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

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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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Contentss of the Introduction

1.. History 9

2.. HIV-1 replication cycle and viral targets used for therapy 11

2.1.. Viral entry

1 1

2.2.. Reverse transcription

1 2

2.3.. Integration

1 7

2.4.. Gene expression and virion assembly 17

3.. HIV-1 infection in vivo

1 8

3.1.. The primary phase of HIV-1 infection 18

3.2.. The chronic phase of HtV-1 infection 19

3.3.. Viral dynamics

1 9

4.. Limitations of current antiretroviral therapy 20

4.1.. Nucleoside and nucleotide analogue reverse transcriptase inhibitors 20

4.2.. Non-nucleoside reverse transcriptase inhibitors 21

4.3.. Protease inhibitors 22

4.4.. Reservoirs of HIV-1 23

4.4.1.. Viral sanctuary sites 24

4.4.2.. Cellular viral reservoirs and viral latency 24

4.5.. Residual HIV-1 replication 26

5.. Interventions to lower HIV-1 reservoirs and residual replication 27

6.. Outline of this thesis 28

1.. History

Sincee the first reports of a new disease now known as acquired

immunodeficiencyy syndrome (AIDS) in 1981 and 1982

1-4

, until the end of

20033 an estimated 26.8 million people died of AIDS. At the end of 2003, an

estimatedd 40 million people were living with HIV, 5 million of whom were

infectedd that year

5

.

Inn 1983 the human immunodeficiency virus 1 (HIV-1) was isolated and found

too be the cause of AIDS

6J

(figure 1). In 1986 another virus, human

immunodeficiencyy virus 2 (HIV-2) was isolated, which is related to but distinct

fromm HIV-1. HIV-2 infections only represent a minority of all HIV infections.

Althoughh HIV-2 is less pathogenic compared with HIV-1, it can still cause

AIDSS

89

. HIV-2 is also contained to certain geographical regions (in particular

Westt Africa). In 1985 the first blood-screening ELISA (enzyme-linked

immunosorbentt assay) antibody test for HIV-1 was approved by the US Food

andd Drug Administration (FDA)

10

.

Thee essence of HIV-1 infection is a slow decline in CD4

+

T cells over time,

suchh that once a threshold of approximately 200 x 10

6

CD4

+

T cells/L is

passed,, overt immune deficiency occurs resulting in opportunistic infections

andd virally-induced tumours. In this case we speak of AIDS

11

.

Inn the early 80s 51% of the patients died within a year after the diagnosis of

AIDSS and after 5 years only 15% was still alive

12

.

Thee first drug active against HIV-1 was the nucleoside analogue reverse

transcriptasee inhibitor (NRTI) zidovudine (ZDV). Treatment with ZDV resulted

inn a decrease in HIV-1 related mortality in clinical studies

13,14

. Because of the

positivee results, it was approved by the FDA in 1987

15

.

Subsequentlyy it became clear that the effect of monotherapy was only

transientt and limited, because of the selection of drug-resistant strains

16

"

20

.

Thee development in the mid-nineties of reliable and sensitive methods to

quantifyy the amount of virus in the blood by measuring the amount of HIV

RNAA in blood plasma

21

~

27

, and the introduction of drugs in two new classes,

thee protease inhibitors and the non-nucleoside reverse transcriptase

inhibitors,, increased our understanding of the replication dynamics of HIV-1. It

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becamee clear that antiretroviral therapy composed of at least three drugs

(highlyy active antiretroviral therapy, HAART) was necessary to avoid

developmentt of viral resistance and to obtain sustained suppression of viral

replicationn to minimal levels

28,29

. The introduction of HAART has led to a

significantt decline in the incidence of AIDS and AIDS-related morbidity and

mortalityy in the developed world

30

"

34

. However, with the currently available

drugss complete eradication of HIV-1 and thus curation of HIV-1 has proven to

bee an elusive target

3S43

. At the end of 2003, 21 drugs in 4 different classes

hadd been approved by the FDA (figure 1)

44

.

2.. HIV-1 replication cycle and viral targets used for therapy

HIV-11 is a lentivirus belonging to the subfamily of retroviruses

45

, that most

likelyy evolved from simian immunodeficiency virus (SIV), crossing from its

predominantt host (chimpanzees) to humans sometime during the first half of

thee 20

th

century

46

'

47

.

2.1.. Viral entry

Uponn entering the body HIV-1 mainly infects T lymphocytes, macrophages

andd dendritic cells containing the CD4 receptor

48,49

(figure 2). In contrast with

otherr retroviruses, HIV-1 infection can take place in both activated and resting

cellss

50

"

53

and preferentially in HIV-specific CD4

+

T cells

54

. Initial viral

attachmentt occurs through the binding of the HIV surface glycoprotein subunit

(gp120)) to the CD4 molecule expressed on the membrane of the cell to be

infected.. Beside the CD4 receptor, HIV-1 also needs the presence of the

co-receptorss CCR5 (R5 strains) or CXCR4 (X4 strains) or both (R5X4 strains) to

triggerr conformational changes needed for membrane fusion and thus

infectionn of a cell

5 5

. These co-receptors are an attractive target for potential

interventionn against HIV infection and the first compounds are being tested in

vitroo

56

*

59

. The first studies demonstrated a potent antiretroviral effect of these

compoundss in vitro and in vivo

60,61

. The binding of the virus to the membrane

iss followed by fusion between the viral transmembrane glycoprotein subunit

(gp41)) and the cell membrane

62

~

64

. Directed against this fusion mechanism is

thee most recent class of antiretroviral drugs available, the fusion inhibitors.

