Multidrug therapies have significantly prolonged life and improved the quality of life of patients infected with HIV-1. With such combined therapies, the viral load of HIV-1 in plasma may be reduced to a level below the limit of detection. However, undetectable plasma levels do not correlate with the viral load in the brain due to the limited penetration of anti-HIV drugs across the blood-brain barrier (BBB). As a result, residual viruses in the brain may constitute a reservoir from where they could repopulate compartments that have been cleared of the virus and are often associated with AIDS dementia complex, characterized by progressive cognitive and motor dysfunctions.
The discovery of HIV protease inhibitors (HPIs) has been a major advance in the treatment of patients infected with HIV. However, these HPIs have a limited penetration into the central nervous system (CNS). One major limitation to their penetration into the brain is active efflux. Several efflux proteins have been discovered in the brain such as P-glycoprotein (P-gp), multidrug resistance–associated proteins (MRPs), and brain multidrug resistance protein (BMDP), while others remain unaccounted for.
This paper reviews available information regarding the cerebral transport of HPIs taking in consideration the chemical properties susceptible to influence penetration from the blood circulation into the brain and the interactions with efflux proteins that can influence efflux from the brain back into the blood circulation.
BRAIN AND HIV ENCEPHALITIS
In the brain, cells responsible for the establishment of the barriers are the capillary endothelial cells, representing the BBB, and the epithelial glial cells representing the blood cerebrospinal fluid barrier. Due to the greater surface area of the BBB compared with the blood cerebrospinal fluid barrier, the BBB has a major role in controlling the transport of endogenous and exogenous compounds from the blood circulation to the brain parenchyma.
The BBB is a lipophilic membrane represented by cerebral capillary endothelial cells and characterized by the presence of tight junctions, low enzymatic metabolism, the lack of intracellular vesicles and of endothelial fenestrae, and the presence of efflux proteins. These properties, specific to the cerebral endothelial cells and not found in other endothelial cells, protect the brain from the free entry of toxins and pathogens coming from the blood circulation.
This intrinsic system of protection may prove itself deleterious in regards to brain bioavailability as it may significantly reduce brain uptake of drugs. This is the case for anti-retroviral agents and in particular HPIs.
A certain number of ATP binding cassette (ABC) proteins have been described as transporters of drugs. Among them, 3 proteins or groups of proteins are known at the present time: P-gp, several MRPs, and the newly discovered BMDP. 1,2 These proteins have been reported inside the epithelium and the endothelium of different organs such as intestine, liver, BBB, and testis. All these proteins are mainly located within the plasma membrane, where they are able to efflux numerous drugs from the cells. P-gp has also been detected on the nuclear envelope and on the membrane of cytoplasmic organelles. 3,4 It has been suggested that the role of these proteins is to protect the CNS and several tissue compartments vulnerable to toxicity from penetration of lipophilic xenobiotics.
Of the 3 majors proteins cited above, P-gp is the most well known. The exact location of P-gp in the BBB is still controversial. 5,6 While most authors showed its presence in endothelial cells of human, rat, mice, bovine, and porcine brain capillaries, Pardridge et al 7 placed the transporter at the astrocyte foot process. P-gp has also been described at the blood–cerebrospinal fluid (CSF) barrier 8 at the apical membrane in the choroid plexus.
Multidrug Resistance–Associated Proteins
MRPs consist of several members and, like P-gp, may be found in different organs. Among MRP isoforms, MRP1 has been observed in various in vitro models of the BBB. It has also been characterized in vivo at the BBB 9 and CSF barrier level, 10 in astrocytes, 11 and in microglia. 12
Brain Multidrug Resistance Protein
This new member of the ATP binding cassette superfamily has been described recently at the porcine BBB (BMDP). 1 This transporter shows similarity with the breast cancer resistance protein ABCG2/BCRP/MXR.
Brain as a Sanctuary Site for HIV During HIV-1 Encephalitis
HIV-associated dementia usually occurs during the late stages of the disease and manifests as a spectrum of neurologic and psychiatric symptoms 13 comprising distraction, confusion, apathy, withdrawal, and emotional disturbances. Some patients may also have parkinsonian tremor, ataxia, and limb weakness.
The presence of large quantities of unintegrated viral DNA in the brain of HIV patients strongly suggests that HIV resides in the brain. 14 HIV-1 enters the brain early in the infection, constitutes a sanctuary site for virus replication, and persists in the brain throughout the course of the disease. Once in the brain, the virus is often protected from anti-HIV drugs that are present in the blood circulation and prevented from crossing the BBB. HIV-1 is also detected in the CSF at the early stages of the disease and the concentration of the virus may be higher in the CSF compared with that in the blood circulation. However, the relation between HIV-1 in the CSF and in the brain remains unknown. HIV-1 probably enters the brain in infected monocytes/macrophages rather than T cells. 15 Dallasta et al 16 have demonstrated that a significant BBB disruption occurs during HIV-1 encephalitis and that this damage of the critical barrier structure serves as the primary portal of entry whereby activated HIV-1-infected cells gain access to the brain.
It has been demonstrated that, even if the viral load is reduced in plasma, the virus is still able to survive and infect cells all over the body and especially the brain.
For this reason, transport of anti-HIV drugs through the BBB into the brain is of particular interest to optimize the treatment of AIDS and avoid HIV encephalitis.
Factors Involved in the Cerebral Transport of HIV Protease Inhibitors
Drugs may be transported through the BBB by passive or active transport.
Regarding the passive transfer through the BBB, the main factors that intervene are ionization of the drug, molecular weight, lipophilicity, and protein binding. 17 All these factors define Fick's law of diffusion and condition the transfer of compounds through biologic membranes. The main physical and chemical properties of HPIs involved in the transport of HPIs through the BBB are reported in Table 1.
Ionization of HPIs
Transport through the BBB is decreased in case of ionization of acidic compounds. Ionization of basic groups has no significant effect. 18 pKa values of anti-HPIs (Table 1) show that most of these compounds are weak basic compounds slightly ionized at physiological pH.
Drug transport through the BBB is inversely proportional to its molecular weight. 19 It has been demonstrated that molecular weight may be a limiting factor for values >600 days. Molecular weights of HPIs approach this limit.
