Limitations of current antiretroviral therapy in HIV-1 infection: the search for new strategies - CHAPTER 11 Summary & Discussion

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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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Potentt therapy for HIV-1 infected patients became available in the

mid-nineties,, with the introduction of highly active antiretroviral therapy (HAART).

Suchh therapy resulted in many patients in a plasma HIV-1 RNA below the limit

off detection, and a restoration of their immune function

'

. This led to a

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

mortalityy in the developed world

'

. Based on early viral decay studies

assumingg total suppression of viral replication in patients using HAART, it was

estimatedd that with this therapy HIV-1 could be eliminated after 2-3 years of

treatment

.. However, in the following years it became clear that with currently

availablee anti-HIV-1 therapy eradication of HIV-1 is not possible

"

.

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

factorss influencing each other. HIV-1 can persist in sanctuary sites and

cellularr reservoirs consisting of latently infected cells and there is ongoing

residuall replication, as described in chapter 1

-

'

-

The cellular

reservoirss are a result of viral latency and might contribute to residual

replication,, which in its turn can refeed the reservoirs. Before eradication and

thuss curation of HIV-1 can be accomplished, these factors need to be

targeted.. This thesis investigates several strategies to do that.

Thee pool of latently infected cells is established early during infection. The

initiationn of a three-drug regimen within 10 days of a primary (acute) infection

cannott prevent this

. We treated patients with a primary HIV-1 infection with

aa five-drug, triple class regimen, which is more potent than a standard

triple-drugg regimen

, to study if such a regimen could affect the size of this

latentlyy infected reservoir. The results are discussed in chapter 2. Although in

patientss treated with a triple class, five-drug antiretroviral regimen the amount

off resting CD4+ T cells containing replication-competent HIV-1 dropped below

thee lower limit of quantification (LLQ), replication competent HIV-1 persisted in

thee body

. This was demonstrated by the rebound of both the number of

latentlyy infected cells and the plasma HIV-1 RNA after the interruption of

HAART. .

wass studied . This study demonstrated that lopinavir has a poor penetration

intoo seminal plasma, with a median concentration of only 0.23 mg/L, while the

minimumm required concentration to inhibit HIV-1 replication is believed to be

5.00 mg/L (Abbott product information). These results were later also confirmed

byy other studies

. Because of this poor penetration of lopinavir into

seminall plasma it would be expected that HIV-1 RNA would be detectable in

seminall plasma, thereby confirming that the male genital tract really is a

sanctuaryy site. However, HIV-1 RNA was undetectable in the seminal plasma

off most of the patients studied. This led us to reconsider the male genital tract

ass being a sanctuary site, and after an extensive review of the literature we

refutedd this site being a sanctuary site

, except in the presence of another

sexuallyy transmitted infection.

Nevertheless,, all data to date confirm that in the male genital tract, as well as

inn other sites such as the central nervous system (CNS), there can be a

suboptimall penetration of drugs. The CNS may be a true sanctuary site

~

19,40-422 j

suboptimal penetration of particular antiretrovirals might partly be

causedd by the action of drug transporters, of which P-glycoprotein (P-gp) is

one,, and this will be discussed further on.

AA possible strategy to lower the residual HIV-1 replication is the use of more

potentt antiretroviral drugs or drug combinations. It has been demonstrated

thatt a triple class, five-drug regimen results in a faster decay of the plasma

HIV-11 RNA and a better long-term suppression of HIV-1 replication than a

standardd dual class, triple regimen

. That the potency of antiretroviral

agentss can be improved upon is demonstrated in the study described in

chapterr 4. Monotherapy during one week with the new non-nucleoside

reversee transcriptase inhibitor (NNRTI) TMC125 resulted in a decay of the

plasmaa HIV-1 RNA which was comparable with that achieved by a triple class

five-drugg regimen

. We suggested that a possible explanation for this potent

effectt might be the higher accumulation of TMC125 in the lymph nodes

comparedd to plasma. This is compatible with the lymph nodes being the major

sitee for HIV-1 replication

. It has recently been demonstrated that

intensificationn of antiretroviral therapy with currently available drugs

acceleratess the decay rate of the latent cellular reservoir and decreases

residuall viral replication, but even this can not completely suppress ongoing

virall replication

. Further studies are necessary to elucidate the effect of

TMC1255 containing anti retroviral therapies on the viral decay rate, and on the

residuall replication, by measuring intracellular unspliced HIV-1 RNA as

discussedd below.