Thee first and at this time only drug in this class is enfuvirtide (T20), which

inhibitss the fusion between gp41 and the cell membrane

64

"

71

.

2.2.. Reverse transcription

Afterr entering the cell HIV-1 is uncoated, releasing genomic RNA, proteins

andd enzymes in the cytoplasm of the cell. Within the cytoplasm viral RNA is,

byy the unique retroviral enzyme reverse transcriptase (RT),

reverse-transcribedd into double-stranded complementary DNA using endogenous

cellularr nucleotides

72

. HIV-1, as other retroviruses, consists of two single

strandedd copies of its RNA genome per virion. If these two RNA molecules

aree not identical a template switch can occur, resulting in the generation of a

novell recombinant DNA genome derived from both parental RNA's

73

. The

highh frequency of genetic recombination, together with the high mutation rate

off HIV-1 reverse transcription (3.4 X 10"

5

per cycle of replication), results in

HIV-11 populations being highly heterogeneous

74

. This gives HIV-1 the ability

too rapidly evade the host immune response and to develop resistance against

antii retroviral drugs in the presence of suboptimal suppression of viral

replicationn or a low genetic barrier of the anti retroviral regimen used against

resistancee development.

Thee RT enzyme is the first step in the HIV-1 life cycle that was targeted by

drugss

75

. Two classes of anti retroviral drugs are targeted against this enzyme:

thee NRTIs and the non-nucleoside reverse transcriptase inhibitors (NNRTIs).

Thee NRTIs (figure 1) were the first class of drugs developed against HIV-1

13

.

Theyy are prodrugs that are phosphorylated by intracellular kinases to their

activee form, dideoxynucleoside triphosphates. The resulting

NRTI-triphosphatess mimic and compete with one of the natural

2-deoxyribonucleosidee triphosphates (dNTP) (table 1) for incorporation in the

developingg proviral DNA chain during reverse transcription. Unlike their

naturall counterpart they lack the ribose 3 -OH group necessary for attachment

off the next dNTP in the sequence, thereby blocking further DNA synthesis

76

~

82

.. Soon after the clinical introduction of the first NRTI, ZDV, in the late

eightiess resistance against ZDV was reported and at this time resistant HIV-1

strainss are known for all NRTIs, with the frequent occurrence of cross-resistancee against more than one NRTI 16,83,84 4

Figuree 2 HIV-1 replication cycle and viral targets used for therapy

F u s i o nn i n h i b i t o r s P r o t e a s ee i n h i b i t o r s Env v RNA A Cyctophilin n

Fusion n

^ 4 ^ C X C R 4 4 CCR5 5 _{Translation!! /} importt I Uncoating g

NRTII + NNRTI

Greyy blocks are the classes of antiretrovirals (modified from Nature Medicine 2003;9:853-8600 85).

Tablee 1 Nucleoside and nucleotide analogues and the natural counterparts Naturall triphosphate deoxythymidinee 5-triphosphatee (dTTP) deoxyadenosinee 5-triphosphatee (dATP) deoxycytidinee 5-triphosphate (dCTP) ) carboxylicc 2-deoxyguanosine Analogue e zidovudinee (ZDV) stavudinee (d4T) didanosinee (ddl)

tenofovirr disoproxil fumarate (TDF) )

zalcitabinee (ddC)

lamivudinee (3TC)

emtricitabine e

abacavirr (ABC)

Activee form analogue

zidovudinee 5-triphosphate 2,3-didehydro-2,3--dideoxythymidine e triphosphatee (d4TTP) 2,3-dideoxyadenosinee 5-triphosphatee (ddATP) PMPAA diphosphate [(R)-9(2- phosphonomthoxy--propyl)adenine,, PMPApp 2,3-dideooxycytidinee 5-triphosphatee (ddCTP) 3TCC 5-triphosphate [(-)-2',3'-dideoxy-5-fluoro-3'--thiacytidine e carbovirr triphosphate

Thee NNRTIs (figure 1) interact specifically with a non-substrate binding site of thee RT of HIV-1, thereby inhibiting RT activity resulting in inhibition of HIV-1 replicationn 87. As with other antiretroviral drugs, in case the replication is not suppressedd adequately the rapid replication rate of HIV-1 inevitably lead to virall variants that exhibit drug resistance. In case of NNRTI resistance there is almostt always cross-resistance, with as consequence that one mutation resultss in resistance against all currently available NNRTIs 88"94.