Lipophilicity is probably one of the most important factors conditioning brain uptake of drugs. 19 It is described as the octanol/phosphate-buffered saline distribution coefficient (log P) corresponding to the partition coefficient of a nonionized compound between an organic solution and an aqueous solution. Transport of a drug through the BBB is generally directly proportional to its lipophilicity. However, too high values of lipophilicity may decrease the rate of transport due to trapping of the compound inside the membrane. Values of log P falling between −0.2 and 1.3 have been described as optimal for the cerebral transport. Within these values, cerebral transfer depends on blood flow and the permeability coefficient. 20,21 Moreover, a good correlation has been described between log P and the permeability coefficient as long as the molecular weight remains <800 daltons. 22 Values of partition coefficients of HPIs sometimes vary according to the studies (Table 1) but are always higher than the upper optimal value (1.3), indicating that these compounds are rather highly lipophilic and could be partially trapped inside the membrane during their transport. 23
Another important factor involved in drug transport through the BBB is protein binding. Logically, due to the size of the protein-drug complex and the characteristics of the BBB, only the free fraction of the drug should be transported through the BBB. This theory and protein binding as limiting factor have been questioned. Several articles have shown that the fraction of a drug that is transported through the BBB is higher than the free fraction. 24 Different hypotheses have been suggested such as the dissociation of the protein-drug complex in the microcirculation following the binding of the protein to a specific endothelial receptor. 25
When considering all these chemical and physical characteristics, the different properties of the HPIs are not very favorable for transport through the BBB. Protein binding is high for most of HPIs, except indinavir. Molecular weight is close to the limit value of 600 daltons for most of them and log P values are much higher than the optimal interval of −0.2 and 1.3.
Polli et al 26 investigated the transport of amprenavir, ritonavir, indinavir, and saquinavir across Caco-2 monolayers. The epithelial cell permeability based on the apparent permeability x surface coefficient (Papp) was at the following rank order: amprenavir = ritonavir > indinavir = saquinavir, showing that amprenavir and ritonavir have better permeation properties and should be more effective at penetrating membrane barriers, when compared with indinavir and saquinavir. However, Glynn and Yazdanian 23 did not succeed in establishing a correlation between in vitro (brain microvessel endothelial cell permeability) and in vivo (brain or CSF/plasma concentration ratios) data. This lack of correlation, mostly based on literature reports, probably comes from differences in experimental conditions.
For some drugs, transfer through the BBB may be higher or lower than that expected from the chemical and physical properties. A facilitated or an active transport can be responsible for a higher rate of transfer. In case of lower rate of transfer, efflux proteins may be involved. When studying the transport of anti-HIV drugs inside Caco-2 cells, the reported absorptive permeabilities (Papp) for HPIs were lower than those that might be expected with their high lipophilicities, suggesting that efflux proteins are involved in their transport. 27
INTERACTIONS OF P-GLYCOPROTEIN AND HIV PROTEASE INHIBITORS
Properties of HPIs in Favor of Interactions With P-Glycoprotein
General properties have been reported as essential for drugs to be either substrate or modulator of P-gp and other efflux proteins, such as lipophilicity, molecular weight, and hydrogen-bonding.
Values of log P octanol-water partition between 3.6–4.5 have been described as optimal for binding to P-gp. 28 The range of lipophilicity of HPIs (2.03–5.2, Table 1) fits relatively well with the one described by Bain and Leblanc. 28
Among 40,000 compounds tested as substrates or inhibitors of P-gp in human colon carcinoma cells, average molecular weight of substrates was 636 daltons while average molecular weight for inhibitors was 558. 29 These 2 molecular weight values are very close to those of HPIs.
Seelig and Landwojtowicz 30 suggested that the dissociation rate of the P-gp-substrate complex is controlled by the number of hydrogen bonds. HPIs present high potential for hydrogen bonding.
It has been previously demonstrated that a basic nitrogen atom was required for a drug to be recognized as P-gp substrate. Tang-Wai et al 31 showed that, for colchicine and analogues, the nitrogen atom of the acetamido group at position 7 was essential for P-gp recognition. All HPIs possess a nitrogen basic atom. However, other studies have reported that drugs without this basic nitrogen atom, such as dexamethasone, could also be P-gp substrates. 32
Characteristics of HPIs (lipophilicity, H bonding, basic N atom) seem to be in favor for being P-gp substrates or modulators. We next focus on the in vitro and in vivo experiments investigating this aspect.
HIV PROTEASE INHIBITORS AS SUBSTRATES OF P-GLYCOPROTEIN
HPIs as Substrates of P-Glycoprotein at the BBB Level
HPIs as Cerebral P-gp Substrates In Vitro
Most of the in vitro experiments used different animal or human cerebral cell culture models to investigate the transport of HPIs with or without P-gp inhibitors.
Glynn and Yazdanian 23 used probenecid and verapamil as efflux pump inhibitors and investigated the effect of these inhibitors on the permeability of different HPIs (indinavir, saquinavir, amprenavir) in a bovine brain microvessel endothelial cell model. After inhibition by probenecid or verapamil, permeability was unchanged or reduced for all these agents, except for saquinavir, which showed a 5-fold increase. These results suggested that the contribution of efflux pumps (inhibited by probenecid or verapamil) to low cellular permeability of these anti-HIV agents was not significant, except for saquinavir.
In an in vitro BBB co-culture model made of bovine brain capillary endothelial cells with rat brain astrocytes, van der Sandt et al 33 showed a polarized transport for the 3 HPIs, amprenavir, ritonavir, and indinavir, but this transport was much lower than that observed on LLC-PK1:MDR1 pig kidney epithelial cells overexpressing P-gp and on Caco-2 cell lines. The P-gp inhibitors verapamil, LY 335979, and valspodar (PSC833) were effective in blocking HPIs' active transport, with verapamil being the strongest.
Using porcine primary brain capillary endothelial cell monolayers, saquinavir was transported by P-gp and this transport was inhibited by ritonavir, saquinavir, indinavir, and valspodar. 34
In a co-culture-based model of human BBB using human brain microvascular endothelial cell with human astrocytes, a pretreatment of cells with the P-gp inhibitor quinidine significantly increased the transport of indinavir from the apical to the basal compartment, with the flux in this direction twice that in the absence of quinidine. 35 This influence of P-gp in indinavir transport was confirmed by uptake studies showing increased accumulation of indinavir in the presence of 5 μM indinavir. Mégard et al 35 used also native membrane vesicles containing P-gp to investigate the interaction between indinavir, vinblastine (a well-known P-gp substrate), and P-gp. Competition experiments showed that binding of indinavir and vinblastine to P-gp was mutually exclusive and that indinavir bound to the vinblastine binding site on P-gp.