Nott only residual replication but also the presence of a pool of latently infected

cells,, even during potent antiretroviral therapy, is a major obstacle in the

treatmentt of HIV-1. In the search for new approaches to reduce the pool of

latentlyy infected cells and (residual) HIV-1 replication, a role for mycophenolic

acidd (MPA), the active metabolite of mycophenolate mofetil (MMF), has been

suggestedd

. By blocking lymphocyte proliferation, MPA can limit the

availabilityy of target cells for HIV-1, thereby reducing HIV-1 replication. By

decreasingg the dGTP production it can also directly inhibit HIV-1 replication,

ass dGTP is necessary for HIV-1 replication. The depletion of endogenous

dGTPP further results in an altered competition between CBVTP (the active

metabolitee of abacavir) and dGTP, thereby increasing the antiretroviral

efficacyy of abacavir. In one study MPA was able to reduce the pool of latently

infectedd cells in patients with a undetectable plasma HIV-1 RNA

. Chapter

5,, 6, 7 and 8 describes the use of MMF in HIV-1 infection. Both patients with a

primaryy and a chronic HIV-1 infection were included in this study (the MAN

study).. They all started therapy with a triple class, five-drug regimen

consistingg of once daily didanosine e.c. 400 mg and twice daily abacavir 300

mg,, lamivudine 150 mg, indinavir 800 mg (boosted with ritonavir 100 mg bid)

andd nevirapine 200 mg (first 2 weeks nevirapine 200 mg once daily). They

weree randomised to a group with or without MMF.

Thee results presented in chapter 5 demonstrate that MMF was well tolerated

inn HIV-1 positive patients starting HAART. However, MPA had no effect on the

decayy rate of the plasma HIV-1 RNA during the first 14 days of therapy, or on

thee decay rate of the pool of latently infected cells during the first 24 weeks of

therapyy

. It is likely that MPA has no additional effect on the HIV-1

replicationn rate when given in combination with such a highly potent regimen

drug,, dual class regimen was used . Surprisingly, the pool of latently

infectedd cells increased temporarily in our study after stopping MMF. A

possiblee explanation is that when therapy is started, MPA blocks the cells in

theirr cell cycle. Upon stopping MMF, these blocked cells can complete their

celll cycle and can temporarily contribute to the pool of latently infected cells.

Inn chapter 6 the immunological effects of MPA in patients treated with HAART

withh or without MMF are described. Although T cell proliferation was

completelyy inhibited in patients using MMF, the changes in the total numbers

off CD4

and CD8

T cells and their subsets were comparable in both patient

groupss

. This may be explained by the fact that the key enzyme in the de

novonovo synthetic pathway inhibited by MPA (inosine 5'-monophosphate

dehydrogenase)) exists in two isoforms. One isoform (type II), mainly used by

activatedd lymphocytes, is much more actively inhibited by MPA than the other

isoformm (type I), used by resting cells

. Since activated T cells are prone to

diee by activation-induced cell death, proliferation within this population

presumablyy does not contribute to maintain the T cell pool.

Further,, there was also no difference in the development of HIV-1 specific

cellularr immunity in patients with and without MMF.

Thee findings in this study combined with a recently published study, in which

MMFF was safely given to HIV-1 patients with a median CD4

T cell count of 34

xx 10

/L

, increases confidence that MMF can be given safely to HIV-1

infectedd patients.

Althoughh the effects on the virological response seem to be minimal, there

mightt be other potential uses of MMF. It has been demonstrated that chronic

hyperactivationn of the immune system in HIV-1 infected patients leads to

enhancedd T-cell turnover and exhaustion of the naive T-cell compartment.

Suppressionn of viral replication by HAART was believed to prevent the loss of

CD4

T cells by the prevention of direct virus mediated killing, but preventing

thee further loss of CD4

T cells might also or even mainly be the result of

reducingg the antigenic trigger that leads to a deleterious immune activation.

Afterr starting HAART an immediate reduction in CD4

and CD8

T-cell

divisionn rates can be seen

~

. Using MMF in therapy naive HIV-1 patients

might,, by inhibiting proliferation and thereby decreasing the level of immune

activation,, lower the rate of CD4

T cell loss, thereby delaying the need for

antii retroviral therapy. Indeed, recent studies demonstrated that stopping

HAARTT in the presence of MMF resulted in improved control of virus

replicationn compared to stopping without MMF

. A study in patients with

multidrugg resistant HIV did not demonstrate viro logica I efficacy but

demonstratedd nevertheless an increase in CD4

T cell counts

. This study

didd not look at the effect of MMF on the immune activation but it is

conceivablee that the immunesuppressive effect of MMF prevented the further

losss of CD4

T cells. Therefore, the use of MMF might be a rational option to

increasee the CD4

T cell count in patients without other treatment options.