Anotherr approach to inhibit HIV-1 replication at the step of reverse

transcriptionn is the use of agents that decrease concentrations of natural

intracellularr nucleosides. Examples of such agents are hydroxyurea and

mycophenolatee mofetil (MMF)

95_105

.

Hydroxyureaa has been widely used over the last 30 years for the treatment of

humann malignancies. It inhibits the cellular enzyme ribonucleotide reductase,

aa rate-limiting enzyme in the synthesis of dNTP. Besides its direct antiviral

effectt it was expected that the decrease in dNTP synthesis might also

increasee the effect of the nucleoside analogues ZDV, ddl and ddC

95,106

.

However,, in patients using hydroxyurea in combination with antiretroviral

therapyy the expected decrease of dNTP, predicted from in vitro studies, was

nott seen

107

. In addition, hydroxyurea can result in serious toxicity and blunts

thee CD4

+

T cell replenishment resulting from effective antiretroviral therapy,

andd its effectiveness in HIV-1 therapy is disappointing

108

.

MMFF is used to prevent organ rejection and to treat acute rejection after organ

transplantationn

109

. It is a prodrug that is rapidly converted into its active

metabolitee mycophenolic acid (MPA). MPA selectively inhibits the enzyme

inosinee 5'-monophosphate dehydrogenase (IMPDH), which is responsible for

thee conversion of inosine 5'-monophosphate (IMP) to guanosine

monophosphatee (GMP) in the de novo synthetic pathway of the purine

guanine.. This is the most important source of GMP in lymphocytes while the

salvagee pathway is more important in other cells

110

'

111

(figure 3). In

lymphocytess blocking this enzyme depletes guanosine triphosphate (GTP)

andd deoxyguanosine triphosphate (dGTP) pools, resulting in inhibition of the

activationn and proliferation of T lymphocytes. In HIV-1 infected patients this

resultss in a limitation of the available number of target cells for HIV-1. The

MPAA mediated decrease of dGTP might also directly decrease reverse

transcriptionn by limiting the production of essential compounds for the viral

replication.. The MPA mediated depletion of endogenous dGTP further results

inn an altered competition between CBVTP (the active metabolite of abacavir

(ABC))) and dGTP, thereby increasing the antiretroviral efficacy of ABC in vitro

105

.. Finally, MPA increases the phosphorylation of ABC to CBVTP 81. In vitro MPAA might also synergistically enhance the activity of didanosine and tenofovir1 0 1. .

Figuree 3 The potential effects of Mycophenol mofetil in HIV-1 infection

Dee Novo Pathway Ribose-5-phoshate e Mveophenolatee Mofetil „ . „ . ee Pathway Guanine e

I I

PRPPPRPP synthetase 5-phosphorfcosyi-11 -pyrophosphate (PRPP) Inosinee monophosphate (IMP) '' Adenosine MP

jsejse (IMPDb^

Guanosmee monophosphate (GMP) ^ ™

Ribonucleotide Ribonucleotide reductase reductase

Deoxyadenoss ine DP Deoxyguanosine DP

1 1

3'-dkfeoxyadenosinee "'- Deoxyadenos ine TP Deoxyguanosine TP

5'-trphosphatee , ,- . . , . . , , ^ _ Carbovrr trphosphate ^ ^ (abacavr) ) (didanosine) ) PMPAA dphosphate [(R>r9(( 2-phosphonomthoxy-propyi)) adenine * * (ïenofovrr disoproxi)

Adenosinee DP Guanoss ine DP LYMPHOCYTEE PROLIFERATION

(TARGETCELLSS FOR HIV)

HIVV REPLICATION

Adenosinee TP ,

" RNA *4~ Guanoss ine TP

thickk line: normal pathway, thick dotted line: inhibition by the respective compounds, thinn line: induction of the pathway, thin dotted line: inhibition of the pathway

2.3.. Integration

Afterr reverse transcription the viral preintegration complex (PIC) is formed,

consistingg of viral double-stranded DNA, the reverse transcriptase, matrix,

integrasee and Vpr proteins

i12

. After the PIC enters the nucleus the viral

double-strandedd DNA needs to be integrated in the cellular DNA. This

integrationn has a preference for genes that are active, although other regions

aree also targeted

113114

. The viral enzyme HIV-integrase is essential for this

process.. There is apparently no functional equivalent of this enzyme in human

cells,, making it therefore a potential target for antiretroviral therapy. At this

timee there are no drugs for clinical use that target this enzyme, but the first

compoundss have been discovered which inhibit HIV-integrase in vitro, and the

firstfirst phase I and II clinical trials are ongoing

115

"

117

.

2.4.. Gene expression and virion assembly

Oncee the viral DNA is integrated into the cellular DNA, replication of new

particless can take place. During activation of the infected cell, DNA, including

virall DNA, is transcribed to mRNA by natural cell mechanisms. This mRNA is

transportedd out of the nucleus to the ribosomes in the cytoplasm.

