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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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Highlyy active antiretroviral therapy with or without
mycophenolatee mofetil in treatment naïve HIV-1
patients s
S.. U.C. Sankatsing, S. Jurriaans, P. van Swieten, F. van Leth, M.. Cornelissen, F. Miedema, J.M.A. Lange, H. Schuitemaker,
J.M.. Prins
Abstract t
Objective:Objective: To study the effect of mycophenolate mofetil (MMF) on the decay ratee of plasma HIV-1 RNA and the latently infected cellular reservoir in
treatmentt naïve patients starting antiretroviral therapy. Design:Design: Randomized trial.
Methods:Methods: 19 HIV-1 infected patients (9 with a chronic and 10 with a primary infection)) starting a triple class antiretroviral drug regimen were randomized to
aa group with or without MMF. Plasma samples for HIV-1 RNA were taken and HLA-DR~CD4++ T cells were co-cultured for HIV-1 isolation. Slopes of plasma HIV-11 RNA and cellular viral load decay were calculated for the first 14 days andd the first 24 weeks of treatment respectively.
Results:Results: The median plasma HIV-1 RNA decay rate (log™ HIV-RNA copies/mUday)) was 0.25 (IQR: 0.18-0.30) and 0.28 (0.22-0.32) in chronically
infectedd patients with and without MMF respectively (p=0.56), and 0.31 (0.31-0.32)) and 0.32 (0.26-0.34) in primary infected patients with and without MMF (p=0.75).. The median decay rate (number of infected cells/106 cells/day) of latentlyy infected cells was 0.017 and 0.004 in patients with and without MMF respectivelyy (p=0.89). The increase in CD4+ T cells was comparable between patientss with and without MMF. After stopping MMF there was an increase of thee cellular reservoir in 6 out of 8 patients.
Conclusion:Conclusion: The addition of MMF to a triple class antiretroviral regimen in treatmentt naive patients does not significantly increase the plasma HIV-1
Introduction n
Thee presence of a pool of latently infected cells with a very long half-life 1_6 andd ongoing low-level replication 7"10 are major obstacles for eradication of HIV-11 by antiretrovirals alone. Even therapy with a triple class antiretroviral regimenn could not accomplish this 11. To further lower the reservoir of latently infectedd cells and the ongoing low-level replication the use of mycophenolate mofetill (MMF) has been proposed 12. MMF is a prodrug that is rapidly convertedd to its active metabolite mycophenolic acid (MPA). MPA selectively suppressess proliferation of T and B lymphocytes through non-competitive, reversiblee inhibition of inosine 5'-monophosphate dehydrogenase (IMPDH), a keyy enzyme in the de novo synthetic pathway of the purine guanine 13,14. Lymphocytess are mainly dependent on this pathway 13. A decrease of availablee guanine results in blocking of cells in the S phase 15, thereby limiting thee availability of target cells for HIV. MPA also suppresses HIV replication itselff through the guanine depletion 16 and increases the efficacy of the guanosinee analogue reverse transcriptase inhibitor abacavir17.
WeWe earlier demonstrated that a triple class, five-drug regimen results in a fasterr initial plasma HIV-1 RNA decline 18 and a stronger long-term suppressionn of viral replication compared to a standard triple drug regimen 19. Too study whether an even stronger suppression of viral replication and a fasterr decay of the latently infected cellular reservoir is possible we randomizedd treatment naive patients to such a regimen with or without MMF.
Methods s
Patients Patients
Nineteenn antiretroviral naive patients, 9 with a chronic and 10 with a primary HIV-11 infection, were recruited from our outpatient clinic. In primary HIV-1 infection,, treatment was started within 2 months after onset of symptoms 20. Alll patients started with didanosine enteric coated 400 mg once daily and abacavirr 300 mg, lamivudine 150 mg, indinavir 800 mg, ritonavir 100 mg and nevirapinee 200 mg twice daily (nevirapine with a lead in of 2 weeks 200 mg QD).. The patients were randomized to either a group with or without MMF. Threee patients started with MMF 500 mg BID and after 4 weeks the dose was
increasedd to 1000 mg BID. Because the plasma levels were well above the minimumm required concentration 21, the dose was decreased to 500 mg BID afterr 12-24 weeks of treatment and further patients randomized to MMF used 5000 mg BID.
Patientss were seen before study entry, at the start of therapy, at day 5 and afterr 1, 2, 4, 8, 12 and 16 weeks of therapy and thereafter every 8 weeks, for assessingg side effects and obtaining blood samples. The Medical Ethics Committeee of our hospital approved the study and written informed consent wass obtained from all patients.
