Limitations of current antiretroviral therapy in HIV-1 infection: the search for new strategies - CHAPTER 9 P-glycoprotein in human immunodeficiency virus type 1 infection and therapy

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Limitations of current antiretroviral therapy in HIV-1 infection: the search for new

strategies

Sankatsing, S.U.C.

Publication date

2004

Link to publication

Citation for published version (APA):

Sankatsing, S. U. C. (2004). Limitations of current antiretroviral therapy in HIV-1 infection: the

search for new strategies.

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P-glycoproteinn in human immunodeficiency virus

typee 1 infection and therapy

S.U.C.. Sankatsing, J.H. Beijnen, A.H. Schinkel, J.MA Lange,

J.M.. Prins

Withh the advent and widespread use of potent antiretroviral therapy in the

mid-1990s,, the clinical course of human immunodefiency virus (HIV) type 1

(HIV-1)) infection has changed dramatically in a substantial proportion of HIV-1

infectedd individuals. This has led to a significant decline in the incidence of

AIDSS and AIDS-related morbidity and mortality in the developed world

~*.

Proteasee inhibitors in combination with inhibitors of HIV-1 reverse

transcriptasee cause a dramatic reduction in plasma viremia, with the plasma

HIV-11 RNA load being below the limit of detection in many patients

.

However,, with the currently available drugs, complete eradication of HIV-1

fromm an infected person is not achieved because of the persistence of latently

infected,, resting CD4

T cells harbouring replication-competent HIV-1, and

becausee of ongoing low-level viral replication

"

. One cause of ongoing viral

replicationn can be suboptimal penetration of drugs into anatomical sanctuary

sitess like the central nervous system. It has been suggested that drug

transporterss like P glycoprotein (P-gp) may contribute to this suboptimal

penetration.. Such drug transporters might also lower intracellular drug levels,

therebyy limiting the therapeutic efficacy of antiviral drugs in peripheral blood

mononuclearr cells (PBMCs)

, including CD4

T cells

.

P-gp,, a plasma membrane protein encoded by the multi drug resistance

(MDR)) gene, was discovered in 1976

and functions as an ATP-dependent

drugg efflux pump

. It is a transporter of a wide range of compounds,

includingg hydrophobic amphiphatic drugs, calcium channel blockers,

antihistamines,, peptides and steroids. The function of P-gp is thoroughly

investigatedd in the oncology field, because of its ability to induce resistance to

antii cancer therapy by pumping the drugs out of tumor cells

. In this

minirevieww we summarize the possible roles of P-gp in HIV-1 infection and

therapy. .

Tissuee distribution of P-gp

P-gpp expression on cell membranes can be detected with monoclonal

antibodiess like MRK16 and UIC2

~

. P-gp function can be studied with the

fluorescentt dye rhodamine 123 (Rh 123). Cells expressing functional P-gp

havee less intracellular accumulation of rhodamine, because of increased efflux

off the dye by P-gp

. The level of intracellular Rh 123 accumulation can

bee measured by flow cytometry.

P-gpp is expressed at high levels in tissues like the gastrointestinal tract, liver

andd kidney and on capillary endothelial cells of the brain and on PBMCs

21,23,24,26-300

_

|

j

|

ed CD4

and CD8

T cells. P-gp

expressionn increased on CD4

and CD8

T cells after stimulation with

phytohemagglutininn (PHA), with almost 100% of CD8

T cells expressing P-gp

afterr stimulation, suggesting that the level of P-gp expression on these cells

increasedd upon immune activation

. An overview of P-gp tissue distribution

iss given in table 1.

Tablee 1 Localization and function of P-gp

Tissuee and cells s Jejunum, , ileum,, colon Liver r Kidney y Pancreas s Lung g Heart t Adrenall gland Brain n Testes s Uterus s Bonee marow Immune e System m Site e Mucosall surface Zó"i'

Biliaryy canalicular surfaces of hepatocytess and apical surfacess of cells lining the smalll biliary ductules 3 Apicall surfaces of epithelial cellss of the proximal tubulus 23 Apicall surfaces of epithelial cellss lining the small ductules

23 3

Capillaryy endothelium ** Endotheliall luminal membraness of cardiac arterioless and capillaries 34,35 Surfacess of cells in the cortex andd medulla 23