Thee early mRNA transcripts of HIV-1 are doubly spliced and produce the viral

regulatoryy proteins Tat, Rev, and Nef. The function of HIV-1 Rev is to facilitate

thee expression of the late transcripts of HIV-1. These can be divided into two

categories:: unspliced and singly spliced, and they are expressed when a

thresholdd level of Rev is attained. The unspliced HIV-1 mRNA is translated

intoo the structural precursor polyproteins of the gag and pol gene. The

differentt singly spliced mRNAs of HIV-1 are translated into the envelope

proteinss gp120 and gp41, as well as Vrf, Vpr and Vpu

118

"

121

. A shift in the

transcriptionall pattern from the expression of predominantly spliced mRNA to

predominantlyy unspliced mRNA is indicative of active viral replication and

thereforee unspliced mRNA can be used as a measurement for active viral

replicationn

39,122

. During antiretroviral therapy, resulting in a decrease of HIV-1

replication,, the ratio of unspliced mRNA to spliced mRNA decreases

39

.

Onn the ribosomes the viral gag and pol proteins are assembled from the

mRNAA template, resulting in large polyproteins that are not infective yet. To

completee the viral replication and generate infective viral particles these gag

andd pol polyproteins need to be proteolytically cleaved by viral (HIV) protease.

Thiss viral protease is part of the virion and is introduced into the cell during

infection.. The cleavage of pol precursor protein by viral protease also

generatess additional viral proteases, which can become active directly, but

whichh can also be incorporated in the newly formed virions which are released

outt of the cell

123

-

126

.

Thiss viral protease is another target for HIV-1 therapy and drugs in the class

off protease inhibitors are since 1995 widely used for the treatment of HIV-1

infectionn

29

'

127128

. By blocking the viral protease, they prevent the assembly of

infectiouss virions. They don't prevent the reverse transcription of HIV-1 or the

incorporationn of viral DNA into the cellular genome.

3.. HIV-1 infection in vivo

3.1.. The primary phase of HIV-1 infection

Thee most common mode of HIV-1 infection is sexual transmission at the

genitall mucosa

129

. Little is known about the pathophysiological events during

thee primary phase of infection in humans. In rhesus macaques infected with

SIV,, which is clinically comparable with HIV-1 infection in humans

13 132

, it

hass been demonstrated that HIV-1 first attaches to or infects Langerhans

cells,, which are mucosal dendritic cells. Within 2 to 5 days after infection

thesee dendritic cells migrate to regional lymph nodes, where other cells with

CD44 receptors are infected through direct cell-to-cell contact. Studies with

positronn emission tomography (PET scan) during acute SIV infection in rhesus

macaquess and acute HIV infection in humans revealed activation of lymphoid

tissuess in the upper body (axillary, cervical and mediastinum lymph nodes).

Thiss was independent of the mode of transmission. At a later stage of

infectionn activity is also seen in lymph nodes at other sites of the body

Duringg the stage of acute infection virus populations double every 6-10 h. This

resultss in a peak plasma viraemia after an average of 6-15 days

Virallyy induced local inflammatory responses actually facilitate viral replication

andd the development of an acute phase of viraemia, leading to dissemination

off infection to other lymphoid tissues and organs. At this phase of infection

patientss are highly infective. During the process of a primary infection between

700 - 90% of the patients develop a mononucleosis-like illness with usually

feverr and skin rash, but also with sweats, malaise, lethargy, anorexia, nausea,

myalgia,, arthralgia, headaches, sore throat, diarrhoea, generalised

lymphadenopathy,, lymphopenia and thrombocytopenia

139

-

145

. There is a

tendencyy to progress faster to AIDS when the symptoms are more severe or

lastt for more than 15 days.

146

-

148

.

3.2.. The chronic phase of HIV-1 infection

Afterr the initial rise the plasma HIV-1 RNA decreases in the following weeks to

aa steady state level due to virus-specific immune responses

27137138)

starting

thee chronic phase of infection. This is the long asymptomatic period which can

lastt for years, between primary infection and the development of clinical

immunee deficiency (AIDS). In this phase there is a quasi-steady state between

virionn production and clearance

149

. The term quasi-steady state is used

becausee on a longer time scale the CD4

+

T-cell count falls, indicating that the

systemm is not at a true steady state. The lower the steady state plasma HIV-1

RNA,, which is a measurement for HIV-1 replication, the better the prognosis

withh a longer duration of a stable CD4

+

T-cell count

150

.

3.3.. Viral dynamics

Mathematicall modelling of data obtained from patients starting HAART

estimatedd a daily HIV-1 virion production of 10.3 x 10

9

virions per day with a

halff live (ti

/2

) of 0.24 days to contain a steady state plasma HIV-1 RNA level.