DecayDecay rate of plasma HIV-1 RNA and the cellular reservoir
EDTA-anticoagulatedd blood was obtained at every visit to measure plasma HIV-11 RNA using the NucliSens HIV-1 QT assay (bioMérieux, Boxtel, The Netherlands).. Adaptation of the protocol allowing ultrasensitive detection resultedd in a lower quantification limit of 5 copies/mL 18. In two patients the Versantt HIV-1 RNA bDNA 3.0 assay (Bayer Corporation, Tarrytown, NY) with aa lower quantification limit of 50 copies/mL was used because of primer mismatches. .
Thee plasma HIV-1 RNA elimination rate constant (first order elimination) duringg the first 14 days of treatment was calculated for all patients as describedd earlier18'22. All samples from start of treatment up to and including thee first undetectable sample were included in this analysis. In case of an undetectablee sample (below 5 copies/mL), the value of the lower limit of detectionn was used.
Thee reservoir of resting CD4+ T cells harboring replication competent HIV-1 wass quantified at baseline and 1, 2, 4, 8 and 24 weeks after starting therapy.
Inn case of a primary HIV-1 infection this was also done 5 days after starting therapyy and in patients stopping MMF on the day of stopping MMF and 4 and 88 weeks thereafter. HIV-1 was isolated from HLA-DR" CD4+ T cells according too a modification of the method described by Finzi et al. 3 and the decay rate off the pool of resting CD4+ T cells harboring replication competent HV-1 was calculatedd as described in detail in a previous study 11.
Sampless between (and including) the first detectable and the first undetectablee sample were used for this analysis when the patient remained undetectable.. If the patient had subsequently a detectable sample again, we usedd the samples between the first detectable and this rebound sample.
Immunology Immunology
AtAt all visits CD4+ and CD8+ T cell numbers were measured in peripheral blood usingg immunofluorescence flow cytometry.
StatisticalStatistical analysis
Dataa were analyzed using SPSS 11.0.1, (SPSS Inc. Chicago, Illinois).
Baselinee characteristics, decay rates of plasma HIV-1 RNA and the number of latentlyy infected cells, and CD4+ T cell counts of the two treatment groups weree compared using the Mann Whitney test.
Thee plasma viral decay rate was analyzed separately for patients with a primaryy infection and those with a chronic infection. For the decay rate of the latentlyy infected cells this distinction was not possible due to the small number off available samples.
Results s
PatientPatient characteristics
Alll patients were male. One patient without MMF temporarily stopped therapy afterr 4 days because of nausea. This patient was excluded from the plasma HIV-11 RNA decay analysis. Baseline characteristics of the remaining 18 patientss are shown in table 1. Side effects of therapy have been described elsewhere21.. Overall, no more side effects were seen in patients using MMF.
DynamicsDynamics of plasma HIV-1 RNA
Inn chronically infected patients plasma HIV-1 RNA dropped below 50 copies/mLL after 2-24 and 3-16 weeks of treatment in respectively patients with andd without MMF, and in patients with a primary infection after 1-25 and 12-20 weekss of treatment respectively.
Thee median plasma HIV-1 RNA decay rate during the first two weeks of therapyy (logi0 HIV-RNA copies/ml/day) in chronically infected patients was
0.255 (IQR 0.18-0.30) with MMF and 0.28 (IQR 0.22-0.32) without MMF (p=0.56)) and in primary infected patients 0.31 (IQR 0.31-0.32) and 0.32 (IQR 0.26-0.34)) respectively (p=0.75)(figure 1).
Tablee 1 Baseline characteristics
Numberr of patients (n) Agee (years) [median (IQR)] CD4++ T cells (x10b/L) [median (IQR)] Plasmaa HIV RNA (log10 copies/mL)
[mediann (IQR)]
Numberr of patients (n) Agee (years) [median (IQR)] CD4++ T cells (x106/L) [median (IQR)]
Plasmaa HIV RNA (log™ copies/mL) [mediann (IQR)] Chronicc infection ++ MMF 4 4 355 (31-46) 365(190-510) ) 4.66(3.20-5.31) ) Primaryy infection ++ MMF 5 5 355 (32-35) 430(390-1050) ) 4.544 (3.48-5.50) Chronicc infection MMF 4 4 333 (27-47) 4500 (398-578) 4.344 (4.00-5.24) Primaryy infection -MMF F 5 5 355 (29-42) 5755 (498-705) 4.700 (4.33-6.23) p-value e 0.87 7 0.49 9 0.69 9 p-value e 0.69 9 0.19 9 0.56 6 ** Mann-Whitney U test;
DynamicsDynamics of the latently infected cellular reservoir
Inn both groups there was a Diphasic decay of the number of latently infected
23 3
cellscells upon start of therapy, similar as we reported previously . The decay ratee (k) of the number of latently infected cells during the first 24 weeks of therapyy (number of infected cells/106/day) was similar in patients with (k= 0.0170)) and without MMF (k=0.0042)(p=0.89).
