Full Text]

10 . Chaillou, S., Durant, J., Garraffo, R. et al. (2002). Intracellular concentration of protease inhibitors in HIV-1-infected patients: correlation with MDR-1 gene expression and low dose of ritonavir. HIV Clinical Trials 3, 493–501.[Medline]

11 . Jones, K., Hoggard, P. G., Khoo, S. et al. (2001). Effect of alpha1-acid glycoprotein on the intracellular accumulation of the HIV protease inhibitors saquinavir, ritonavir and indinavir in vitro. British Journal of Clinical Pharmacology 51, 99–102.[CrossRef][ISI][Medline]

12 . Kashuba, A. D., Dyer, J. R., Kramer, L. M. et al. (1999). Antiretroviral-drug concentrations in semen: implications for sexual transmission of human immunodeficiency virus type 1. Antimicrobial Agents and Chemotherapy 43, 1817–26.[Free Full Text]

13 . Gao, W. Y., Cara, A., Gallo, R. C. et al. (1993). Low levels of deoxynucleotides in peripheral blood lymphocytes: a strategy to inhibit human immunodeficiency virus type 1 replication. Proceedings of the National Academy of Sciences, USA, 90, 8925–8.

14 . Jemal, M., Rao, S., Gatz, M. et al. (2003). Liquid chromatography-tandem mass spectrometric quantitative determination of the HIV protease inhibitor atazanavir (BMS-232632) in human peripheral blood mononuclear cells (PBMC): practical approaches to PBMC preparation and PBMC assay design for high-throughput analysis. Journal of Chromatography B 795, 273–89.

15 . Colombo, S., Telenti, A., Guignard, N. et al. (2004). Differences in intracellular/plasma concentration ratio and intracellular drug accumulation between PIs and NNRTIs. 5th International Workshop on Clinical Pharmacology of HIV Therapy, Rome, Italy, 2004. Abstract 52, p. 36. Virology Education, Utrecht, The Netherlands.

16 . Ford, J., Cornforth, D., Hoggard, P. G. et al. (2004). Intracellular and plasma pharmacokinetics of nelfinavir and M8 in HIV infected patients; relationship with P-glycoprotein expression. Antiviral Therapy 9, 77–84.[Medline]

17 . van Heeswijk, R. P. G., Cohen Stuart, J. W. T., Burger, D. M. et al. (2001). Long-term suppression of viral replication despite low plasma saquinavir concentrations in the CHEESE study. British Journal of Clinical Pharmacology 53, 211–4.[CrossRef][ISI]

18 . Ford, J., Boffito, M., Wildfire, A., et al. (2004). Intracellular and plasma pharmacokinetics (PKs) of saquinavir/ritonavir (SQV/r) administered once daily in HV infected patients. 11th Conference on Retroviruses and Opportunistic Infections, Sans Francisco, USA, 2004. Abstract 601, p. 283. Foundation for Retrovirology and Human Health, Alexandra, VA, USA.

19 . Peytavin, G., Landman, R., Lamotte, C. et al. (2001). Saquinavir (SQV). plasma and intracellular concentrations in a once daily dosing combination FORTOVASE (SQV-SGC)-low dose Ritonavir (RTV) in a prospective study (IMEA 015) in HIV-infected patients (pts). 2nd International Workshop of Clinical Pharmacology of HIV Therapy, Noordwijk, the Netherlands, 2001. Abstract 3.16. Virology Education, Utrecht, The Netherlands.

20 . Armbruster, C., Vorbach, H., Steindl, F. et al. (2001). Intracellular concentration of the HIV protease inhibitors indinavir and saquinavir in human endothelial cells. Journal of Antimicrobial Chemotherapy 47, 487–90.[Abstract/Free Full Text]

21 . Hoggard, P., Meaden, E., Tjia, J., et al. (2002). The intracellular accumulation of lopinavir (LPV) and ritonavir (RTV) is influenced by the expression of efflux transporters P-glycoprotein and MRP1 in HIV infected patients receiving Kaletra. Sixth International Congress on Drug Therapy in HIV Infection, Glasgow, UK, 2002. Abstract PL 8.2, p. 10. Gardiner-Caldwell, Tytherington, UK.