HPIs as Cerebral P-gp Substrates In Vivo
In vivo experiments used either wild-type animals pre-treated or not with a P-gp inhibitor or P-gp-deficient animals, described by Polli et al 26 as “chemical” or “genetic” knockout animals, respectively.
Using microdialysis in rats, Edwards et al 36 observed that brain-to-blood ratio of amprenavir was significantly increased in the presence of the efflux inhibitor elacridar (GF120918), compared with the vehicle (0.617 vs. 0.076, respectively).
Also using GF120918, Savolainen et al 37 showed in rats that the P-gp inhibitors can increase nelfinavir brain concentrations.
In their experiments, Polli et al 26 showed that GF120918 increased brain concentrations of amprenavir by a 13-fold factor, whereas in double mdr1a−/−/1b−/− knockout mice, they reported a 27-fold increase.
In mice treated with nelfinavir, a pretreatment with the P-gp inhibitor LY335979 increased brain levels by 25-fold, whereas plasma concentrations were increased by only 1.8-fold. The modulation by LY335979 was dose dependent. Comparison of nelfinavir transport in P-gp-deficient mice and mice pretreated with LY335979 showed that P-gp was inhibited by about 75% with a LY335979 dose of 50 mg/kg. 38
After IV administration of indinavir, nelfinavir, or saquinavir to mdr1a−/− mice and their wild type, brain concentrations were 8-10-fold higher in mdr1a−/− for indinavir and saquinavir and about 40-fold higher for nelfinavir when compared with wild-type mice. 39
Brain penetration of saquinavir was much higher (1.8-fold factor) in mdr1a−/−/1b−/− mice when compared with wild-type mice. High-dose ritonavir did not abrogate P-gp-mediated cerebral transport of saquinavir. 40
When either injected IV or administered orally, saquinavir brain concentrations were increased in P-gp-deficient mice when compared with wild type by a factor of 5 (for IV route) and 10 (for oral gavage). 41
After IV injection of indinavir (2 mg/kg) in wild-type and mdr1a−/− knockout mice, brain concentrations in knockout mice were 5.8 times higher after 6 minutes, twice as high after 2 hours, and unchanged after 4 and 24 hours, when compared with wild-type mice, while plasma concentrations remained unchanged in both groups. 35
[14C]amprenavir was administered orally at the dose of 50 mg/kg to “chemical” P-gp knockout mice (treated with the P-gp inhibitor GF120918) and “genetic” knockout mice (mdr1a−/−/1b−/−) and brain concentrations were determined and compared with mice treated with the vehicle and wild-type mice, respectively. 26 Co-administration with GF120918 led to a 13-fold increase in the brain while a 27-fold increase in the brain concentrations of amprenavir was observed in mdr1a−/−/1b−/− mice, compared with wild-type.
HPIs as Substrates of P-Glycoprotein in Other Barriers
Most of the studies investigating HPIs as P-gp substrates and conducted in other barriers confirmed the results obtained at the BBB level.
On HCT-8 human intestinal adenocarcinoma cells, expressing high levels of P-gp, saquinavir showed a P-gp-mediated transport across the epithelial monolayer. 42
Accumulation of saquinavir was higher in MES-SA, P-gp-negative cells derived from human uterine sarcoma than in Dx5, P-gp-positive cells derived from MES-SA cells grown with doxorubicin, 41 indicating that saquinavir is a substrate of P-gp.
Comparing 3 different models to test drugs as P-gp substrates (monolayer efflux, ATPase activity, and calcein-AM assay), Polli et al 43 also investigated amprenavir, nelfinavir, saquinavir, and ritonavir. The 4 anti-HIV compounds tested positive for the 3 assays and were described as “unambiguous substrates.” Polli et al 26 investigated the effect of the P-gp inhibitor GF120918 on the transport of amprenavir, ritonavir, indinavir, and saquinavir across Caco-2 monolayers. The addition of GF120918 abolished any significant directionality in transport rates, indicating that these 4 HPIs are substrates of P-gp.
Saquinavir and ritonavir transport was investigated in peripheral blood mononuclear cells (PBMCs) isolated from HIV patients. 44 Ritonavir, but not saquinavir, correlated with the expression of P-gp in these cells.
Using LLC-PK1:MDR1 pig kidney epithelial cells over-expressing P-gp and different P-gp inhibitors (verapamil, LY 335979, and valspodar), van der Sandt et al 33 investigated the interactions between P-gp and the HPIs amprenavir, ritonavir, and indinavir. They observed that the effects of these blockers were different. The active transport of amprenavir was similarly inhibited at 50–60% by the 3 inhibitors. LY 335979 was a much stronger inhibitor of the transport of ritonavir when compared with verapamil and valspodar. Indinavir transport was inhibited by LY 335979 and, surprisingly, increased by valspodar.
Placental P-gp is also able to efflux saquinavir as demonstrated by Smit et al. 45 Using mice fetuses of 3 different genotypes (mdr1a+/+/1b+/+, mdr1a+/−/1b+/−, mdr1a−/−/1b−/−), they demonstrated that IV administration of [14C]saquinavir to pregnant dams resulted in much higher concentrations of the protease inhibitor in mdr1a−/−/1b−/− fetuses than in wild-type fetuses. Similar results were obtained in mice treated with the P-gp inhibitors valspodar or GF120918 compared with mice treated with the vehicle.
In summary, regarding HPIs as P-gp substrates, many studies have investigated saquinavir at the BBB level 23,34,39–42 as well as in other barriers 26,42,43,45 and concluded that this HPI is a substrate of P-gp, with the exception of one study that was conducted in PBMCs from HIV patients. 44 Studies showed that ritonavir also acted as a P-gp substrate. 33,34,43 For indinavir, most of the studies concluded that this HPI was a substrate, 26,33–35,39 with 2 studies indicating that P-gp inhibitors such as verapamil 23 and valspodar 33 had no effect on its permeability in cells expressing P-gp. For amprenavir, all studies except one 23 reported this HPI as a substrate 26,33,36,43 with a 27-fold increase in transport after P-gp inhibition. 26 Nelfinavir was also found to act as a P-gp substrate 43 with very high increase ratios up to 25 46 or 40 39 after P-gp inhibition.