Onee patient stopped MMF after 16 weeks of therapy at his own request, and noo cells were isolated from this patient for further analysis. Given the lack of effectt of MMF on the decay rate of the cellular reservoir, we also stopped MMFF in the other 8 patients on MMF.
Att that moment, they were on MMF for a median of 57 (range 32-100) weeks. Fourr weeks after stopping MMF there was an increase in the number of latentlyy infected resting HLA-DR" CD4+ T cells in 6 of the 8 patients. In one
patientt there was a decrease, in another patient there was no change (figure 2).. Stopping MMF had no effect on the plasma HIV-1 RNA.
Immunology Immunology
Theree were no differences in absolute CD4+ T cell increase between chronicallyy infected patients with or without MMF 24.
Figuree 1 Plasma HIV-1 RNA during first two weeks of therapy
i-r i-r Chronicc Infection ++ M M F (n=4) 00 2 4 6 8 10 12 14 Dayss on therapy >> -II g Chronicc Infection - M M F ( n = 4 ) ) 00 2 4 6 8 10 12 14 Dayss on therapy 3 , , Primaryy Infection ++ M M F ( n = 5 ) 00 2 4 6 8 10 12 14 Dayss on therapy to to Primaryy Infection - M M F ( n = 5 ) ) 00 2 4 88 10 12 14 Dayss on therapy
Figuree 2 Latently infected cellular reservoir in patients stopping MMF 244 48 72 96 W e e k ss on t h e r a p y 1Stopp MMF week 100 «« o ££ o
11 1
244 48 72 96 120 144 W e e k ss o n t h e r a p y Stopp MMF week 6 5 488 72 96 120 144 W e e k ss o n t h e r a p y Stopp MMF week 6 5 TJJ uj 244 48 72 96 W e e k ss o n t h e r a p y Stopp M M F week 41 II 8 244 48 72 96 120 W e e k ss on t h e r a p y ' S t o pp M M F week 4 8 2Stopp HAART week 56
<^\ \ 00 24 48 72 96 W e e k ss on t h e r a p y ' S t o pp MMF week 33 ££ o 244 48 72 96 120 144 W e e k ss o n t h e r a p y Stopp M M F week 82 00 24 48 72 96 W e e k ss on t h e r a p y 1Stopp M M F week 32
openn circles: proportion of cells containing replication competent HIV-1 quantifiable, black circles:: proportion of cells containing replication competent HIV-1 below the detection limit of thee assay.
Discussion n
Thee purpose of our study was to determine whether in therapy naive patients addingg MMF to a triple class regimen results in a better suppression of HIV-1 replicationn and a faster decay of the cellular reservoir.
Whereass in vitro MPA inhibits HIV-1 replication 12,16, we found no difference in thee plasma HIV-1 RNA decay rate in patients with or without MMF. The MPA levelss were all above the minimum required concentration 21 and lymphocyte proliferationn was inhibited, as evidenced by a low thymidine incorporation in wholee blood after stimulation with anti-CD3 24, thereby excluding the possibilityy of a too low dosage of MMF. Although MPA lowered the plasma concentrationn of nevirapine it remained above the minimum required concentrationn 21. The absence of an additive effect of MPA on the plasma HIV-11 RNA decay in our patients could be due to the fact that the potent triple classs regimen used already suppressed HIV-1 replication effectively. In theory MMFF might have an effect on the plasma HIV-1 RNA decay rate after the 14 dayss studied. However, the sharpest decay of plasma HIV-1 RNA occurs duringg the first 14 days of treatment 25 and further, the time to reach an undetectablee plasma viral load (<50 copies/ml) was also not different for patientss with or without MMF.
Thee decay of the latently infected cellular reservoir was not different for patientss with or without MMF. Chapuis et al. reported a reduction of the numberr of latently infected cells in patients treated with MMF 12. This was explainedd by increased apoptosis of latently infected and by a further suppressionn of the ongoing low-level replication 12. Low-level replication might refeedd the latent reservoir26,27. In that study patients had been treated with a duall class drug regimen for 36 weeks when MMF was added. It is conceivable thatt MMF has no added value in patients in whom the low-level replication is alreadyy better suppressed with the more potent triple class regimen 18,19. Whetherr MPA indeed increases apoptosis of CD4+ T cells is also questionable
28
.. If the reduction of the latent reservoir would be partially the result of apoptosiss we would have seen a decrease of this reservoir even with our potentt regimen.