Thee pharmacological interactions between MPA and HAART were studied in

chapterr 7, demonstrating that there are no interactions between MPA and

abacavirr or indinavir. However, MPA lowered the plasma concentration of

nevirapinee significantly, possibly by interfering with the enterohepatic recycling

off nevirapine

.

Thee supposed working mechanism of MPA is the inhibition of dGTP

productionn and an increase in the formation of CBVTP, as discussed in

chapterr 1. We therefore measured intracellular dGTP and CBVTP

concentrationss in our study patients. Surprisingly, no differences were seen in

intracellularr dGTP concentrations between patients with or without MMF

.

Onee might argue that MMF dosages used were suboptimal, but plasma

concentrationss were above the minimum required concentration of 0.14 ug/mL

622

and lymphocyte proliferation was completely blocked in patients using

MMF.. One possible explanation for this lack of effect on dGTP concentrations

couldd be the interpatient variability in tri phosphorylation or, alternatively, that

proliferationn assays are more sensitive than dGTP measurement. Another

possibilityy is that cells might compensate the effect of MPA by using other

meanss for dGTP production. dGTP measurement over time might shed some

lightt on whether cells can compensate the effect of MPA. Such a study is

currentlyy underway in kidney transplant patients starting MMF.

twoo months after starting therapy the concentration of his serum anti-HIV-1

antibodiess decreased, resulting in seronegativity, which persisted for a year

.

AtAt that time the patient stopped therapy, resulting in a detectable but very low

plasmaa HIV-1 RNA within 6 days and the re-emergence of anti-HIV-1

antibodiess within one month. Probably, the early potent antiviral therapy, and

thee MMF treatment together resulted in decreased viral antigen exposure and

abrogationn of antibody formation.

Itt has been demonstrated that all HIV-1 protease inhibitors are substrates for

P-gpp

~

. A well-known effect of P-gp in HIV infection is that P-gp can limit

thee penetration of these drugs into the CNS and therefore might contribute to

thee existence of sanctuary sites

-

. Chapter 9 is a review of the

potentiall role of P-gp in HIV-1 infection and therapy. It is clear that P-gp can

potentiallyy lower the penetration of the protease inhibitors in CD4

T

lymphocytess

and the different anatomical sites, given its distribution in the

bodyy

. A logical step therefore could be to inhibit the P-gp activity, thereby

increasingg the penetration of protease inhibitors. However, it is necessary to

knoww what the effect in vivo is, because P-gp can also protect cells against

HIV-11 replication

.

Thiss was elucidated in the study described in chapter 10. In this study, naive

andd memory CD4

T cells were collected from 27 patients, 13 therapy naive

andd 14 on a protease inhibitor containing regimen including either nelfinavir or

indinavir.. Based on their P-gp activity cells were sorted into a P-gp

and a

P-gp

fraction. In these fractions unspliced HIV-1 RNA was measured, as

markerr of active viral replication

. HIV-1 replication was lower in patients

onn successful treatment, as could be expected, but intracellular HIV-1 RNA

wass still detectable, as has been reported in the past

. in patients on a

PII containing regimen unspliced HIV-1 RNA was significantly lower in P-gp

naivee cells than in P-gp

naïve cells and DNA was significantly lower in

P-gp

memory cells than in P-gp

memory cells. These data make clear that

thee protective effect of P-gp activity against HIV-1 replication is more

importantt in vivo than its negative effect on the penetration of the Pis

.

Therefore,, contrary to previous suggestions, even during Pl-therapy not P-gp

inhibition,, but P-gp induction might reduce HIV-1 replication.

Thee studies described in this thesis demonstrate that there are stilt new

strategiess and mechanisms that can be used to further optimise antiretroviral

therapyy or that warrant further study.

Ass stated earlier the loss of CD4

T cells is a result of the chronic

hyperactivationn of the naive T cell compartment

~

. There is ample evidence

thatt T regulatory (or suppressor) cells (Tregs) are responsible for the down

regulationn of immune responses, resulting in suppression of proliferative T-cell

responsess

. At this time there are no data on Tregs in HIV-1 patients, but

givenn the chronic hyperactivation of the immune system during HIV-1 infection

itt seems likely that Treg activity might be decreased in HIV-1 patients. On the

otherr hand one could wonder if Treg function is not higher in HIV-1 infected

patients,, because of the observed decrease of the HIV specific T-cell

responses.. If the role of Tregs in HIV-1 infection is better understood, they

mightt be a target for manipulating the immune system.