Capillaryy endothelium M Capillariess A

Placentaa if

Hematopoieticc stem cells u

Naturall killer cells a

BB lymfocytes iV

CD4++ T cells 31 CD8++ T cells 31

(Possible)) function Preventionn of uptake and facilitation of excretionn across the mucosa 32 Excretionn of xenobiotics in bile JJ

Excretionn of xenobiotics in urine ió

Unknown n

Unknown n

ProtectionProtection against cardiac toxicity of certainn drugs 35

Secretionn of Cortisol and aldosterone in thee cortex36

ProtectionProtection against toxic substances M ProtectionProtection against toxic substances ** ProtectionProtection of the fetus against toxic substancess 37"39

ProtectionProtection against toxic substances ""; a rolee in differentiation and proliferation of stemm cells by influencing regulatory substancess 2; secretion of certain growth factorss and cytokines28

NKK cell mediated toxicity *, ,*w Unknown n

Secretionn of cytokines a

P-gpp function

Itt is not clear whether P-gp expression is necessary for a normal life. P-gp

knockoutt mice do not express P-gp but still have normal viability and fertility

andd a normal life span

-

'

"*

. it has been suggested that in vivo P-gp

protectss cells against toxic substances by efflux of these compounds. In the

gastrointestinall tract (duodenum, jejunum, ileum, cecum and colon), high

levelss of P-gp are located only on the mucosal surfaces of these tissues. This

suggestss that the role of P-gp is to prevent the uptake of toxic substrates and

perhapss to facilitate excretion across the mucosa of the gastrointestinal tract

23,32,466

_gp

p

j j

| j

and kidney could be responsible for the

secretionn of xenobiotics into the bile and the urine

. The role of P-gp in the

pancreass and lungs is not known so far.

P-gpp in the blood-brain barrier could prevent the uptake of toxic substances

intoo the brain

-

-

and the absence of P-gp in mice resulted in increased

levelss of accumulation of many drugs in the brain

-

-

^

. Therefore, the

absencee of P-gp or the inhibition of P-gp might increase the central nervous

systemm toxicities of some drugs. This was indeed demonstrated for ivermectin,

domperidone,, loperamide and ritonavir in mice

-

-

. p-gp expressed in the

placentaa plays an important role in the protection of the developing fetus

againstt toxic substances

.

P-gpp expression on bone marrow stem cells might protect them from toxic

substancess and could also be responsible for the transport of certain growth

factorss and cytokines produced by stem cells

. It is unclear whether P-gp

alsoo has this function in other cell types.

Itt has been suggested that NK-cell cytotoxicity requires P-gp function for the

effluxx of lytic products. Inhibition of P-gp resulted in a decreased cytotoxicity of

NKK cells

. The decrease in the lytic activities of NK cells in the presence of

R-verapamil,, an inhibitor of P-gp, was dose dependent

. However, no

differencee in cytotoxic T-cell function was found between wild-type mice and

P-gpp knockout mice

. It is possible that P-gp knockout mice develop some

compensationn mechanism for the lack of P-gp.

Polymorphismm of P-gp

Thee MDR-1 (or ABCB1) gene encodes P-gp. So far, 28 single nucleotide

polymorphismss have been found at 27 positions

, and these are

sometimess linked to each other

. A C/T polymorphism in exon 26 (C3435T)

correlatess in a significant manner with P-gp expression and activity in the

duodenumm

. The polymorphism in exon 26 appears to be linked to single

nucleotidee polymorphisms in exon 12 (C1236T) and exon 21 (G2677T)

.

Individualss with the homozygous T genotype at position 3435 showed a P-gp

functionn higher than that of individuals with the homozygous C genotype

,

butt this is in contrast to the findings of another study, which demonstrated the

contraryy

. It has been suggested that the discrepancy between these studies

mightt reflect linkage to an as yet undefined promotor or enhancer region

polymorphism(s)) or nucleotide sequences that are important for messenger

RNAA processing

.

Thesee genetic differences might explain the differences in P-gp expression

betweenn individuals and, thereby, the differences in individual drug response

inn patients

"

. There is an ethnic difference in allele frequencies. Africans or

peoplee of African descent have a lower frequency of the T/T genotype and a

higherr frequency of the C/C genotype than Caucasians

.