Most of the experimental models used to investigate HPIs as substrates of efflux proteins show limitations that may introduce bias. Cell lines used in in vitro experiments show overexpression of P-gp, with levels probably much higher than levels observed in physiological barriers. P-gp-deficient mice also have limitations as they present an altered expression of CYP3A4 47 and BCRP. 48 Finally, most of the P-gp inhibitors are also inhibitors of other efflux proteins. This is the case with valspodar, also inhibitor or MRP1 and MRP2, and of elacridar (GF120918), inhibitor of both P-gp and BCRP.
However, even if the models used to investigate HPIs as substrates of P-gp have limitations, most of them strongly suggest that HPIs are substrates of P-gp and other efflux proteins.
HPIs as Inhibitors of P-Glycoprotein
HPIs as Inhibitors of P-Glycoprotein at the BBB Level
Most in vitro and in vivo cerebral studies investigating the potency to inhibit P-gp were limited to ritonavir, with differing results.
Brain penetration of saquinavir was much higher (1.8-fold factor) in mdr1a−/−1b−/− mice when compared with wild-type mice. In vivo, high-dose ritonavir did not abrogate P-gp-mediated cerebral transport of saquinavir. 40
In 6 patients treated with indinavir, the addition of ritonavir increased by 2.4 the ratio of CSF to blood concentrations 1 hour after drug administration. 49 However, area under the concentration curve (AUC)CSF/(AUC)blood ratio, considered as gold standard for drug entry in CSF, was not evaluated.
Tayrouz et al 50 investigated in humans the effect of ritonavir on the pharmacokinetics and tolerance of the antidiarrheal drug loperamide. Loperamide is a peripherally acting opioid receptor agonist that presents a low penetration of the BBB due to extrusion by P-gp from the brain endothelial cells and is highly metabolized by CYP3A. Two groups of HIV patients were treated with loperamide, one group associated with ritonavir and the other receiving the corresponding placebo. Loperamide pharmacokinetics were studied and central opioid effects were measured by evaluation of pupil diameter, cold pressor test, and transcutaneous PCO2 and PO2. Ritonavir increased the AUC of loperamide, delayed the formation of the major desmethyl metabolite, and increased the urinary metabolic ratio while the total loperamide and metabolite urinary excretion remained unchanged. Moreover, no additional central effects due to loperamide were observed after administration of ritonavir when compared with placebo. According to the authors, these results were in favor of an inhibitory effect of CYP by ritonavir without any effect on P-gp. Effectively, an inhibition of P-gp would have increased the central concentrations of loperamide and should have increased the rate of CNS adverse effects.
The inhibitory potency of 3 HPIs (saquinavir, ritonavir, and indinavir) was investigated in vitro in a model of microglia cell line (MLS-9) using radiolabeled digoxin as P-gp substrate. The accumulation of digoxin was increased with pretreatment of the cell line with HPIs. The P-gp specificity of these inhibitors was evidenced by an increase in the rate of accumulation in the overexpressing line but not in the corresponding parent line. 51
Drewe et al 34 showed on a brain capillary endothelial cell model that ritonavir could inhibit saquinavir transport.
In Vitro/In Vivo Comparison
The 3 following studies investigated ritonavir as a P-gp inhibitor both in an in vitro cell model and in the animal brain.
The inhibitory effect of HPIs on P-gp has been investigated on pig-kidney LLC-PK1 cells overexpressing mdr genes and in mdr1a−/−/1b−/− knockout mice. 40 On cell models, saquinavir and ritonavir were actively transported by human MDR1 and mouse mdr1a P-gp, and both HPIs were moderate P-gp inhibitors. In mdr1a−/−/1b−/− mice, saquinavir brain transport 40 was mediated by P-gp and was not inhibited by high-dose ritonavir co-administration.
Polli et al 26 investigated the effect of a pretreatment with ritonavir on the permeability and distribution of amprenavir. Pretreatment of Caco-2 cells with 25 μM ritonavir increased the permeability of amprenavir and pretreatment of mice with ritonavir for 4 days increased the concentrations of amprenavir in testis, blood, and muscle by a 1.8–2.4 factor while not modifying brain concentrations, while pretreatment of mice with the P-gp inhibitor GF120918 increased cerebral concentrations by a 13-fold factor.
The tissue distribution of DPC 681, a potent and selective inhibitor of HIV-1, was investigated with and without ritonavir using quantitative whole-body autoradiography (QWBA) in rats 52 and using transport studies in Caco-2 cells with the efflux inhibitor GF120918. In Caco-2 cells, ritonavir at 5–10 μM significantly inhibited the P-gp-mediated transport of DPC 681. In rats, QWBA analysis following administration of DPC 681, with or without ritonavir, showed very low levels of DPC 681 in the brain and higher concentrations in the CSF. Combinations with ritonavir did not significantly modify concentrations in the brain, although they increased CSF concentrations by a 4-fold factor. These results indicate that CSF data alone should be interpreted with caution and cannot be used alone as a surrogate for CNS penetration. 52
HPIs as Inhibitors of P-Glycoprotein in Other Barriers
On HCT-8 human intestinal adenocarcinoma cells, addition of 5–20 μM saquinavir resulted in a dose-dependent reduction of the basolateral to apical transport of rhodamine 123, a P-gp substrate, across the membrane, 42 indicating that saquinavir is a P-gp inhibitor.
In MDR-expressing Dx5 cells, derivative from human uterine sarcoma MES-SA cell line, the accumulation of the P-gp substrates vinblastine, daunorubicin, or paclitaxel increased in a dose-dependent manner in the presence of 50 μM of ritonavir, saquinavir, or nelfinavir, indicating inhibition of P-gp. Higher concentrations of indinavir, up to 250 μM, were required to obtain the same level of P-gp inhibition. 53
In LLC-PK1:MDR1 cells, amprenavir, ritonavir, and indinavir were transported by P-gp with no effect on their own transport. 33 HPIs at concentrations of 1, 10, or 100 μg/mL did not influence their transport at the concentration of 10 μg/mL, indicating that there was no competitive interaction of HPIs at the level of P-gp binding sites at the given concentrations.