T1/22 of virus producing cells is believed to be 1.6 days with the HIV-1 life cycle

beingg 1.2 days, resulting in a complete replacement of the virus population in

144 days

149

-

151

-

154

. Mathematical modelling of recently obtained data with more

potentt regimens shows an even shorter mean t

V2

of 30 minutes for HIV-1

vironss and 0.7 days for virus producing cells

155156

. Upon starting potent

antii retroviral therapy there is an immediate drop in circulating plasma HIV-1

RNAA as a result of the clearance of free virions from plasma and the loss of

virus-producingg cells. This indicates that the vast majority of circulating

plasmaa virus derives from continuous rounds of newly infected cells,

replicationn and cell turnover, and not from cells that produce virus chronically

orr are latently infected and become activated

151

. This very high replication

rate,, combined with a mutation rate of 3.4 x 10"

5

mutations per base-pair per

replicationn cycle

7

\ results in the occurrence of every single-point mutation

betweenn 10

4

and 10

5

times per day

157

.

4.. Limitations of current anti retroviral therapy

Antii retroviral drugs need to be available at those locations in the body where

HIV-11 replication is taking place to have an antiretroviral effect. This means

thatt the NRTIs, NNRTIs and Pis must pass the wall of the gastrointestinal

tractt and through the liver further into the blood plasma to come in contact

withh infected cells. Furthermore, NRTIs are prodrugs that need to be

metabolisedd intracellularly before an antiretroviral effect can be accomplished

82

.. The NNRTIs and Pis have to survive metabolism in the liver during the first

passs effect and Pis are also challenged by drug transporters

158164

.

4.1.. Nucleoside and nucleotide analogue reverse transcriptase inhibitors

Ass described in section 2.2. the NRTIs (table 1) are intracellularly

phosphorylatedd to their active forms, which mimic and compete with one of

thee natural 2'-deoxyribonucleoside triphosphates. Incorporation of the

NRTI-triphosphatee in the developing proviral DNA results in blocking of the DNA

synthesiss

76

~

82

. Cellular enzymes and state of activation influence the

intracellularr activation of NRTIs. An example is the activation of ZDV. The

productionn of the active ZDV triphosphate is determined by two rate-limiting

steps,, thymidine kinase and nucleoside diphosphate kinase, leading to an

accumulationn of the mono- and diphosphate ZDV derivates

165166

. NRTI

activationn is also dependent on whether the cell is active or resting

(post-mitotic))

167

'

168

. Active cells preferentially phosphorylate thymidine analogues

(ZDVV and stavudine (d4T)), while resting cells preferentially phosphorylate

adenosinee (didanosine (ddl)) and cytidine analogues (zalcitabine (ddC) and

lamivudinee (3TC))

167_172

.

Thee phosphorylation of ABC depends on IMP as phosphate donor. IMP is also

importantt for the formation of GMP and GTP. In vitro, the addition of MPA

givess an accumulation of IMP by blocking its transformation to GMP, resulting

inn an increased phosphorylation of ABC (figure 3)

8 1

.

NRTIss are predominantly excreted by the renal system (tubular secretion) and

interactionss due to cytochrome P450 activity are not regularly encountered

173

.

Plasmaa concentrations of the NRTIs can be measured but do not correlate

withh the in vivo effect and therefore seems to be of no clinical use at this time

82,174 4

Theree is a competition between the NRTI-triphosphates and the natural dNTP

forr incorporation in the developing DNA chain. The higher the ratio is in favour

off the NRTI-triphosphates, the better the antiretroviral effect. For CBVTP this

cann be accomplished by the use of MMF, as discussed earlier in section 2

105

.

Itt has been demonstrated that in patients failing on a 3TC containing regimen

thee ratio of 3TCTP:dCTP is significantly lower than in patients performing well

onn such a regimen

107

.The intracellular phosphorylation of the NRTIs depends

onn several intracellular metabolic parameters

175_177

. To optimise therapy with

NRTIss it might therefore be relevant to measure the intracellular active

triphosphates,, but this is too complicated to perform in daily clinical practice.

Theree is a difference in penetration of the NRTIs into other compartments than

thee blood, which can contribute to the maintenance of anatomical reservoirs

ass discussed further on. ZDV, d4T, 3TC and ABC achieve concentrations in

thee cerebrospinal fluid (CSF) that are above the minimum required

concentrationn necessary to inhibit viral replication

178

-

180

, while ddl and ddC

achievee concentrations lower than the minimum required concentration

178

.

Theree are no data available at this time regarding the penetration of

emtricitabinee and TDF into the CSF. In seminal plasma the concentrations of

ZDV,, d4T, 3TC, ABC and ddl are above the minimal required concentration

181-1844

N o d a t a a r e ava

j|ai3|

e o n t n e

penetration of ddC, TDF and emtricitabine

intoo seminal plasma.