Mechanismm of action

Thee most prevalent theory of the mechanism of action of P-gp favors a direct

pumpp mechanism

"

. The P-gp pump recognizes substrates through a

complexx substrate recognition region and directly pumps drugs out of the cell

byy using molecular mechanisms that are not yet well understood. ATP

hydrolysiss by P-gp provides the energy for this active process. The question

is,, how are the drugs actually pumped out of the cell? One proposed

mechanismm is that P-gp detects drugs and ejects them before they reach the

cytoplasmm by removing the drugs directly from the plasma membrane

'

.

Anotherr possibility is that P-gp acts as a flippase, carrying its substrate from

thee inner leaflet of the lipid bilayer to the outer leaflet

.

P-gpp inhibition

P-gpp inhibitors, also called reversal agents or P-gp modulators, inhibit the

effluxx of P-gp transported drugs in vitro and in mice

"

.

P-gpp inhibitors modulate P-gp function by competing with the binding and

transportt of the drug or through a non-competitive binding by binding either to

thee drug interaction site or to another modulator binding site, which leads to

allostericc changes

. This is consistent with the finding that P-gp has at

leastt two independent drug-binding sites

.

Theree are three groups of P-gp inhibitors or modulators (table 2). The first

groupp of inhibitors are therapeutic agents. In vivo they function as a P-gp

inhibitorr only at concentrations higher than those required for therapeutic

activity.. Therefore, these agents cannot be used as P-gp inhibitors in vivo

becausee of their potential toxic effects. The second group of P-gp modulators

aree analogues of the first group of modulators. They are more potent and less

toxic.. For example emopamil, gallopamil and Ro11-2933 are analogues of

verapamil;; and PSC 833 is a non-immunosuppressive cyclosporin analogue.

Thee third group of modulators are developed and targeted against specific

MDRR mechanisms

. Clinical trials have demonstrated the beneficial effect of

P-gpp inhibitors for the treatment of cancer. One trial with breast cancer

patientss and one trial with lung cancer patients demonstrated a survival

benefitt with the addition of the P-gp inhibitor verapamil to chemotherapy

.

Inn another study, the combination of the P-gp inhibitor cyclosporine with

chemotherapyy statistically improved the relapse-free survival rate and the

overalll survival rate

. The proposed mechanism for this beneficial effect was

reversall of the P-gp induced drug efflux out of tumor cells. P-gp inhibitors can

causee side effects. The first group of P-gp inhibitors must be used at high

dosess to accomplish the inhibition of P-gp and can therefore cause side

effects.. In addition, side effects may be caused by the accumulation of

substratess of P-gp. The second and third groups of P-gp inhibitors are used at

muchh lower doses to accomplish the desired effect, and the side effects are

probablyy mainly caused by the accumulation of P-gp substrates

.

Tablee 2 Example of P-gp modulators

Firstt group Secondd group Thirdd group dexverapamil l Emopamil81 1 Gallopamil8186 6 PSCC 833 (valspodar) R011-293392 2 amiodaronee '9,8U bepridil83 3 caroverinee 85 clomipramine85 5 cyclosporinn A 90,91 diltiazemm ** felodipinee 95 isradipinee 95 nicardipinee M quinidine96 6 97 quinine e trifluoperazinee 85,98 II 85,99 verapamil l #

firstt generation modulators: therapeutic agents with a P-gp inhibitor effect at higher concentrationss than those required for therapeutic activity; &second generation modulators:: analogues of first generation modulators; +third generation modulators aree developed and targeted against specific MDR mechanisms.

GFF 120918c LYY 335979 6 OC144-093' ' VX-71089 9 XR90511 93

Relationn of P-gp and CYP3A4

Cytochromee P450 (CYP) mixed function oxidases account for the majority of oxidativee biotransformations of xenobiotics and endogenous compounds. Moree than 30 different human CYP enzymes have been identified, of which CYP3A44 appears to be one of the most important, as it contributes to the biotransformationn of approximately 60% of therapeutic drugs. Most of the activityy of CYP3A4 is located in the liver and in the small intestine, and CYP3A44 is responsible for the first-pass drug metabolism 1 0 . Some drugs, for examplee vinblastine, are substrates for both CYP3A4 and P-gp. Other drugs, forr example, verapamil are substrates for CYP3A4 and inhibitors of P-gp, Yet otherr drugs, for example ketoconazole, inhibit both CYP3A4 and P-gp 101/l02. Stt John's Wort contains compounds that are substrates for both P-gp and CYP3A44 and, in addition, induce intestinal P-gp and intestinal and hepatic CYP3A44 in humans 103'104.