22 . Breilh, D., Pellegrin, I., Rouzes, A. et al. (2004). Virological, intracellular and plasma pharmacological parameters predicting response to lopinavir/ritonavir (KALEPHAR Study). AIDS 18, 1305–10.[CrossRef][ISI][Medline]

23 . Biedler, J. L. & Riehm, H. (1970). Cellular resistance to actinomycin D in Chinese hamster cells in vitro: cross-resistance, radioautographic, and cytogenetic studies. Cancer Research 30, 1174–84.[ISI][Medline]

24 . Endicott, J. A. & Ling, V. (1989). The biochemistry of P-glycoprotein-mediated multidrug resistance. Annual Review of Biochemistry 58, 137–71.[CrossRef][ISI][Medline]

25 . Gottesman, M. M. & Pastan, I. (1988). The multidrug transporter, a double-edged sword. Journal of Biological Chemistry 263, 12163–6.[Free Full Text]

26 . Lee, C. G., Gottesman, M. M., Cardarelli, C. O. et al. (1998). HIV-1 protease inhibitors are substrates for the MDR1 multidrug transporter. Biochemistry 37, 3594–601.[CrossRef][ISI][Medline]

27 . Kim, R. B., Fromm, M. F., Wandel, C. et al. (1998). The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors. Journal of Clinical Investigation 101, 289–94.[Abstract/Free Full Text]

28 . Chen, C. J., Chin, J. E., Ueda, K. et al. (1986). Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells. Cell 47, 381–9.[ISI][Medline]

29 . Higgins, C. F., Callaghan, R., Linton, K. J. et al. (1997). Structure of the multidrug resistance P-glycoprotein. Seminars in Cancer Biology 8, 135–42.[CrossRef][ISI][Medline]

30 . Loo, T. W. & Clarke, D. M. (1999). The human multidrug resistance P-glycoprotein is inactive when its maturation is inhibited: potential for a role in cancer chemotherapy. Federation of American Societies for Experimental Biology 13, 1724–32.

31 . Deeley, R. G. & Cole, S. P. (1997). Function, evolution and structure of multidrug resistance protein (MRP). Seminars in Cancer Biology 8, 193–204.[CrossRef][ISI][Medline]

32 . Litman, T., Druley, T. E., Stein, W. D. et al. (2001). From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance. Cellular and Molecular Life Sciences 58, 931–59.[ISI][Medline]

33 . Gottesman, M. M., Fojo, T. & Bates, S. E. (2002). Multidrug resistance in cancer: role of ATP-dependent transporters. Nature Reviews Cancer 2, 48–58.[CrossRef][ISI][Medline]

34 . Doyle, L. A. & Ross, D. D. (2003). Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2). Oncogene 22, 7340–58.[CrossRef][ISI][Medline]

35 . Chan, L. M., Lowes, S. & Hirst, B. H. (2004). The ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability. European Journal of Pharmaceutical Sciences 21, 25–51.[CrossRef][ISI][Medline]

36 . Huai-Yun, H., Secrest, D. T., Mark, K. S. et al. (1998). Expression of multidrug resistance-associated protein (MRP) in brain microvessel endothelial cells. Biochemical and Biophysical Research Communications 243, 816–20.[CrossRef][ISI][Medline]

37 . Kruh, G. D. & Belinsky, M. G. (2003). The MRP family of drug efflux pumps. Oncogene 22, 7537–52.[CrossRef][ISI][Medline]

38 . Borst, P., Evers, R., Kool, M. et al. (2000). A family of drug transporters: the multidrug resistance-associated proteins. Journal of the National Cancer Institute 92, 1295–302.[Abstract/Free Full Text]

39 . Kage, K., Tsukahara, S., Sugiyama, T. et al. (2002). Dominant-negative inhibition of breast cancer resistance protein as drug efflux pump through the inhibition of S-S dependent homodimerization. International Journal of Cancer 97, 626–30.[CrossRef][ISI]

40 . Luckie, D. B., Wilterding, J. H., Krha, M. et al. (2003). CFTR and MDR: ABC transporters with homologous structure but divergent function. Current Genomics 4, 109–21.

41 . Hoetelmans, R. M. (1998). Sanctuary sites in HIV-1 infection. Antiviral Therapy 3, Suppl. 4, 13–17.

42 . Jette, L., Tetu, B. & Beliveau, R. (1993). High levels of P-glycoprotein detected in isolated brain capillaries. Biochimica et Biophysica Acta (BBA) — Reviews on Cancer 1150, 147–54.

43 . Neyfakh, A. A., Serpinskaya, A. S., Chervonsky, A. V. et al. (1989). Multidrug-resistance phenotype of a subpopulation of T-lymphocytes without drug selection. Experimental Cell Research 185, 496–505.[ISI][Medline]

44 . Chaudhary, P. M., Mechetner, E. B., Roninson, I. B. et al. (1992). Expression and activity of the multidrug resistance P-glycoprotein in human peripheral blood lymphocytes. Blood 80, 2735–9.[Abstract]

45 . Wang, X., Furukawa, T., Nitanda, T. et al. (2003). Breast cancer resistance protein (BCRP/ABCG2) induces cellular resistance to HIV-1 nucleoside reverse transcriptase inhibitors. Molecular Pharmacology 63, 65–72.[Abstract/Free Full Text]