Althoughh it is clear that even the most potent combinations of current

antiretroviralss cannot clear the reservoir of latently infected cells, it has been

demonstratedd that more potent drug regimes can further decrease its size

30,45

.. Furthermore, even in the presence of potent antiretroviral therapy,

resultingg in a plasma HIV-1 RNA below the lower limit of quantification, still

somee degree of HIV-1 replication takes place ii.i3.i4.25.26.8M6_

f j

j

moree potent combinations of antiretroviral therapy accelerate the decay of the

pooll of latently infected cells again proofs that residual replication is an

importantt factor for the maintenance of this pool, because the antiretrovirals

usedd are only effective during active replication

'

. Therefore more potent

antiretrovirall drugs than currently available are needed. The results with

TMC1255 show that this is possible

.

Thee availability of a new potent class of antiretrovirals, the fusion inhibitors

*

95

,, gives us the opportunity to study the antiretroviral effects of a four-class

drugg regimen. Given the previous results, the expected increased

antiretrovirall potency might further improve the inhibition of viral replication

thatt are targeted against the CCR5 co-receptor, which is necessary for viral

attachment

,, and against HIV-integrase, which is necessary for integration of

thee virat DNA in the cellular DNA

~

. When these drugs become available

wee might even think about inhibiting HIV replication at six different targets at

thee same time to fully suppress the ongoing residual replication.

Off interest is the fact that TMC125 and the NNRTI efavirenz have the same

antivirall potency in vitro but not in vivo. This might be explained by the 4-fold

accumulationn of TMC125 in lymph nodes as compared to blood, as was

demonstratedd in dogs

. Because the lymph nodes are the most important

sitee where HIV-1 replication is taking place

'

jt is important that

antiretrovirall drugs penetrate these sites well. At this time not much is known

aboutt the accumulation of antiretroviral drugs in lymph nodes. It was recently

demonstratedd that the lymph node / blood plasma ratio for ritonavir, nelfinavir

andd lopinavir were 0.64, 0.58 and 0.21 respectively

. However, the potency

off antiretroviral drugs cannot fully be explained by the penetration in the lymph

nodes.. Although nelfinavir has a better penetration in lymph nodes than

lopinavir,, the last one is more potent in a clinical setting, probably because

plasmaa lopinavir levels are more stable than those of nelfinavir

. The lymph

nodee / blood plasma ratio for indinavir was investigated in two studies. In one

studyy a ratio of 2.07 was seen

but in the second study this was only 0.25

m

.. The penetration of other drugs in the lymph nodes and possible strategies

too improve it should be studied because this might be a target to further

optimisee currently used antiretrovirals.

Too improve the (bio)availability of antiretroviral drugs, the clinical relevance of

drugg transporters in HIV-1 patients using HAART needs to be further studied.

Althoughh we demonstrated that the drug efflux function of P-gp is not that

importantt during PI treatment, it is clear that other drug transporters might be

off importance. Other drug transporters, especially transporters of the

multidrugg resistance protein (MRP) drug transport family, have also been

identifiedd as a transporters for Pis

-

-

and NRTIs

. Further studies

aree necessary to clarify what their effects in vivo are, as it seems likely that

theree is some combined effect between P-gp and the other drug transporters.

Thee pool of latently infected cells consists of CD4

T cells (mainly resting

memoryy cells) containing replication competent, integrated provirus, which is a

resultt of viral latency 91012,21,22,24,119,120

|

tly infected

cellss results again in replication and thus in production of HIV-1 virions

.

Becausee this pool of latently infected cells cannot be completely eradicated

withh current available therapy we have to look for other strategies to minimise

thee contribution of this reservoir to HIV-1 replication. Such a strategy could be

too interfere with the transcription of the provirus to the complete virion. This

mightt be accomplished by using the mechanism of RNA interference (RNAi)

122-1300

h

j

f g

regulation in which target mRNAs are

degradedd in a sequence-specific manner with small interfering RNAs

(siRNAs).. It was already known that such a mechanism existed in plants, but

onlyy recently it was also discovered in animals

. in vitro studies

demonstratedd that siRNAs targeted against specific HIV mRNA sequences

resultedd in inhibition of HIV-1 replication

-

>

.

The problem is how to

deliverr an si RNA in a patient at those sites where replication is taking place.

Vectorss based on lentiviruses are promising candidates to introduce siRNA in

cells,, which then become able to produce the siRNA themselves

-

-

. But

thee use of viruses as a vector can be dangerous, as was seen in a study in

whichh a retrovirus was used to transfer a gene in 10 patients with X-linked

severee combined immunodeficiency (SCID). Two of the 10 treated patients

developedd an uncontrolled exponential clonal proliferation of mature cells

.

Furthermore,, it was recently demonstrated that HIV-1 can escape from

RNAi-mediatedd inhibition by selecting mutations in the viral target sequence

.

Thee potential usage of siRNA is not only limited to HIV-1 therapy, but might

alsoo be used in other fields and therefore warrants further research.