P-gpp might enhance the metabolic effect of CYP3A4 in the small intestine in threee ways. First, P-gp can limit the uptake of substrates in the small intestine

therebyy limiting the amount of substrate that needs to be transformed by

CYP3A4.. Second, P-gp can increase the extent of metabolism by CYP3A4

throughh repeated cycles of intracellular uptake and efflux, thereby increasing

thee level of exposure of a drug to CYP3A4 before absorption in the systemic

circulation.. Finally, P-gp might preferentially remove drug metabolites

catalyzedd by CYP3A4, thereby limiting competitive inhibition

.

P-gpp expression and function during HIV-1 infection

Thee level of P-gp expression on T-cell and monocytic cell lines increases

uponn infection with HIV-1

. By using monoclonal antibody MRK-16, an

increasedd level of P-gp expression was established on CD4

T cells from

HIV-11 infected patients

. The level of P-gp expression on CD4

T cells

increasedd with the progression of HIV-1 infection

. Later studies did not

confirmm these findings, however

. in cells with increased levels of P-gp

expression,, one would expect a lower level of accumulation of Rh 123.

However,, CD4

, CD8

, CD16

and CD19

cells from HIV-1 infected patients

accumulatedd more Rh 123 than cells from healthy controls

. in the

presencee of cyclosporin, a P-gp inhibitor, the level of intracellular

accumulationn of Rh 123 increased in CD4

and CD8

T cells from healthy

controls,, but in HIV-1 infected cells, the level of intracellular accumulation of

Rhh 123 did not increase further

. This suggests that even when the level of

P-gpp expression is increased in HIV-1-infected patients, the pump function of

P-gpp is decreased.

AA significantly reduced P-gp function was found in CD16

NK cells from HIV-1

infectedd patients compared with that found in cells from healthy individuals.

Thiss reduced P-gp function became more prominent with the progression of

thee HIV infection and was significantly correlated with a decreased NK-cell

cytotoxicc function

-

. This is consistent with the decreased NK-cell

cytotoxicityy in HIV-1 infected patients

. In vitro the reduced level of P-gp

expressionn and function of NK cells of HIV-1 infected individuals could be

recoveredd by stimulating them with IL-15

.

P-gpp expression and anti retroviral agents

Theree is clear evidence for interactions between P-gp activity and

antiretrovirall agents.

Onlyy a few studies have addressed the effect of P-gp on the behavior of

nucleosidee reverse transcriptase inhibitoirs (NRTIs). Early in vitro studies

demonstratedd a decreased level of accumulation of the NRTI zidovudine in

HIV-11 infected cells compared to that in uninfected cells, which was correlated

withh an increased level of P-gp expression on HIV-1 infected cells

.

Zidovudinee was less effective in inhibiting HIV-1 replication in cells expressing

P-gpp

. It may be possible that this effect was mediated by another drug

transporter,, such as MRP4, which was discovered later

. Zidovudine,

didanosinee and zalcitabine do not appear to induce P-gp expression or

functionn in the cells analyzed

.

Thee non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine,

efavirenzz and delavirdine are not substrates for transport by P-gp in Caco-2

celll lines. All are able to induce P-gp expression and function, resulting in

increasess in the levels of P-gp expression of 3.5, 1.75 and 2.35 fold,

respectivelyy

and reduced levels of accumulation of Rh123 by 72%, 8 1 %

andd 85%, respectively

. Delavirdine is not only an inducer of P-gp but also

ann inhibitor of P-gp

.