46 . Mouly, S., Lown, K. S., Kornhauser, D. et al. (2002). Hepatic but not intestinal CYP3A4 displays dose-dependent induction by efavirenz in humans. Clinical Pharmacology and Therapeutics 72, 1–9.[CrossRef][ISI][Medline]

47 . Eagling, V. A., Profit, L. & Back, D. J. (1999). Inhibition of the CYP3A4-mediated metabolism and P-glycoprotein-mediated transport of the HIV-1 protease inhibitor saquinavir by grapefruit juice components. British Journal of Clinical Pharmacology 48, 543–52.[CrossRef][ISI][Medline]

48 . Wacher, V. J., Silverman, J. A., Zhang, Y. et al. (1998). Role of P-glycoprotein and cytochrome P450 3A in limiting oral absorption of peptides and peptidomimetics. Journal of Pharmaceutical Sciences 87, 1322–30.[CrossRef][ISI][Medline]

49 . Mouly, S. & Paine, M. F. (2003). P-glycoprotein increases from proximal to distal regions of human small intestine. Pharmaceutical Research 20, 1595–9.[CrossRef][ISI][Medline]

50 . 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, 1623–7.[CrossRef][ISI][Medline]

51 . Srinivas, R. V., Middlemas, D., Flynn, P. et al. (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, 3157–62.[Abstract/Free Full Text]

52 . Speck, R. R., Yu, X. F., Hildreth, J. et al. (2002). Differential effects of p-glycoprotein and multidrug resistance protein-1 on productive human immunodeficiency virus infection. Journal of Infectious Diseases 186, 332–40.[CrossRef][ISI][Medline]

53 . Owen, A., Chandler, B., Ford, J. et al. (2003). Differential expression of human immunodeficiency virus coreceptors, by CEM, CEMVBL and CEM E1000 Cells. Journal of Infectious Diseases 187, 874–5.[CrossRef][ISI][Medline]

54 . Bleiber, G., May, M., Suarez, C. et al. (2004). MDR1 genetic polymorphism does not modify either cell permissiveness to HIV-1 or disease progression before treatment. Journal of Infectious Diseases 189, 583–6.[CrossRef][ISI][Medline]

55 . Walker, J., Martin, C. & Callaghan, R. (2004). Inhibition of p-glycoprotein function by XR9576 in a solid tumour model can restore anticancer drug efficacy. European Journal of Cancer 40, 594–605.[CrossRef][ISI][Medline]

56 . Meaden, E. R., Hoggard, P. G., Newton, P. et al. (2002). P-glycoprotein and MRP1 expression and reduced ritonavir and saquinavir accumulation in HIV-infected individuals. Journal of Antimicrobial Chemotherapy 50, 583–8.[Abstract/Free Full Text]

57 . Perloff, M. D., von Moltke, L. L., Fahey, J. M. et al. (2000). Induction of P-glycoprotein expression by HIV protease inhibitors in cell culture. AIDS 14, 1287–9.[CrossRef][ISI][Medline]

58 . Huang, L., Wring, S. A., Woolley, J. L. et al. (2001). Induction of P-glycoprotein and cytochrome P450 3A by HIV protease inhibitors. Drug Metabolism and Disposition 29, 754–60.[Abstract/Free Full Text]

59 . Chandler, B., Almond, L., Ford, J. et al. (2003). The effect of protease inhibitors and nonnucleoside reverse transcriptase inhibitors on P-glycoprotein expression in peripheral blood mononuclear cells in vitro. Journal of Acquired Immune Deficiency Syndromes 33, 551–6.[Medline]

60 . Ford, J., Meaden, E. R., Hoggard, P. G. et al. (2003). Effect of protease inhibitor-containing regimens on lymphocyte multidrug resistance transporter expression. Journal of Antimicrobial Chemotherapy 52, 354–8.[Abstract/Free Full Text]

61 . Bossi, P., Legrand, O., Faussat, A. M. et al. (2003). P-glycoprotein in blood CD4 cells of HIV-1-infected patients treated with protease inhibitors. HIV Medicine 4, 67–71.[CrossRef][Medline]

62 . Huisman, M. T., Smit, J. W., Crommentuyn, K. M. et al. (2002). Multidrug resistance protein 2 (MRP2) transports HIV protease inhibitors, and transport can be enhanced by other drugs. AIDS 16, 2295–301.[CrossRef][ISI][Medline]

63 . Laupeze, B., Amiot, L., Payen, L. et al. (2001). Multidrug resistance protein (MRP) activity in normal mature leukocytes and CD34-positive hematopoietic cells from peripheral blood. Life Sciences 68, 1323–31.[CrossRef][ISI][Medline]

64 . Gupta, A., Zhang, Y., Unadkat, J. D. et al. (2004). HIV protease inhibitors are inhibitors but not substrates of the human breast cancer resistance protein (BCRP/ABCG2). Journal of Pharmacology and Experimental Therapeutics 310, 334–41.[Abstract/Free Full Text]

65 . Marzolini, C., Paus, E., Buclin, T. et al. (2004). Polymorphisms in human MDR1 (P-glycoprotein): recent advances and clinical relevance. Clinical Pharmacology and Therapeutics 75, 13–33.[CrossRef][ISI][Medline]

66 . Hoffmeyer, S., Burk, O., von Richter, O., 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, 3473–8.