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CHAPTERR 11

Infectiee met het "human immunodeficiency virus" (HIV) leidt tot een langzame

afbraakk van het afweersysteem van de mens. In het bijzonder treedt er een

dalingg op van het aantal CD4

T cellen (een bepaald type witte

bloedlichaampjes).. Dit leidt uiteindelijk tot het optreden van ernstige infecties

enn kanker. Op dat moment spreken we van "acquired immunodeficiency

syndrome"syndrome" (AIDS).

Inn 1983 werd het HIV-1 ontdekt als verwekker van AIDS. In 1986 werd er een

tweede,, verwant virus ontdekt als verwekker van AIDS, namelijk HIV-2. Dit

viruss komt echter veel minder voor, zodat het meeste onderzoek zich

concentreertt op HIV-1, zoals ook alle studies in dit proefschrift. In 1987 kwam

hett eerste middel beschikbaar dat de vermenigvuldiging van HIV-1 kon

remmen.. Het bleek al snel dat het virus zich kan aanpassen, waardoor het

ongevoeligg (resistent) wordt voor dit middel. In de jaren die hierna volgden

werdenn er nieuwe middelen ontwikkeld om de HIV-1 vermenigvuldiging te

remmen.. Op dit moment zijn er vier verschillende klassen van anti-HlV

medicijnenn (antiretrovirale middelen), waarbij elke klasse op een andere

manierr de HIV-1 vermenigvuldiging remt. Halverwege de jaren negentig bleek

datt de HIV-1 vermenigvuldiging alleen effectief geremd kan worden door met

driee antiretrovirale middelen te behandelen (triple therapie). Deze combinaties

staann bekend als "HAART" (highly active antiretroviral therapy). Behandeling

mett deze medicijncombinaties resulteert in de meeste patiënten in een

onmeetbaarr lage hoeveelheid HIV-1 in het bloed plasma (uitgedrukt in HIV-1

RNAA deeltjes per mL bloed plasma), en een herstel van hun afweer systeem

12

.. Dit heeft geleid tot een significante daling in het voorkomen van AIDS en

AIDSS gerelateerde aandoeningen en sterfte in de Westerse wereld

"

.

Berekeningenn aan de hand van de daling van de hoeveelheid virus deeltjes in

hett bloed na start van de behandeling met drie antiretrovirale middelen wekte

dee hoop dat HIV geheel uit een patiënt zou kunnen verdwijnen na 2 tot 3 jaar

therapie.. Hierbij werd er van uitgegaan dat deze combinatie van middelen de

HIVV vermenigvuldiging geheel remt

. Het bleek echter in de jaren hierna dat

mett de huidige beschikbare antiretrovirale behandeling HIV niet geheel

verdwijntt uit een patient

"

.

Hett aanwezig blijven van HiV in het lichaam is een dynamisch proces dat

afhankelijkk is van verschillende factoren die elkaar beïnvloeden, zoals

uitgebreidd wordt beschreven in hoofdstuk 1. HIV kan aanwezig blijven in

schuilplaatsenn in het lichaam, zoals het centraal zenuwstelsel, omdat

bepaaldee geneesmiddelen daar moeilijk komen. Ook is er een cellulair

reservoir,, welk bestaat uit rustende cellen waarin zich HIV bevindt, de

zogenaamdee latent geïnfecteerde cellen. Verder blijft er ondanks het gebruik

vann de antiretrovirale middelen toch in beperkte mate vermenigvuldiging van

hett HIV plaats vinden ("ongoing residual replication")

-

.

-

. Het

cellulairee reservoir is een gevolg van virale latentie. Virale latentie is het

vermogenn van een virus om voor langere tijd in een cel aanwezig te blijven

zonderr zich te vermenigvuldigen. De antiretrovirale middelen hebben dan

geenn vat op het virus. Deze virale latentie kan op zich ook weer bijdragen aan

dee residual replication, als latent geïnfecteerde cellen geactiveerd worden en

weerr virus gaan produceren. De residual replication kan op zijn beurt weer de

cellulairee reservoirs aanvullen. Voordat we HIV compleet uit een patiënt zullen

kunnenn verwijderen, zullen we eerst deze factoren moeten aanpakken.

Inn dit proefschrift worden nieuwe strategieën beschreven die de factoren

welkee eradicatie van HIV in de weg staan mogelijk zouden kunnen

beïnvloeden. .

Latentt geïnfecteerde cellen die het cellulaire reservoir vormen ontstaan al in

hett begin van een HIV infectie, tijdens de primaire of acute fase van de

infectie.. Het starten van behandeling met antiretrovirale middelen binnen 10

dagenn na de manifestatie van een primaire infectie kan dit niet voorkomen

.