Alll presently available HIV-1 protease inhibitors are substrates for P-gp

51,70,71,117-1233

j j

t

t

jth p.gp with an affinity in the order ritonavir >

nelfinavirr > amprenavir > indinavir > saquinavir

. CEM cells expressing

P-gpp have reduced intracellular concentrations of the protease inhibitors

ritonavir,, indinavir, saquinavir and nelfinavir compared with those in CEM cells

nott expressing P-gp

. The intracellular accumulation of saquinavir and

otherr protease inhibitors increased in the presence of the P-gp inhibitors

verapamill and GF 120918

. In patients, the concentrations of protease

inhibitorss in PBMCs was inversely correlated with the amount of MDR-1

mRNAA expression

. This explains why the protease inhibitors ritonavir,

indinavirr and saquinavir are less effective against HIV-1 replication in cells

expressingg P-gp

, although this effect of P-gp was refuted by other

investigatorss

. The different cell lines used in those studies might explain

thiss difference.

Furtherr in vitro studies demonstrated that ritonavir, indinavir, saquinavir and

nelfinavirr have weak inhibitory effects on P-gp, with ritonavir being the most

potentt one 24,51,70,118,122-124,126

|

j

navir is a sixfold

moree potent P-gp inhibitor than the cyclosporine analog SDZ PSC 833, which

iss assumed to be one of the most potent inhibitors

. However, that study

usedd cultured pig brain endothelial cells, in which the transporter(s) affected

wass not unambiguously identified. On the other hand, the simultaneous

administrationn of more than one protease inhibitor did not result in a

decreasedd P-gp efflux function in contrast to that after exposure to

LY-335979,, a more potent P-gp inhibitor

. This suggests that ritonavir is only a

moderatee P-gp inhibitor

. Nelfinavir and, to a lesser extent, its metabolite M8

aree also inhibitors of P-gp function on CD4

and CD8

T cells

. Protease

inhibitorss are also able to increase the level of P-gp expression. Lopinavir, for

instance,, is both an inhibitor and inducer of P-gp, but the overall effect of

lopinavirr seemed to be induction

.

Givenn the tissue distribution of P-gp (Table 1), it might lower the bioavailability

off protease inhibitors and could be responsible for the existence of sanctuary

sites,, such as the brain and the testes, by limiting the levels of accumulation

off protease inhibitors in these tissues. In P-gp knockout mice, the levels of

indinavir,, saquinavir and nelfinavir in plasma were 2- to 5-fold higher after oral

administrationn compared with the levels in wildtype mice, and the

concentrationss in the brain were 7- to 36-fold higher after intravenous

administrationn and 10-fold higher after oral administration compared with the

levelss in wildtype mice

. in wiidtype mice intravenous administration of

thee potent P-gp inhibitor LY-335979 resulted in a dose dependent increase in

thee

C-labeled nelfinavir, amprenavir, indinavir and saquinavir concentrations

inn the brain and an increase in the

C-labeled nelfinavir in the testes

.

Likewise,, in the same model, an increase in plasma saquinavir concentrations

andd improved penetration into the brain and testes were seen after

co-exposuree to the potent P-gp inhibitor GF120918

. The penetration of

amprenavirr into the brains of rats increased in the presence of GF120918

.

Thesee data support the idea that P-gp expression in the gastrointestinal tract

cann limit the bioavailabilities of these drugs and that P-gp can contribute to the

decreasedd concentrations of these drugs in sanctuary sites like the brain and

thee male genital tract

-

.

Inn vivo, the addition of low-dose ritonavir increased indinavir plasma trough

concentrationss and indinavir concentrations in seminal plasma and

cerebrospinall fluid

. Inhibition of CYP3A4 by ritonavir decreases the

metabolismm of indinavir, but this could not fully explain the increased indinavir

concentrationss in seminal plasma and cerebrospinal fluid

. Therefore,

ritonavirr might also influence the blood-brain and blood-testis barrier by its

P-gpp inhibitory function

even though ritonavir being only a moderate P-gp

inhibitor

. .

Effectt of P-gp on HIV-1 replication

Finally,, P-gp can influence infectivity and replication of HIV-1.

Inn vitro, P-gp expression on T cells inhibited HIV-1 fusion with the plasma

membranee and also inhibited virus replication at a later step in the viral life

cycle.. This reduction in the level of HIV-1 replication correlated with the level

off P-gp expression

. Mutations of P-gp at the ATP utilization site, which

therebyy inactivated ATP hydrolysis and resulted in an inactive P-gp pump

function,, still resulted in decreased HIV-1 infectivity. This suggests that the

P-gpp function is not necessary to block the infectivity of HIV-1

. On the other

hand,, when CD4

T cells were incubated with quinidine or PSC 833 to inhibit

thee P-gp function but not P-gp expression, the levels of HIV-1 production in

thesee cells increased. In summary, P-gp expression and function can inhibit

HIV-11 infectivity and replication capacity.