The effect of ritonavir on the pharmacokinetics of a P-gp substrate, docetaxel, was investigated, showing that the 50-fold increase in systemic exposure of oral docetaxel, when combined with ritonavir, was mainly explained by a decrease in metabolism and that the inhibition of P-gp did not seem to contribute to this increase at the intestinal level. 54
Lopinavir showed inhibitory properties in the transport of rhodamine in Caco-2 monolayer cells. 55
Comparison in Inhibitory Potencies Between HPIs In Vitro
The following studies compared the potencies of HPIs as P-gp inhibitors in different in vitro models. As to investigating the accumulation effects of radiolabeled digoxin in Caco-2 cells, various HPIs were tested as inhibitors of P-gp. Nelfinavir proved to be the most potent inhibitor followed by ritonavir, saquinavir, and indinavir. 38
Ritonavir, indinavir, nelfinavir, and valspodar (SDZ PSC 833) increased the net uptake of saquinavir into porcine primary brain capillary endothelial cells, with ritonavir showing a >50 times higher inhibitory potency than nelfinavir and indinavir and an approximately 6-fold higher potency than valspodar. 34
In T cells, HPIs have also been described as efflux protein inhibitors. With the use of calcein flux assays in T-lymphocytic cell line CEM/VLB100 expressing MDR1, 4 HPIs were found to increase calcein fluorescence, indicating their interaction with MDR1. The resulting affinities were as follows: ritonavir > nelfinavir > indinavir > saquinavir. 56 Interactions of HPIs with MDR1 were also investigated by the same authors using the same cell lines with focus on the cytotoxicity of doxorubicin for these cells in presence and absence of HPIs. Cytotoxicity of doxorubicin against MDR1+ cells was increased by a 3- to 7-fold factor in the presence of 5 μM of saquinavir, ritonavir, or nelfinavir but remained unchanged in the presence of indinavir.
The inhibitory effect of HPIs on P-gp has been studied in peripheral blood lymphocytes, using rhodamine 123 efflux studies. The inhibition was dose dependent and suggested the following order of potency: ritonavir > saquinavir > nelfinavir > indinavir. A reduction in P-gp function was also observed in peripheral blood lymphocytes isolated from AIDS patients receiving treatment with HPIs. 57
The accumulation of radiolabeled saquinavir, used as a substrate for P-gp, in P-gp-positive Dx5 cells, derived from uterine sarcoma MES-SA cells, was reduced by ritonavir and unlabeled saquinavir to a level similar to that observed in MES-SA, P-gp-negative cells. This reduction of accumulation was limited to 55 and 42% with nelfinavir and indinavir, respectively, indicating that ritonavir and saquinavir were more potent inhibitors of P-gp than nelfinavir and indinavir. 41
In summary, regarding HPIs as P-gp inhibitors, many studies have investigated the ability of ritonavir to act as an inhibitor of P-gp. Contradictory conclusions have been reported, especially when comparing in vitro and in vivo studies, the main difficulty being to separate the inhibition of P-pg from the inhibition of cytochrome metabolism, especially when investigated at the intestinal level where there is a high overlap both in substrates and inhibitors of P-gp and CYP450. 50
The other HPIs seem to show inhibitory properties for P-gp in vitro. By comparison among HPIs in vitro, ritonavir appears to have the higher inhibitory potency 34,41,57 and indinavir the lowest. 34,41,46,56,57
Most of the studies investigating the inhibitory effect of HPIs on P-gp, at the BBB level and in other barriers, used non-specific substrates of P-gp, with the risk of interferences with other efflux proteins.
Moreover, in vivo studies investigating HPIs as inhibitors of P-gp in the brain mainly concerned ritonavir. They did not give any evidence that ritonavir inhibits P-gp at the BBB level.
In conclusion, all the studies investigating HPIs as inhibitors of P-gp suggest that ritonavir is probably able to inhibit efflux proteins (and among them, possibly P-gp) in vitro with no or limited consequences in vivo. 26,40,52
HPIs as Inducers of P-Glycoprotein
In vitro, several articles have reported that HPIs were able to induce the expression and the functionality of P-gp with different results according to the cell line and the HPI.
An increase in P-gp expression was reported in human PBMCs treated with nelfinavir. 58,59 A concentration-dependent induction of P-gp expression was observed in LS174T human colon carcinoma cells treated with amprenavir, nelfinavir, and ritonavir 60; in LS180V human colon carcinoma treated with ritonavir 61 and lopinavir 55; and in human CD4 T-lymphoblastoid CEMrev cells treated with saquinavir. 62 In the case of increased expression of P-gp in PBMCs treated with nelfinavir, no differences were observed between genotypes (CC, CT, TT). 58 On the contrary, no differences of P-gp expression were observed in PBMCs treated with saquinavir, ritonavir, indinavir, amprenavir, and lopinavir. 59
A significant increase of P-gp functionality was also described in LS180V cells exposed to high concentrations of rito-navir 61 or chronically exposed to lopinavir 55 with a reduction in accumulation of the P-gp substrate, rhodamine 123.
In vivo, multiple doses of amprenavir or nelfinavir produced changes in P-gp expression. 60 Treatment of rats with either oral amprenavir or nelfinavir resulted in a dose-dependent increase of P-gp expression in the liver and the intestine after 14 days of administration. The level of P-gp in intestine and liver returned to normal 5 days after the last dose. This increase was not observed after 7 days of treatment. On the contrary, no difference was observed in P-gp expression on total lymphocytes from patients receiving an HPI-containing regimen with saquinavir, ritonavir, nelfinavir, indinavir, or the combination lopinavir/ritonavir. 59
In summary, regarding HPIs as inducers of P-glycoprotein, several in vitro studies reported inducing effects of HPIs on P-gp expression whereas others did not. Only one in vivo study demonstrated that nelfinavir and amprenavir were able to increase the expression of P-gp in the liver and the intestine. No investigation of HPIs as inducers of P-gp has been conducted at the BBB level, either in vitro or in vivo.
INTERACTIONS OF MULTIDRUG RESISTANCE ASSOCIATED PROTEINS AND HIV PROTEASE INHIBITORS
Similar substrate recognition principle as that described for P-gp (see above) has been described for MRP1. 30
Several articles have reported interactions between MRPs and HPIs either as substrate or inhibitors.