Daaromm hebben wij patiënten met een primaire HIV infectie behandeld met vijf

antiretroviralee middelen uit drie verschillende klassen, wat een krachtiger

combinatiee is dan een standaard medicijncombinatie bestaande uit drie

middelenn

. Vervolgens keken we of een dergelijk combinatie effect heeft

opp de grootte van het latent geïnfecteerde reservoir. De resultaten van deze

studiee worden besproken in hoofdstuk 2. Deze studie toont aan dat in de

patiëntenn die behandeld werden met vijf antiretrovirale middelen het aantal

cellenn dat "levensvatbaar" HIV bevat onder de detectie grens van onze

bepalingsmethodee daalt. Maar toch blijft HIV aanwezig in het lichaam, zoals

blijktt uit het terugkeren van het HIV-1 RNA en de latent geïnfecteerde cellen

inn het bloed na het stoppen van de medicatie

.

Zoalss eerder gezegd zijn er bepaalde plekken in het lichaam waarin de

antiretroviralee middelen niet voldoende doordringen, zodat deze plekken een

schuilplaatss voor HIV kunnen zijn. Van een aantal antiretrovirale middelen is

bekendd dat ze slecht in het mannelijk genitaal stelsel kunnen komen, en

daaromm in het sperma niet aantoonbaar zijn

-

-

. |

hoofdstuk 3 wordt de

penetratiee van de protease remmer lopinavir in het sperma beschreven, als

maatt voor de beschikbaarheid in het mannelijk genitaal stelsel

. Het blijkt dat

slechtss weinig lopinavir het sperma bereikt, met een mediane concentratie

vann maar 0,23 mg/L, terwijl de minimum vereiste concentratie voor een goede

remmingg van de HIV-1 vermenigvuldiging 5,0 mg/L is (Abbott product

informatie).. Deze resultaten werden later bevestigd in andere studies

.

Vanwegee de slechte beschikbaarheid van lopinavir in het sperma zou men

verwachtenn dat het HIV-1 RNA in sperma meetbaar zou zijn, waarmee

bevestigdd zou worden dat het mannelijk genitaal stelsel een schuilplaats is

voorr HIV tijdens therapie. Maar het bleek dat ondanks de lage lopinavir

spiegelss HIV-1 RNA onmeetbaar was in sperma. Na een uitgebreid onderzoek

vann de beschikbare literatuur kwamen wij tot de conclusie dat het mannelijk

genitaall stelsel geen schuilplaats voor HIV is tijdens therapie, behalve als de

patiëntt ook andere seksueel overdraagbare aandoeningen heeft

.

Desalnietteminn is het duidelijk dat er andere plekken zijn in het lichaam waar

dee verminderde beschikbaarheid van antiretrovirale middelen kan leiden tot

doorgaandee virus vermenigvuldiging tijdens therapie, zoals bijvoorbeeld het

centraall zenuwstelsel

. De verminderde beschikbaarheid van

antiretroviralee middelen op die plaatsen zou gedeeltelijk veroorzaakt kunnen

wordenn door de activiteit van drugtransport eiwitten, waar P-glycoprotein

(P-gp)) een voorbeeld van is. Dit wordt verderop uitgebreider besproken.

Eenn mogelijke strategie om de residual replication te verminderen is het

gebruikk van krachtiger antiretrovirale middelen. In het verleden is aangetoond

medicijncombinatiee bestaande uit vijf antiretrovirale middelen uit drie

verschillendee klassen na start van de behandeling een snellere daling gaf van

dee hoeveelheid HIV-1 RNA in het bloed, en een betere remming van de HIV

vermenigvuldigingg op de langere termijn

. In hoofdstuk 4 beschrijven we

datt nog krachtiger antiretrovirale middelen mogelijk zijn. Monotherapie

gedurendee 1 week met de nieuwe non-nucleoside reverse transcriptase

inhibitorr (NNRTI) TMC125 gaf een daling van de hoeveelheid HIV-1 RNA in

hett bloed welke vergelijkbaar was met de daling die bereikt wordt met vijf

antiretroviralee middelen uit drie klassen

. Een mogelijke verklaring voor dit

krachtigee effect zou de grotere beschikbaarheid van TMC125 in de

lymfeklierenn vergeleken met het bloed kunnen zijn. Dit is in overeenstemming

mett het feit dat de lymfeklieren de belangrijkste plek zijn waar HIV

vermenigvuldigingg plaats vindt

.

Recentt is aangetoond dat het versterken van de anti-HIV therapie met meer

middelenn de daling van het cellulair reservoir versnelt en ook een daling geeft

vann de residual replication, maar deze toch niet geheel remt

.