4.2.. Non-nucleoside reverse transcriptase inhibitors

Thee NNRTIs are a group of compounds that induce allosteric changes in the

HIV-11 RT, thereby rendering the enzyme incapable of converting RNA to

DNA.. Unlike NRTIs, they don't need to be phosphorylated to become active

87

'.. However, all NNRTIs are metabolised to some degree by the cytochrome

P4500 (CYP) system of enzymes, making them prone to clinically significant

drugg interactions, as they act either as inducers or inhibitors of CYP

158

. Over

300 different human CYP enzymes have been identified, of which CYP3A4

appearss to be the most important one as it contributes to the

biotransformationn of approximately 60% of therapeutic drugs

185

. Delavirdine

(DLV)) is an inhibitor of CYP3A4 metabolism. Efavirenz (EFV) is an inhibitor of

CYP2C99 and CYP2C19 and seems to be both an inhibitor and inducer of

CYP3A44 metabolism. Nevirapine (NVP) is an inducer of both CYP3A4 and

CYP2B66 metabolism

158

'

186

.

Mostt important are the pharmacokinetic interactions between the NNRTIs and

thee Pis. For example, DLV, EFV and NVP can increase the plasma

concentrationss of nelfinavir. DLV and EFV can increase the plasma

concentrationss of ritonavir (RTV) and DLV can increase the plasma

concentrationn of saquinavir (SQV) and indinavir (IDV). This increase in plasma

concentrationss can further contribute to the side effects of the protease

inhibitorss that can already occur with normal concentrations of those drugs

159,1877

A m o r e

j

m

p

0 r

t

a n

t problem in the face of antiretroviral therapy is the

loweringg of the plasma concentrations of IDV, SQV, RTV, amprenavir (APV)

andd lopinavir (LPV) by EFV and that of IDV, SQV, RTV and LPV by NVP

158,188-1900 5

e c a u s e

this might result in the selection of resistant HIV-1 strains.

Ass with the NRTIs, NNRTIs also have a different penetration in other

compartmentss than blood. NVP and EFV penetrate well into the CNS,

achievingg adequate CSF levels

191193

. NVP, but not EFV penetrates in the

malee genital tract

182194

. No data are available on the penetration of DLV into

thee CNS or the male genital tract.

4.3.. Protease inhibitors

Forr effective anti-HIV activity of the protease inhibitors (Pis) adequate plasma

concentrationss are required

195,196

. All Pis are substrates and inhibitors of

CYP3A44 in the liver and small intestine

197

~

206

. Inducers of CYP3A4 like the

NNRTIss can therefore decrease the plasma concentrations of the Pis. The

inhibitoryy effect of the Pis, with RTV being the most potent one, can influence

thee metabolism of other CYP3A4 substrates and can result in interactions

betweenn the Pis

204

-

205

. These interactions can also be used to optimise

therapyy including Pis. For example, the co-administration of a low dose of

RTVV with IDV improves the overall pharmacokinetics of IDV by inhibiting the

metabolismm of IDV, and allows the IDV intake twice daily instead of three

timess daily

207

-

209

.

Alll Pis are also substrates for the drug transporter P-glycoprotein (P-gp)

158

"

164

.. This drug transporter can lower the uptake of the Pis in the

gastrointestinall tract and in the cells

16321 212

. As P-gp itself also seems to

decreasee HIV-1 replication

213214

, the overall effect in vivo is unclear at the

moment. .

Memberss of the multidrug-resistance protein (MRP) family have also been

recognisedd as transporters of Pis

215216

. These drug transporters might

thereforee limit the antiretroviral effect of Pis.

Ass with the NRTIs and the NNRTs, there is a difference in the accumulation of

Piss in the blood and in other compartments of the body. IDV is the only PI

knownn to achieve optimal concentrations in the CSF

217218

. (_PV, nelfinavir

(NFV),, RTV, SQV and APV don't achieve effective concentrations in the CSF

219-2233

L

j

k e w i s e

|

D V

penetrates well into the seminal plasma

219

-

223

-

224)

j

n

contrastt to NFV, RTV, SQV and APV

219224

-

225

. We demonstrated that LPV

poorlyy penetrates into seminal plasma

226

. At the present time no data are

availablee on the penetration of atazanavir into CSF and into seminal plasma.

4.4.. Reservoirs of HIV-1

Thee terms "compartment", "reservoir" and "sanctuary site" are not well defined

andd are used interchangeably

227233

. For clarity a standard definition is

needed,, which was recently suggested by us

234

.

AA viral compartment is an anatomical site in which the virus in untreated

patientss evolves distinctively from other anatomical sites or the main pool of

infectedd cells, due to differences between the major cell types sustaining viral

replicationn

228

. A viral reservoir is a cell type or anatomical site in which a

replication-competentt virus persists much longer than in the main pool of

infectedd cells that sustain the infection, and this cell type or anatomical site

cann replenish the pool of infected cells

232233

. A viral sanctuary site is an

anatomicall site which is highly impermeable to (some) antiretroviral drugs,

andd in which viral replication continues during treatment, thus allowing

developmentt and/or selection of drug-resistant strains.