HPIs as Substrates of MRPs
Williams et al 63 showed that saquinavir was a substrate of MRP1 and MRP2. They used canine kidney MDCKII cells as wild-type or as transfectant overexpressing MRP1 or MRP2 or P-gp in which P-gp and BCRP was continuously blocked by the inhibitor GF120918. Efflux ratios of saquinavir in these 3 types of cells were reduced in a concentration-dependent manner by addition of the MRP inhibitor MK-571. They also showed that the transport of saquinavir by these 3 pumps had the apparent rank order of P-gp > MRP2 ≫ MRP1.
In LLC-PK1:MRP1 pig kidney epithelial cells overexpressing MRP1, a polarized transport was observed for ritonavir and indinavir but not for amprenavir. 33 In these cells, the P-gp inhibitors LY 335979 and valspodar did not modify the transport ratio, while the MRP1 inhibitor probenecid almost completely inhibited this transport. However, the transport ratio in LLC-PK1:MRP1 cells was much smaller for ritonavir and indinavir than that observed in the LLC-PK1:MDR1 over-expressing P-gp, indicating the rank order P-gp > MRP1 for these 2 HPIs.
Comparing human lymphocytes, either wild-type CEM or overexpressing P-gp CEM (VBL) or overexpressing MRP1 CEM (E1000) cell lines, Jones et al 64,65 observed that saquinavir and ritonavir were substrates of MRP1.
Saquinavir and ritonavir efflux by MRP1 was investigated in PBMCs isolated from HIV patients. 44 Ritonavir and saquinavir efflux correlated with the expression of MRP1.
The transport of saquinavir, ritonavir, and indinavir was investigated in MDCKII cells lines expressing human MRP1, MRP2, MRP3, MRP5, and murine Bcrp1. 66 MRP2 but not MRP1, MRP3, and MRP5 efficiently transported these 3 HPIs.
HPIs as Inhibitors of MRPs
As described above for P-gp, Srinivas et al 56 investigated the interactions between HPIs and the efflux protein MRP1, using T-lymphocytic cell line CEM/VM-1-5 overexpressing MRP1. In these MRP1+ cells, saquinavir and ritonavir but not indinavir and nelfinavir increased by 3-fold the cytotoxicity of doxorubicin, indicating that saquinavir and ritonavir were inhibitors of MRP1.
The inhibitory effect of ritonavir and saquinavir was also investigated in isolated pig brain microvessels using confocal microscopy expressing both MRP1 and MRP2. 67 Ritonavir and saquinavir were able to inhibit the transport of sulforhodamine 101, substrate of MRPs.
Using killifish renal proximal tubules, Gutmann et al 68 observed that saquinavir, and especially ritonavir, were potent inhibitors of MPR2-mediated transport.
The inhibitory effect of the HPIs indinavir, amprenavir, and ritonavir on MRP1 function was investigated using UMCC-1/VP cell lines overexpressing specifically MRP1 and the etoposide cytotoxicity assay. 69 MRP1 is able to protect MRP1 overexpressing cells from etoposide toxicity. Ritonavir, and not the other 2 HPIs, was able to block MRP1 activity with an increased cytotoxicity of etoposide at a level similar to that obtained with the known MRP1 inhibitor, probenecid.
HPIs as Inducers of MRPs
A 3-day exposure of human colon adenocarcinoma cells to increasing concentrations of ritonavir increased the expression of MRP1 in a concentration-dependent manner by 3-fold at a concentration of 30 μM. At this concentration, the accumulation of the MRP1 substrate carboxyfluorescein decreased by 30%. This decrease in accumulation was partially reversed by the MRP1 inhibitor indomethacin. 61
In conclusion, regarding interactions between MRPs and HPIs, only in vitro studies have been conducted to investigate the involvement of MRPs in the transport of HPIs. Results were contradictory, some showing that ritonavir and saquinavir were both substrates 33,63–65 and inhibitors 56,67–69 of MRP1 while others demonstrated that MRP1 did not transport saquinavir and ritonavir. 66 Conversely, other HPIs like nelfinavir, amprenavir, and indinavir did not seem to interact with MRP1 or MRP2. These results should be confirmed in vivo at the BBB level. Moreover, the expression and the functionality of MRP1 and other MRPs at the BBB level require further clarification to evaluate their real involvement in transport of HPIs across the BBB.
Interactions with P-gp and MRPs have been extensively studied but many questions remain unanswered. Interactions with the recently discovered efflux protein BMDP or BCRP at the BBB level remain to be conducted. Only one study, on MDCKII-Bcrp1 cells, indicated that the murine Bcrp1 was not a good transporter of HPIs, without showing any data. 66
To combat HIV infection and inhibit viral replication in the brain, anti-HIV agents must cross the BBB. Drug-drug interactions are generally considered as undesirable. However, when dealing with efflux proteins, inhibition of these efflux proteins may lead to increased transport of antiretroviral agents through the BBB and may be advantageous to treat CNS diseases, such as HIV encephalitis.
Except in some cases, all HPIs were described as P-gp substrates, either at the BBB level or in other barriers, as could be expected given the chemical and physical properties (lipophilicity, molecular weight, H bonding) in favor of interactions with P-gp. Consequently, all HPIs may benefit from combination therapy with an inhibitor of efflux to increase the cerebral uptake.
The potential of HPIs for inhibiting efflux proteins is less clear. Out of all the HPIs, ritonavir has been the most studied. It is described as a “booster” able to increase exposure levels for various HPIs such as indinavir, amprenavir, saquinavir, nelfinavir, and lopinavir. However, this “booster” effect is mainly due to inhibition of the metabolizing enzyme CYP3A4. Such an involvement of P-gp inhibition remains controversial according to the authors and may not be the predominant effect when compared with CYP3A4 inhibition. Moreover, after prolonged contact in cells and repeated administration in rats, some HPIs were inducers of P-gp. However, this induction and its consequences are to be proven in humans.
Due to the low inhibitory potencies of HPIs for efflux proteins, combination of anti-HIV drugs is not optimal in terms of inhibition of efflux proteins, especially at the BBB level. Combination of HPIs with more potent efflux inhibitors could be a better way to clear the brain from the virus.