Vervolgstudiess zijn nodig om te kijken naar het effect van TMC125 bevattende

antiretroviralee combinaties op de daling van het HIV-1 RNA in het bloed en op

dee residual replication. Dat laatste kan beoordeeld worden door intracellulair

unsplicedd HIV-1 RNA te meten, zoals verderop wordt besproken.

Niett alleen residual replication, maar ook het bestaan van latent geïnfecteerde

cellenn tijdens de HIV behandeling vormt een probleem. Tijdens de zoektocht

naarr nieuwe strategieën om de residual replication en de hoeveelheid latent

geïnfecteerdee cellen te verminderen ontstond het idee om mycophenolaat

mofetill (MMF) te gebruiken

~

. MMF wordt gebruikt bij de preventie en

behandelingg van afstotingsreacties na orgaantransplantaties, zoals

niertransplantaties.. MMF wordt in het lichaam omgezet in de werkzame stof

mycophenolzuurr (MPA), die het immuunsysteem remt door selectief de deling

vann T cellen te remmen. HIV kan zich alleen vermenigvuldigen in de T cellen

enn dan met name in T cellen die geactiveerd zijn. Door de deling van T cellen

tete remmen kan MPA ervoor zorgen dat er minder cellen beschikbaar zijn

waarinn HIV zich kan vermenigvuldigen. MPA verlaagt de productie van dGTP,

eenn van de bouwstoffen van DNA en dus nodig voor de HIV

vermenigvuldiging.. Daardoor kan MPA de HIV vermenigvuldiging ook

rechtstreekss remmen. De verlaging van de hoeveelheid natuurlijk dGTP zorgt

err verder voor dat het antiretrovirale middel abacavir beter werkt. MMF was in

eenn eerdere studie in staat de hoeveelheid latent geïnfecteerde cellen te

verlagenn bij patiënten die onder antiretrovirale therapie een onmeetbaar lage

hoeveelheidd virus deeltjes hadden in hun bloed

. In hoofdstuk 5, 6, 7 en 8

werdd het gebruik van MMF bij de behandeling van HIV infectie bestudeerd

(MANN studie). Patiënten met een primaire of een chronische HIV-1 infectie

werdenn in deze studie behandeld met vijf antiretrovirale middelen uit drie

beschikbaree klassen, te weten eenmaal daags didanosine 400 mg en

tweemaall daags abacavir 300 mg, lamivudine 150 mg, indinavir 800 mg (met

ritonavirr 100 mg) en nevirapine 200 mg (eerste 2 weken nevirapine 200 mg

eenmaall daags). De patiënten lootten voor het wel of niet gebruiken van MMF

naastt hun antiretrovirale medicatie.

Dee resultaten in hoofdstuk 5 tonen aan dat MMF goed werd verdragen door

HIVV patiënten die begonnen met antiretrovirale middelen. Er bleek echter

geenn effect van MMF op de snelheid van daling van de hoeveelheid HIV-1

RNAA in het bloed gedurende de eerste 14 dagen van de behandeling en ook

niett op de daling van de hoeveelheid latent geïnfecteerde cellen

. Zeer

waarschijnlijkk heeft MMF geen toegevoegd effect op de remming van de HIV

vermenigvuldigingg indien het wordt gebruikt in combinatie met een zeer

krachtigee combinatie zoals in onze studie. In een eerdere studie waarbij er wel

eenn effect van MMF werd gezien op de daling van het cellulaire reservoir

werdenn de patiënten behandeld met twee antiretrovirale middelen uit twee

klassenn

. Verrassend genoeg nam het aantal latent geïnfecteerde cellen in

onzee patiënten tijdelijk toe na het stoppen van MMF. Een mogelijke verklaring

hiervoorr is dat MPA geïnfecteerde cellen blokkeert in hun delingscyclus op het

momentt dat er gestart wordt met therapie. Op het moment dat MMF gestopt

wordtt kunnen deze geblokkeerde cellen hun celcyclus afmaken en kunnen

daardoorr tijdelijk een toename geven van het reservoir van latent

geïnfecteerdee cellen.

Inn hoofdstuk 6 werden de immunologische effecten bestudeerd bij de

patiëntenn die wel of geen MMF gebruikten naast hun antiretrovirale middelen.