P-gpp polymorphisms in HIV-1

Polymorphismss in MDR1 alleles might be of clinical importance in HIV

treatment,, although controversy remains about the effects of the different

polymorphismss on P-gp expression and function. As discussed earlier, it is not

clearr whether a C or a T allele at position 3435 in exon 26 is associated with a

higherr level of P-gp function. Patients with a homozygous T genotype at exon

266 had, on average, a lower concentration of nelfinavir in plasma compared

withh that in the plasma of patients with the homozygous C genotype

. This

findingss suggests that the T allele is associated with a higher level of P-gp

function,, but this was difficult to reconcile with the fact that the level of P-gp

mRNAA transcription in PBMCs was lower in these patients. In that study

plasmaa efavirenz levels were also lower in patients with the homozygous T

genotype,, although efavirenz is not a substrate for P-gp. The patients with the

homozygouss T genotype had a greater rise in CD4

T-cell count

. This was

explainedd by the protective function of P-gp on pluripotent stem cells

.

P-gpp and cytochrome P450 3A4 during HIV treatment

Antii retroviral drugs can not only influence and be influenced by P-gp, but they

mayy also be substrates for and influenced by CYP3A4 activity. The resulting

effectt is not always predictable.

Thee NNRTI delavirdine, which is an inducer and inhibitor of P-gp, is also an

inhibitorr of CYP3A4

. Likewise, nevirapine and efavirenz are inducers not

onlyy of P-gp but also of CYP3A4

'

.

Nott only are the protease inhibitors substrates for P-gp, but all of them are

alsoo substrates for CYP3A4 in the liver and small intestine, where they are

metabolisedd by at least 80 to 95%

. In vitro studies revealed, in addition,

CYP3A44 inhibitory capacities of the protease inhibitors, with ritonavir being the

mostt potent inhibitor, followed by indinavir, nelfinavir, amprenavir and

saquinavir

"

. .

Inn vitro, P-gp facilitated the removal of the metabolites of indinavir, thereby

preventingg competition for CYP3A4 by the metabolites and indinavir itself

.

Itt has been suggested that this might increase the level of metabolism by

CYP3A44

but that result could not be confirmed

. That study

supportedd the suggestion that the mechanism of increased drug metabolism

byy CYP3A4 is through repeated cycles of intracellular uptake and efflux by

P-gp.. In vivo, low-dose ritonavir increased the bioavailability of indinavir by

inhibitingg metabolism by CYP3A4 and probably by inhibiting drug transport by

P-gp.. This combination proved to be clinically relevant

.

Otherr drug transporters relevant for HIV therapy

Memberss of the MDR protein (MRP) family, which belongs to the group of

ATP-bindingg cassette drug transporters, have also been recognized as

transporterss of nucleoside-based antiretroviral drugs

and protease

inhibitorss

-

-

. For example, MRP4 overexpression impairs the

antivirall efficacies of adefovir and zidovudine in vitro

, and MRP 5

overexpressionn in vitro results in the efflux of adefovir from cells

. In vitro

studiess demonstrated that cells expressing MRP1 have a reduced intracellular

concentrationn of saquinavir and ritonavir compared with those in cells not

expressingg MRP1, but the intracellular concentrations of nelfinavir and

indinavirr were not influenced by MRP1 expression

. Also, saquinavir,

ritonavirr and indinavir are effectively transported in vitro by MRP2

but not

byy MRP1, MRP3 or MRP5. The conflictingg data regarding the effect of MRP1

onn saquinavir, ritonavir and indinavir might be explained by the different cell

liness used in these studies. MRP drug transporters seem to play a minor role

inn the transport of ritonavir and indinavir across the blood-brain barrier

. In

vivo,, expression of MRP 1 on lymphocytes was correlated with lower levels of

accumulationn of saquinavir and ritonavir in these lymphocytes

.