1. Eisenblatter T, Huwel S, Galla HJ. Characterisation of the brain multidrug resistance protein (BMDP/ABCG2/BCRP) expressed at the blood-brain barrier. Brain Res
2. Litman T, Druley TE, Stein WD, et al. From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance. Cell Mol Life Sci
3. Molinari A, Calcabrini A, Meschini S, et al. Subcellular detection and localization of the drug transporter P-glycoprotein in cultured tumor cells. Curr Protein Pept Sci
4. Bendayan R, Lee G, Bendayan M. Functional expression and localization of P-glycoprotein at the blood brain barrier. Microsc Res Tech
5. Sun H, Dai H, Shaik N, et al. Drug efflux transporters in the CNS. Adv Drug Deliv Rev
6. Demeule M, Régina A, Jodoin J, et al. Drug transport to the brain: key roles for the efflux pump P-glycoprotein in the blood-brain barrier. Vascul Pharmacol
7. Pardridge WM, Golden PL, Kang YS, et al. Brain microvascular and astrocyte localization of P-glycoprotein. J Neurochem
8. Rao VV, Dahlheimer JL, Bardgett ME, et al. Choroid plexus epithelial expression of MDR1 P glycoprotein and multidrug resistance-associated protein contribute to the blood-cerebrospinal-fluid drug-permeability barrier. Proc Natl Acad Sci U S A
9. Zhang Y, Han H, Elmquist WF, et al. Expression of various multidrug resistance-associated protein (MRP) homologues in brain microvessel endothelial cells. Brain Res
10. Wijnholds J, de Lange EC, Scheffer GL, et al. Multidrug resistance protein 1 protects the choroid plexus epithelium and contributes to the blood-cerebrospinal fluid barrier. J Clin Invest
11. Decleves X, Regina A, Laplanche JL, et al. Functional expression of P-glycoprotein and multidrug resistance-associated protein (Mrp1) in primary cultures of rat astrocytes. J Neurosci Res
12. Dallas S, Zhu X, Baruchel S, et al. Functional expression of the multidrug resistance protein 1 in microglia. J Pharmacol Exp Ther
13. Anderson E, Zink W, Xiong H, et al. HIV-1-associated dementia: a metabolic encephalopathy perpetrated by virus-infected and immune-competent mononuclear phagocytes. J Acquir Immune Defic Syndr
. 2002;31(suppl 2):S43–S54.
14. Pang S, Koyanagi Y, Miles S, et al. High levels of unintegrated HIV-1 DNA in brain tissue of AIDS dementia patients. Nature
15. Vitkovic L, Tardieu M. Neuropathogenesis of HIV-1 infection: outstanding questions. C R Acad Sci III
16. Dallasta LM, Pisarov LA, Esplen JE, et al. Blood-brain barrier tight junction disruption in human immunodeficiency virus-1 encephalitis. Am J Pathol
17. Suzuki H, Terasaki T, Sugiyama Y. Role of efflux transport across the blood-brain barrier and blood-cerebrospinal fluid barrier on the disposition of xenobiotics in the central nervous system. Adv Drug Deliv Rev
18. Salminen T, Pulli A, Taskinen J. Relationship between immobilised artificial membrane chromatographic retention and the brain penetration of structurally diverse drugs. J Pharm Biomed Anal
19. Levin VA. Relationship of octanol/water partition coefficient and molecular weight to rat brain capillary permeability. J Med Chem
20. Meulemans A, Paycha F, Hannoun P, et al. Measurement and clinical and pharmacokinetic implications of diffusion coefficients of antibiotics in tissues. Antimicrob Agents Chemother
21. Boaziz C, Breau JL, Morere JF, et al. [The blood-brain barrier: implications for chemotherapy in brain tumors.]Pathol Biol (Paris)
22. Bodor N, Buchwald P. Recent advances in the brain targeting of neuropharmaceuticals by chemical delivery systems. Adv Drug Deliv Rev
23. Glynn SL, Yazdanian M. In vitro blood-brain barrier permeability of nevirapine compared to other HIV antiretroviral agents. J Pharm Sci
24. Urien S, Pinquier JL, Paquette B, et al. Effect of the binding of isradipine and darodipine to different plasma proteins on their transfer through the rat blood-brain barrier: drug binding to lipoproteins does not limit the transfer of drug. J Pharmacol Exp Ther
25. Pardridge WM. Receptor-mediated peptide transport through the blood-brain barrier. Endocr Rev
26. Polli JW, Jarrett JL, Studenberg SD, et al. Role of P-glycoprotein on the CNS disposition of amprenavir (141W94), an HIV protease inhibitor. Pharm Res
27. Williams GC, Sinko PJ. Oral absorption of the HIV protease inhibitors: a current update. Adv Drug Deliv Rev
28. Bain LJ, Leblanc GA. Interaction of structurally diverse pesticides with the human MDR1gene product P-glycoprotein. Toxicol Appl Pharmacol
29. Scala S, Akhmed N, Rao US, et al. P-glycoprotein substrates and antagonists cluster into two distinct groups. Mol Pharmacol
30. Seelig A, Landwojtowicz E. Structure-activity relationship of P-glycoprotein substrates and modifiers. Eur J Pharm Sci
31. Tang-Wai DF, Brossi A, Arnold LD, et al. The nitrogen of the acetamido group of colchicine modulates P-glycoprotein-mediated multidrug resistance. Biochemistry (Mosc)
32. Ueda K, Okamura N, Hirai M, et al. Human P-glycoprotein transports cortisol, aldosterone, and dexamethasone, but not progesterone. J Biol Chem
33. van der Sandt IC, Vos CM, Nabulsi L, et al. Assessment of active transport of HIV protease inhibitors in various cell lines and the in vitro blood–brain barrier. AIDS
34. Drewe J, Gutmann H, Fricker G, et al. HIV protease inhibitor ritonavir: a more potent inhibitor of P-glycoprotein than the cyclosporine analog SDZ PSC 833. Biochem Pharmacol
35. Mégard I, Garrigues A, Orlowski S, et al. A co-culture-based model of human blood-brain barrier: application to active transport of indinavir and in vivo-in vitro correlation. Brain Res
36. Edwards JE, Brouwer KR, McNamara PJ. GF120918, a P-glycoprotein modulator, increases the concentration of unbound amprenavir in the central nervous system in rats. Antimicrob Agents Chemother
37. Savolainen J, Edwards JE, Morgan ME, et al. Effects of a P-glycoprotein inhibitor on brain and plasma concentrations of anti-human immunodeficiency virus drugs administered in combination in rats. Drug Metab Dispos
38. Choo E, Leake B, Wandel S, et al. Pharmacological inhibition of P-glycoprotein transport enhances the distribution of HIV-1 protease inhibitors into brain and testes. Drug Metab Dispos
39. Kim RB, Fromm MF, Wandel C, et al. The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors. J Clin Invest
40. Huisman MT, Smit JW, Wiltshire HR, et al. P-glycoprotein limits oral availability, brain, and fetal penetration of saquinavir even with high doses of ritonavir. Mol Pharmacol
41. Washington CB, Wiltshire HR, Man M, et al. The disposition of saquinavir in normal and P-glycoprotein deficient mice, rats, and in cultured cells. Drug Metab Dispos
42. Kim AE, Dintaman JM, Waddell DS, et al. Saquinavir, an HIV protease inhibitor, is transported by P-glycoprotein. J Pharmacol Exp Ther
43. Polli JW, Wring SA, Humphreys JE, et al. Rational use of in vitro P-glycoprotein assays in drug discovery. J Pharmacol Exp Ther
44. Meaden ER, Hoggard PG, Newton P, et al. P-glycoprotein and MRP1 expression and reduced ritonavir and saquinavir accumulation in HIV-infected individuals. J Antimicrob Chemother
45. Smit JW, Huisman MT, van Tellingen O, et al. Absence or pharmacological blocking of placental P-glycoprotein profoundly increases fetal drug exposure. J Clin Invest
46. Choo EF, Leake B, Wandel C, et al. Pharmacological inhibition of P-glycoprotein transport enhances the distribution of HIV-1 protease inhibitors into brain and testes. Drug Metab Dispos
47. Schuetz EG, Umbenhauer DR, Yasuda K, et al. Altered expression of hepatic cytochromes P-450 in mice deficient in one or more mdr1 genes. Mol Pharmacol
48. Cisternino S, Mercier F, Bourasset F, et al. Transport and expression of abcg2 at the mice blood-brain barrier. Paper presented at: AAPS Annual Meeting and Exposition; October 26–30, 2003; Salt Lake City.