Alhoewell de T cel deling compleet geremd was bij patiënten die MMF

gebruikten,, was de toename van het totaal aantal CD4

en CD8

T cellen en

hunn sub-sets tijdens therapie vergelijkbaar voor patiënten die wel of geen

MMFF gebruikten

. Dit kan verklaard worden door het feit dat het enzym dat

geremdd wordt door MPA, inosine 5'-monophosphate dehydrogenase, in twee

variantenn voorkomt. De ene vorm, type II, wordt vooral gebruikt door

geactiveerdee T cellen, en dit type II wordt meer geremd door MPA dan de

anderee vorm, type I, die vooral gebruikt wordt door rustende cellen

. Daar

geactiveerdee T cellen uiteindelijk gedoemd zijn te sterven zal de deling van

dezee cellen niet bijdragen aan het instandhouden van de totale groep van

CD4

T cellen, en heeft remming van deze deling dus evenmin veel invloed.

Err waren verder ook geen verschillen in de ontwikkeling van HIV specifieke

afweerr tussen patiënten met en zonder MMF.

Dee bevindingen van deze studie, samen met die van een recent

gepubliceerdee studie, waar MMF veilig gegeven werd aan HIV patiënten met

eenn mediaan CD4

T cel aantal van 34 x 10

/L

, tonen aan dat het gebruik

vann MMF in HIV patiënten relatief veilig is.

Alhoewell het erop lijkt dat het antiretrovirale effect van MMF minimaal is, zijn

err toch bepaalde situaties denkbaar waarin behandeling met MMF wel

toegevoegdee waarde zou kunnen hebben. Het idee was vroeger dat het

verliess van CD4

T cellen het directe gevolg was van de

virusvermenigvuldigingg in de cel. Door virusvermenigvuldiging te remmen met

antiretroviralee middelen kon dan het verlies aan CD4

T cellen worden

voorkomen.. Het is inmiddels aangetoond dat de chronische hyperactivatie van

hett immuunsysteem bij HIV patiënten leidt tot een verhoogde T cel activeit en

daardoorr een verkorte gemiddelde levensduur van de CD4

T cellen. Deze

verkortee levensduur leidt tot een verhoogde turnover van de CD4

T cellen en

ditt leidt uiteindelijk tot een daling van het totaal aantal van deze cellen doordat

dee aanmaak van deze cellen het verlies niet kan compenseren. De beperking

vann het verlies van CD4

T cellen na start van therapie zou dus ook mogelijk

eenn gevolg kunnen zijn van het feit dat het remmen van de

virusvermenigvuldigingg leidt tot minder prikkeling van het afweersysteem. Na

hett starten met antiretrovirale middelen wordt er direct een daling gezien van

dee deling van CD4

en CD8

T cellen als uiting van deze verminderde

immuunactivatiee

"

.

Hett gebruik van MMF bij patiënten die nog geen antiretrovirale therapie nodig

hebbenn zou het verlies van CD4

T cellen kunnen beperken door hun deling

tee remmen en daardoor zou MMF ervoor kunnen zorgen dat patiënten langer

kunnenn wachten met het starten van antiretrovirale middelen. Recent werd

inderdaadd aangetoond dat het stoppen met antiretrovirale therapie in de

aanwezigheidd van MMF een betere controle gaf van de

virusvermenigvuldigingg dan stoppen zonder MMF

~

. Een studie met MMF

bijj patiënten met resistentie tegen verschillende middelen kon geen

virologischh effect van MMF aantonen, maar er werd wel een stijging van het

aantall CD4

T cellen gezien

. In deze studie werd niet gekeken naar het

effectt van MMF op de chronische immuunactivatie, maar het is aannemelijk

datt deze geremd was door MMF, met als gevolg een afname van het verlies

vann het aantal CD4

T cellen. Daarom zou het gebruik van MMF een

mogelijkheidd kunnen zijn om het aantal CD4

T cellen te laten stijgen bij

patiëntenn die geen andere behandelingsopties hebben.

Dee farmacologische interacties tussen MPA (het werkzame deel van MMF) en

dee antiretrovirale middelen werden bestudeerd in hoofdstuk 7. Er bleek dat

err geen interacties waren tussen MPA en abacavir of indinavir maar het bleek

well dat MPA de concentratie van nevirapine in het bloed kon verlagen,

mogelijkk door het beïnvloeden van de enterohepatische kringloop van dit

middell

. De enterohepatische kringloop is het proces waarbij een

geneesmiddell in de darm wordt opgenomen, en vervolgens door de lever via

hett gal weer in de darm wordt uitgescheiden, waarna het weer wordt

opgenomen.. Als dit proces wordt verstoord, zoals in dit geval waarschijnlijk bij

nevirapine,, zal er uiteindelijk minder van het middel worden opgenomen.

Hett veronderstelde werkingsmechanisme van MPA bestaat eruit dat het de

productiee van dGTP verlaagt, zoals uitgebreid besproken is in hoofdstuk 1.

Daaromm bepaalden wij de intracellulaire dGTP concentratie bij onze studie