4.4.1.. Viral sanctuary sites

Thee CNS is considered a viral sanctuary site. Arguments in favour are: the

slowerr HIV-1 RNA decay in CSF than in blood plasma

235,236

, the lower levels

off NNRTIs and Pis in CSF compared with blood plasma

178

and the observed

differentt resistance patterns in the CNS strains compared with blood plasma

strainss

237239

. Another site considered being a viral sanctuary site for HIV-1 is

thee male genital tract

230

'

240241

. Whether this is a true sanctuary site is

howeverr questionable, except in case of another sexually transmitted infection

234 4

4.4.2.. Cellular viral reservoirs and viral latency

Thee most important cells of the cellular viral reservoir are the latently infected

restingg memory CD4

+

T cells containing replication competent, integrated

proviruss

3536242

>

243

. A significant portion of these cells are HIV-specific

memoryy CD4

+

T cells

244

. But HIV-1 can also persist in other cells, like the

naivee CD4

+

T cells

243,245

, CD8

+

T cells

246 247

, monocytes and macrophages

248-2511

a n c

|

n a t u r a

| killer cells

252

. There are indications that dendritic cells and

BB cells might also contribute to the cellular reservoir, but it is not clear whether

HIV-11 persist intracellularly in the cell or that HIV-1 particles are bound to their

celll membrane

253

"

256

.

Thee establishment of the cellular reservoir is a result of viral latency, which is

aa common feature of all retroviruses and which is one of the most important

hostt cell-virus interactions whereby the virus can survive and persist in the

facee of antiretroviral therapy. HIV-1 latency is a consequence of the normal

physiologyy of CD4

+

T lymphocytes. Most of the CD4

+

T cells are in a resting

(Go)) state, with half of them being naive, having yet to encounter an

appropriatee antigen. The remaining cells are memory cells that have

previouslyy responded to an antigen. Upon encountering an antigen, a burst of

cellularr proliferation and differentiation occurs, giving rise to effector cells.

Mostt of them die quickly, but a subset survives and reverts back to the resting

Goo state, persisting as memory cells

257258

. Uninfected CD4

+

T memory cells

dividee or die with a half live of 6 months, while naïve cells divide or die with a

halff life of 3.5 years

259

. Upon infection with HIV-1, there is an increased T-cell

activationn with the virus preferentially replicating in activated and HIV specific

CD4

++

T cells. For a long time it was not understood by which mechanisms the

CD4

++

T cells are killed s

4

-

151

-

152260

. The last years it became clear that the loss

off CD4

+

T cells is a result of the enhanced T-cell turnover as a result of the

chronicc immune activation by HIV

261264

Theree are two forms of latency: pre-integration (the stage that viral DNA is

presentt in the host cell but not integrated in the host DNA) and

post-integrationn (the stage that viral DNA is integrated in the host DNA)

231

. Upon

bindingg and fusion of virions with CD4

+

T cells, pre-integration latency can

occur.. This doesn't require the cell to be activated or to be in a dividing state

265,2666 |

t h a s b e e n

hypothesised that some activated cells become infected at

thee moment they are in the process of reverting to a resting state. These cells

mightt be permissive for early steps in the viral life cycle up to integration, but

nott for virus gene expression. Upon activation of these cells further

completionn of the viral life cycle can occur

267268

. Activation of these resting

cellss can also be triggered by HIV-1 Nef protein, which may even be produced

directlyy in pre-integration infected CD4

+

T cells

269270

. The pre-integration

stagee of the virus is labile with a half-life of only 1 day

2 7

\ but the pool of cells

containingg pre-integration virus is much larger than that of cells containing

post-integrationn virus

35

'

267

.

Afterr integration of proviral DNA a state of post-integration latency can be

established.. It has been demonstrated that replication competent HIV can be

recoveredd from these latently infected cells, which are mainly resting memory

CD4

++

T-cells

3638

'

41272

. Pro-virus can also be found at lower levels in resting

naivee CD4

+

T-cells

245

-

272273

. Two mechanisms might contribute to latency in

naïvee CD4

+

T-cells. One possibility is that some resting memory cells with

integratedd HIV-1 DNA revert to the naïve phenotype, as has been observed in

normall uninfected human and rodent systems

259

-

274

-

275

. Another possibility is

infectionn of naïve T-cells during thymopoiesis in the thymus

276

.

Thee pool of latently infected cells is established during primary HIV infection,

whichh can't be prevented even when HAART is started before

sero-conversionn

277

. Given the long half life of CD4

+

cells this pool can persist

duringg HAART for years, with an estimated half-life of 44 months which results

inn an estimated need for at least 73 years of therapy for eradication of this

virall reservoir

36

-

38

-

41278

-

280

.

Virall latency is an important obstacle for HIV-1 treatment, because current

HIVV treatment is only effective during active HIV-1 replication and doesn't

targett latently infected cells.

4.5.. Residual HIV-1 replication

Itt is clear that although currently used combinations of antiretroviral therapy

cann suppress HIV-1 replication below the limit of currently available assays,

residuall or low level ongoing replication still takes place in patients

successfullyy treated with antiretroviral therapy. This is demonstrated by the

presencee of intracellular HIV-1 RNA even during the use of HAART with

plasmaa HIV-1 RNA levels below the limit of detection

373940

.