Furthermore,, saquinavir and ritonavir are also inhibitors of MRP1 and MRP2

mediatedd drug transport

'

. it seems likely that there is a combined

effectt of P-gp and the MRP-drug transporter family on the concentrations of

antiretrovirall drugs that results in the decreased bioavailabilities of these

drugs. .

MRP11 also directly influences HIV-1 replication. Overexpression of MRP1 on

CEMM cells resulted in an increase in the level of HIV-1 replication by a factor

1600 compared to that in control CEM cells

. The mechanism is unknown so

far. .

Breastt Cancer Resistance Protein (BCRP) is another drug transporter with the

abilityy to influence intestinal (re)uptake and hepatobiliary excretion of

transportedd drugs

. Preliminary data showed that BCRP is not an efficient

transporterr of the protease inhibitors saquinavir, ritonavir and indinavir

but

cann transport zidovudine

.

Summaryy and conclusions

Manyy of the data to date is generated from in vitro studies with different cell

lines.. The use of different cell lines might explain in part why some of the data

conflict,, and the observed effects might be different for different tissues in

vivo.. Nevertheless, some aspects of P-gp relevant for HIV-infection and

therapyy have become clear. The potential effects of P-gp activity relevant for

HIV-11 treatment include decreasing the uptake of protease inhibitors together

withh CYP3A4 in the small intestine, decreasing the entry of protease inhibitors

intoo the central nervous system, decreasing the entry of protease inhibitors

intoo the testis, and decreasing the intracellular accumulation of protease

inhibitorss and protecting against HIV-1 infectivity and replication in CD4

T

cells.. P-gp activity is also potentially necessary for the function of natural

killerr cells.

Byy limiting the penetration of protease inhibitors into anatomical sites like the

centrall nervous system, P-gp contributes to the maintenance of sanctuary

sites.. P-gp function also reduces intracellular protease inhibitor concentrations

andd could therefore result in intracellular concentrations too low to block HIV-1

replicationn completely. These sub-optimal concentrations in sanctuary sites

andd in cells could contribute to ongoing low level HIV-1 replication. Moreover,

P-gpp can be an important limiting factor in the oral bioavailability of protease

inhibitors. .

Itt must be kept in mind that P-gp is not the only drug transporter of protease

inhibitors.. Other drug transporters, especially transporters of the MRP drug

transportt family, have been identified. The relative contribution of each of the

transporterss to the overall effect needs to be clarified.

Furthermore,, the additional effect of CYP3A4 on protease inhibitor

concentrationss is also important, although the resulting effect is not always

predictable. .

Thee clinical relevance of P-gp during treatment with NNRTIs is less clear.

However,, it is conceivable that in a regimen consisting of protease inhibitors

andd NNRTIs the induction of P-gp expression and function by the NNRTIs

mightt contribute to lower concentrations of the protease inhibitors in plasma

andd cells.

Theree are variations in P-gp expression and function among patients. These

variationss are explained at least in part by polymorphisms in the MDR-1 gene

encodingg P-gp, and this could contribute to the interpatient variabilities in

plasmaa protease inhibitor concentrations. There is still controversy about the

exactt effects of the different polymorphisms on P-gp function. Insight into the

significancee of these polymorphisms might be of clinical use. If the MDR-1

genotypee in patients is known before the start of treatment with protease

inhibitors,, one could predict which patients are at risk for having low plasma

proteasee inhibitor levels. This could justify a dose adjustment at the start of

treatment. .

Thee bioavailability and the intracellular concentrations of protease inhibitors

cann be increased in the presence of potent P-gp inhibitors. Therefore a logical

stepp in order to increase the concentrations of protease inhibitors in plasma,

cellscells and sanctuary sites is to inhibit P-gp function, which can be done with

availablee potent P-gp inhibitors. Inhibition of P-gp function might, however,

leadd to increased toxicities of other drugs, for example, loperamide and

domperidone.. Moreover, P-gp expression appears to decrease HIV-1

infectivityy and replication in T cells, and P-gp inhibition might therefore be

counterproductive. .

Beforee manipulation of P-gp function is considered in anti-HIV-1 therapy, the

questionn of which effect of P-gp is clinically more important must be

answered:: P-gp as a drug efflux pump or P-gp as a protector against HIV

infectivityy and replication.

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