67 . Fellay, J., Marzolini, C., Meaden, E. R. et al. (2002). Response to antiretroviral treatment in HIV-1-infected individuals with allelic variants of the multidrug resistance transporter 1: a pharmacogenetics study. Lancet 359, 30–6.[CrossRef][ISI][Medline]

68 . Johne, A., Kopke, K., Gerloff, T. et al. (2002). Modulation of steady-state kinetics of digoxin by haplotypes of the P-glycoprotein MDR1 gene. Clinical Pharmacology and Therapeutics 72, 584–94.[CrossRef][ISI][Medline]

69 . Kim, R. B., Leake, B. F., Choo, E. F. et al. (2001). Identification of functionally variant MDR1 alleles among European Americans and African Americans. Clinical Pharmacology and Therapeutics 70, 189–99.[CrossRef][ISI][Medline]

70 . Colombo, S., Buclin, T., Tauber, B. L., et al. (2004). Are MDR1 genotypes (3435C > T and 2677G > T) associated with total plasma and intracellular disposition of nelfinavir (NFV) and efavirenz (EFV)? 5th International Workshop on Clinical Pharmacology of HIV Therapy, Rome, Italy, 2004. Abstract 50, p. 35. Virology Education, Utrecht, The Netherlands.

71 . Tanabe, M., Ieiri, I., Nagata, N. et al. (2001). Expression of P-glycoprotein in human placenta: relation to genetic polymorphism of the multidrug resistance (MDR)-1 gene. Journal of Pharmacology and Experimental Therapeutics 297, 1137–43.[Abstract/Free Full Text]

72 . Dresser, M. J., Leabman, M. K. & Giacomini, K. M. (2001). Transporters involved in the elimination of drugs in the kidney: organic anion transporters and organic cation transporters. Journal of Pharmaceutical Sciences 90, 397–421.[CrossRef][ISI][Medline]

73 . Lee, W. & Kim, R. B. (2004). Transporters and renal drug elimination. Annual Review of Pharmacology and Toxicology 44, 137–66.[CrossRef][ISI][Medline]

74 . Zhang, L., Gorset, W., Washington, C. B. et al. (2000). Interactions of HIV protease inhibitors with a human organic cation transporter in a mammalian expression system. Drug Metabolism and Disposition 28, 329–34.[Abstract/Free Full Text]

75 . Su, Y., Zhang, X., Sinko, P. J. et al. (2003). Human organic anion-transporting polypeptide OATP-A (SLC21A3) acts in concert with P-glycoprotein and multidrug resistance protein 2 in the vectorial transport of saquinavir in Hep G2 cells. Molecular Pharmaceutics 1, 49–56.[CrossRef]

76 . Tirona, R. G., Leake, B. F., Merino, G. et al. (2001). Polymorphisms in OATP-C: identification of multiple allelic variants associated with altered transport activity among European- and African-Americans. Journal of Biological Chemistry 276, 35669–75.[Abstract/Free Full Text]

77 . Sweet, D. H., Bush, K. T. & Nigam, S. K. (2001). The organic anion transporter family: from physiology to ontogeny and the clinic. American Journal of Physiology — Renal Physiology 281, F197–F205.

78 . Garraffo, R., Lavrut, T., Simonet, P., et al. (2004). Intracellular concentration of indinavir (IDV) in patients with AIDS receiving two different doses of indinavir/ritonavir (IDV/rtv). 5th International Workshop on Clinical Pharmacology of HIV Therapy, Rome, Italy, 2004. Abstract 82, p. 58. Virology Education, Utrecht, The Netherlands.

79 . Lamotte, C., Landman, R., Peytavin, G. et al. (2004). Once-daily dosing of saquinavir soft-gel capsules and ritonavir combination in HIV-1-infected patients (IMEA015 study). Antiviral Therapy 9, 247–56.[Medline]

80 . Hagenbuch, B., Meier, P. J. et al. (2003). The superfamily of organic anion transporting polypeptides. Biochimica et Biophysica Acta (BBA)—Reviews on Cancer 1609, 1–18.