2

8

1

-283

Residuall replication is confirmed further by studies demonstrating modest

evolutionn over time in HIV-1 proviral sequences in peripheral blood

mononuclearr cells while plasma HIV-1 RNA was below the detection limit

284

"

286

.. The residual replication might be the biggest obstacle for the eradication

off HIV-1, as it can refeed the cellular reservoir. On the other hand, both

sub-optimall penetration of HAART in the sanctuary sites and the cellular reservoir

consistingg of latently infected cells can contribute to the residual replication.

Uponn activation of the latently infected cells new infectious virus can be

released,, which can infect new cells

287

. It is believed that these latently

infectedd cells are mainly present in the lymph nodes. Therefore, the lymph

nodess might be the most important site for residual replication

288

.

5.. Interventions to lower HIV-1 reservoirs and residual replication

Thee persistence of HIV-1 is a dynamic process that depends on viral

sanctuaryy sites, the cellular viral reservoir consisting of latently infected cells,

andd residual replication. Therefore three targets can be identified to further

optimisee HIV-1 treatment: improving the penetration of currently used HAART

inn anatomical and cellular reservoirs, increasing the decay of latently infected

cellss and finally, the use of more potent antiretroviral drugs or combinations to

lowerr residual viral replication.

Forr example, to improve the penetration of indinavir into the CNS ritonavir has

beenn successfully used

223

. However, whether the use of drugs with a good

penetrationn in viral sanctuary sites is clinically relevant to prevent viral

resistancee is not clear at the moment.

Too lower the reservoir of latently infected cells the combination of anti-CD3

andd recombinant interleukin 2 (IL-2) in the presence of HAART has been

used.. This strategy was unsuccessful and caused severe side effects

281,289,2900 Another approach to lower the reservoir of latently infected cells

mightt be the use of MMF. In one study the addition of MMF to HAART

loweredd this reservoir in patients

103

. This was thought to be a result of an

increasedd apoptosis of these cells induced by MMF.

Ass MMF also selectively inhibits activation and proliferation of CD4

+

T cells it

limitss the number of target cells for HIV-1 infection. That, combined with the

synergisticc effect on ABC, might result in a more potent suppression of viral

replicationn and might therefore contribute to a decrease in the residual

replication. .

Too lower residual replication, a triple class five-drug regimen has been used.

Thiss regimen resulted in a faster decay of the plasma HIV-1 RNA compared

withh a standard duo class, triple regimen

291

, and a better long term

suppressionn of HIV-1 replication

292

. This strategy however, was not able to

clearr the reservoir of latently infected cells

43

.

Hydroxyureaa was used in an attempt to further improve antiretroviral therapy

ass it was expected to inhibit the activation of CD4

+

T cells, thereby limiting the

numberr of target cells for HIV-1. Furthermore, hydroxyurea might improve the

effectt of ddl and might therefore decrease residual replication. However, the

resultss were disappointing and hydroxyurea caused a lot of side effects

293

.

6.. Outline of this thesis

Inn this thesis new approaches are studied and described to further improve

antiretrovirall therapy in HIV-1 infected patients with the purpose to lower the

reservoirss of HIV-1 and the residual replication as discussed in chapter 1.

Chapterr 2 describes a study which investigated whether starting a triple class

five-drugg regimen during primary HIV-1 infection can lower the reservoir of

latentlyy infected cells, and whether this effect persists after stopping therapy.

Inn chapter 3 the penetration of the potent protease inhibitor lopinavir into

seminall plasma of HIV-1-infected men was studied, because especially during

sexuall transmitted infections the male genital tract can be considered a

sanctuaryy site for HIV-1.

Ass discussed in the Introduction, one of the possible strategies to lower

residuall replication is the use of more potent antiretroviral drugs. In chapter 4

thee antiretroviral potency of a new NNRTI is compared with that of a triple

class,, five-drug regimen, during one week of treatment.

Ass discussed in chapter 1 MMF can limit the availability of target cells for

HIV-1,, it can increase the antiretroviral activity of abacavir and it might decrease

thee pool of latently infected cells. Therefore in chapter 5, 6, 7 and 8 the

resultss of a study are described in which primary and chronic infected HIV-1

patientss were treated with a triple class five-drug regimen and randomised to

alsoo receive MMF or not. In chapter 5 the effect of MMF on the plasma HIV-1

RNAA and on the reservoir of latently infected cells is described. Chapter 6

discussess the immunological effects of MMF in combination with HAART in

HIV-11 patients starting therapy. Chapter 7 discusses the pharmacokinetics of

MMFF and the interactions of MMF with antiretroviral therapy, and the effect of

MMFF on the intracellular triphosphates. In chapter 8 a patient with a primary

HIV-11 infection is described who became seronegative during treatment with

HAARTT and MMF.

Chapterr 9 reviews the literature regarding the possible effects of P-gp in

HIV-11 replication and can limit the bioavailability and penetration into cells of the

proteasee inhibitors. In chapter 10 we studied whether P-gp function indeed

influencess the HIV-1 replication in vivo.

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