Wee demonstrated previously the strong correlation between plasma HIV-1
RNAA and the proportion of latently infected cells in primary infection 23. Becausee there was no difference in plasma HIV-1 RNA decay between patientss with a primary or a chronic infection we did not differentiate between primaryy and chronic infection for our analysis of the effect of MMF on the cellularr reservoir.
Afterr stopping MMF 6 of the 8 patients showed a transient increase in the proportionn of dividing cells and an increase of their cellular reservoir. Upon startingg HAART and MMF, MPA blocks lymphocytes in their cell cycle, without subsequentt apoptosis. The ensuing decrease of the cellular reservoir during antiretrovirall therapy is accomplished by inhibition of refeeding of the latent reservoir,, in combination with the natural decay, which was comparable for patientss with or without MMF. Upon stopping MMF, cells that were blocked duringg proliferation can complete the cell cycle and can temporarily contribute too low-level replication and refeeding of the latent reservoir. This mechanism mightt also partially explain the reported decline of the latent reservoir in the studyy by Chapuis.
Ourr data demonstrate that adding MMF to a triple class regimen in treatment naivee patients has no immediate beneficial effect. This does not exclude the possibilityy that in other circumstances MMF might be beneficial. MMF might playy a role in salvage therapy 29. An interesting option might be the use of MMFF in treatment naive patients without an indication for HAART or in patientss who are stopping HAART. In such patients limitation of the number of targett cells might limit viral replication and might extent the time until HAART iss necessary30.
Acknowledgement t
Wee thank Marian Nievaard, Research Nurse, for excellent patient care and Marijkee Roos, Sanquin Research and Landsteiner Laboratory, University of Amsterdam,, Amsterdam, the Netherlands, for measuring lymphocyte
non-commerciall foundation that wishes not to be named and by a grant from the Dutchh AIDS fund (project nr 6005).
References s
1.. Chun TW, Carruth L, Finzi D et al. Quantification of latent tissue reservoirs andd total body viral load in HIV-1 infection. Nature. 1997;387:183-188. 2.. Chun TW, Finzi D, Margolick J et al. In vivo fate of HIV-1-infected T cells:
quantitativee analysis of the transition to stable latency. Nat Med. 1995;1:1284-1290. .
3.. Finzi D, Hermankova M, Pierson T et al. Identification of a reservoir for HIV-1 inn patients on highly active antiretroviral therapy. Science. 1997;278:1295-1300. .
4.. Finzi D, Blankson J, Siliciano JD et al. Latent infection of CD4+ T cells providess a mechanism for lifelong persistence of HIV-1, even in patients on effectivee combination therapy. Nat Med. 1999;5:512-517.
5.. Chun TW, Stuyver L, Mizell SB et al. Presence of an inducible HIV-1 latent reservoirr during highly active antiretroviral therapy. Proc Natl Acad Sci USA. 1997;94:13193-13197. .
6.. Wong JK, Hezareh M, Gunthard HF et al. Recovery of replication-competent HIVV despite prolonged suppression of plasma viremia. Science. 1997;278:1291-1295. .
7.. Dornadula G, Zhang H, VanUitert B et al. Residual HIV-1 RNA in blood plasmaa of patients taking suppressive highly active antiretroviral therapy.
JAMA.JAMA. 1999;282:1627-1632.
8.. Zhang L, Ramratnam B, Tenner-Racz K et al. Quantifying residual HIV-1 replicationn in patients receiving combination antiretroviral therapy. N Engl J
Med.Med. 1999;340:1605-1613.
9.. Furtado MR, Callaway DS, Phair JP et al. Persistence of HIV-1 transcription inn peripheral-blood mononuclear cells in patients receiving potent antiretrovirall therapy. N EnglJ Med. 1999;340:1614-1622.
10.. Sharkey ME, Teo I, Greenough T et al. Persistence of episomal HIV-1 infectionn intermediates in patients on highly active anti-retroviral therapy. Nat Med.. 2000;6:76-81.
11.. van Rij R, van Praag R, Prins JM et al. Persistence of viral HLA-DR- CD4 T-celll reservoir during prolonged treatment of HIV-1 infection with a five-drug regimen.. Antivir Ther. 2002;7:37-41.