49. van Praag RM, Weverling GJ, Portegies P, et al. Enhanced penetration of indinavir in cerebrospinal fluid and semen after the addition of low-dose ritonavir. AIDS
50. Tayrouz Y, Ganssmann B, Ding R, et al. Ritonavir increases loperamide plasma concentrations without evidence for P-glycoprotein involvement. Clin Pharmacol Ther
51. Lee G, Schlichter L, Bendayan M, et al. Functional expression of P-glycoprotein in rat brain microglia. J Pharmacol Exp Ther
52. Solon EG, Balani SK, Luo G, et al. Interaction of ritonavir on tissue distribution of a [(14)c]L-valinamide, a potent human immunodeficiency virus-1 protease inhibitor, in rats using quantitative whole-body autoradiography. Drug Metab Dispos
53. Washington CB, Duran GE, Man MC, et al. Interaction of anti-HIV protease inhibitors with the multidrug transporter P-glycoprotein (P-gp) in human cultured cells. J Acquir Immune Defic Syndr Hum Retrovirol
54. Bardelmeijer H, Ouwehand M, Buckle T, et al. Low systemic exposure of oral docetaxel in mice resulting from extensive first-pass metabolism is boosted by ritonavir. Cancer Res
55. Vishnuvardhan D, Mottke LL, Richert C, et al. Lopinavir: acute exposure inhibits P-glycoprotein; extended exposure induces P-glycoprotein. AIDS
56. Srinivas RV, Middlemas D, Flynn P, et al. 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. Antimicrob Agents Chemother
57. Lucia MB, Rutella S, Leone G, et al. In vitro and in vivo modulation of MDR1/P-glycoprotein in HIV-infected patients administered highly active antiretroviral therapy and liposomal doxorubicin. J Acquir Immune Defic Syndr
58. Chandler B, Almond L, Ford J, et al. The effects of protease inhibitors and nonnucleoside reverse transcriptase inhibitors on p-glycoprotein expression in peripheral blood mononuclear cells in vitro. J Acquir Immune Defic Syndr
59. Ford J, Meaden ER, Hoggard PG, et al. Effect of protease inhibitor-containing regimens on lymphocyte multidrug resistance transporter expression. J Antimicrob Chemother
60. Huang L, Wring SA, Woolley JL, et al. Induction of P-glycoprotein and cytochrome P450 3A by HIV protease inhibitors. Drug Metab Dispos
61. Perloff MD, Von Moltke LL, Marchand JE, et al. Ritonavir induces P-glycoprotein expression, multidrug resistance-associated protein (MRP1) expression, and drug transporter-mediated activity in a human intestinal cell line. J Pharm Sci
62. Dupuis ML, Flego M, Molinari A, et al. Saquinavir induces stable and functional expression of the multidrug transporter P-glycoprotein in human CD4 T-lymphoblastoid CEMrev cells. HIV Med
63. Williams GC, Liu A, Knipp G, et al. Direct evidence that saquinavir is transported by multidrug resistance-associated protein (MRP1) and canalicular multispecific organic anion transporter (MRP2). Antimicrob Agents Chemother
64. Jones K, Hoggard PG, Sales SD, et al. Differences in the intracellular accumulation of HIV protease inhibitors in vitro and the effect of active transport. AIDS
65. Jones K, Bray PG, Khoo SH, et al. P-Glycoprotein and transporter MRP1 reduce HIV protease inhibitor uptake in CD4 cells: potential for accelerated viral drug resistance?AIDS
66. Huisman MT, Smit JW, Crommentuyn KM, et al. Multidrug resistance protein 2 (MRP2) transports HIV protease inhibitors, and transport can be enhanced by other drugs. AIDS
67. Miller DS, Nobmann SN, Gutmann H, et al. Xenobiotic transport across isolated brain microvessels studied by confocal microscopy. Mol Pharmacol
68. Gutmann H, Fricker G, Drewe J, et al. Interactions of HIV protease inhibitors with ATP-dependent drug export proteins. Mol Pharmacol
69. Olson DP, Scadden DT, D'Aquila RT, et al. The protease inhibitor ritonavir inhibits the functional activity of the multidrug resistance related-protein 1 (MRP-1). AIDS
70. Flexner C. HIV-protease inhibitors. N Engl J Med
71. Livington DJ, Pazhanisamy S, Porter DJ, et al. Weak binding of VX-478 to human plasma proteins and implications for anti-human immunodeficiency virus therapy. J Infect Dis. 1995;172:1238–1245.
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