12.. Chapuis AG, Paolo RG, D'Agostino C et al. Effects of mycophenolic acid on humann immunodeficiency virus infection in vitro and in vivo. Nat Med. 2000;6:762-768. .
13.. Allison AC, Eugui EM. Mycophenolate mofetil and its mechanisms of action.
Immunopharmacology.Immunopharmacology. 2000 ;47:85-118.
14.. Allison AC, Eugui EM. Immunosuppressive and other effects of mycophenolic acidd and an ester prodrug, mycophenolate mofetil. Immunol Rev. 1993; 136:5-28. .
15.. Cohn RG, Mirkovich A, Dunlap B et al. Mycophenolic acid increases apoptosis,, lysosomes and lipid droplets in human lymphoid and monocytic celll lines. Transplantation. 1999;68:411-418.
16.. Ichimura H, Levy JA. Polymerase substrate depletion: a novel strategy for inhibitingg the replication of the human immunodeficiency virus. Virology. 1995;211:554-560. .
17.. Margolis D, Heredia A, Gaywee J et al. Abacavir and mycophenolic acid, an inhibitorr of inosine monophosphate dehydrogenase, have profound and synergisticc anti-HIV activity. J Acquir Immune Defic Syndr. 1999;21:362-370. 18.. Weverling GJ, Lange JM, Jurriaans S et al. Alternative multidrug regimen
providess improved suppression of HIV-1 replication over triple therapy. AIDS. 1998;12:F117-F122. .
19.. van Praag RM, Wit FW, Jurriaans S et al. Improved long-term suppression of HIV-11 replication with a triple-class multidrug regimen compared with standardd of care antiretroviral therapy. AIDS. 2002;16:719-725.
20.. Tindall B, Barker S, Donovan B et al. Characterization of the acute clinical illnesss associated with human immunodeficiency virus infection. Arch Intern
Med.Med. 1988;148:945-949.
21.. Sankatsing SUC, Hoggard PG, Hurtema ADR et al. Effect of mycophenolate mofetill on the pharmacokinetics of antiretroviral drugs and the intracellular nucleosidee triphosphate pools. Clinical Pharmacokinetics. 2004;Accepted for publication. .
22.. Sankatsing SU, Weverling GJ, Peeters M et al. TMC125 exerts similar initial antivirall potency as a five-drug, triple class antiretroviral regimen. AIDS. 2003;17:2623-2627. .
23.. Sankatsing SU, van Praag RM, van Rij RP et al. Dynamics of the pool of infectedd resting CD4 HLA-DR- T lymphocytes in patients who started a triple classs five-drug antiretroviral regimen during primary HIV-1 infection. Antivir
Ther.Ther. 2003;8:137-142.
24.. Vrisekoop N, Sankatsing SUC, Jansen CA et al. Normal immune reconstitutionn in acute and chronic HIV-1 infected patients with combined
treatmentt of MMF and HAART. 10th Conference on Retroviruses and Opportunisticc Infections, Boston, USA, February 2003, Abstract 657.
25.. Perelson AS, Essunger P, Cao Y et al. Decay characteristics of HIV1 -infectedd compartments during combination therapy. Nature. 1997;387:188-191. .
26.. Ramratnam B, Mittler JE, Zhang L et al. The decay of the latent reservoir of replication-competentt HIV-1 is inversely correlated with the extent of residual virall replication during prolonged anti-retroviral therapy. Nat Med. 2000;6:82-85. .
27.. Fraser C, Ferguson NM, Anderson RM. Quantification of intrinsic residual virall replication in treated HIV-infected patients. Proc Natl Acad Sci USA. 2001;98:15167-15172. .
28.. Quemeneur L, Flacher M, Gerland LM et al. Mycophenolic acid inhibits I n -dependentt T cell proliferation, but not IL-2-dependent survival and sensitizationn to apoptosis. J Immunol. 2002;169:2747-2755.
29.. Margolis DM, Kewn S, Coull JJ et al. The addition of mycophenolate mofetil to antiretrovirall therapy including abacavir is associated with depletion of intracellularr deoxyguanosine triphosphate and a decrease in plasma HIV-1 RNA.. J Acquir Immune Defic Syndr. 2002;31:45-49.
30.. Tambussi G, Rizzardi GP, Lazzarin A et al. Patterns of Viral Rebound and CD44 Slopes in patients with Primary HIV-1 Infection with Persistent Undetectablee Viremia Treated with Supervised Treatment Interruption Plus Mycophenolatee Mofetil. 10th Conference on Retroviruses and Opportunistic Infections,, Boston, USA, February 2003, Abstract 658.
