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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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Effectt of mycophenolate mofetil on the
pharmacokineticss of antiretroviral drugs and the
intracellularr nucleoside triphosphate pools
S.U.C.. Sankatsing, P.G. Hoggard, A.D.R. Huitema, R.W.. Sparidans, S. Kewn, K.M.L. Crommentuyn, J.M.A.. Lange, J.H. Beijnen, D.J. Back, J.M. Prins
Abstract t
Objective:Objective: To study the effect of mycophenolate mofetil (MMF) therapy on the
pharmacokineticc parameters of a number of antiretroviral drugs, and on the intracellularr deoxycytidine triphosphate (dCTP) and deoxyguanosine triphosphatee (dGTP) pools and triphosphate concentrations of lamivudine (3TCTP)) and abacavir (CBVTP).
Design:Design: Randomised pharmacokinetic study.
Participants:: 19HIV-1 infected patients.
Methods:Methods: Antiretroviral naive men starting treatment with didanosine 400 mg
QD,, lamivudine 150 mg BID, abacavir 300 mg BID, indinavir 800 mg BID, ritonavirr 100 mg BID and nevirapine 200 mg BID were randomized to a group withh or without MMF 500 mg BID. After 8 weeks of therapy the plasma pharmacokineticc profiles of mycophenolic acid (MPA, the active metabolite of MMF),, abacavir, indinavir and nevirapine, and the triphosphate concentrations (dCTP,, dGTP and 3TCTP) in peripheral blood mononuclear cells (PBMCs) weree determined.
Results:Results: Nine of the 19 patients received MMF. There was no difference in
plasmaa clearance of indinavir or abacavir between the 2 groups. The clearancee of nevirapine was higher in patients using MMF (p=0.04). In 12 patients,, of whom 5 also received MMF, intracellular triphosphates were measured.. There was no significant difference in intracellular dCTP, dGTP or 3TCTPP concentrations between the 2 groups. Due to assay interference CBVTPP could not be determined.
Conclusion:Conclusion: In this small cohort of patients MMF therapy reduced the plasma
concentrationn of nevirapine but had no effect on plasma indinavir and abacavir concentrations.. In contrast to our hypothesis, there were no consistent effects off MPA on the intracellular concentrations of dCTP, dGTP or 3TCTP.
Introduction n
Generallyy accepted therapy for HIV-1 consists of at least 3 different antiretrovirall drugs, usually from two different classes. We have demonstrated previouslyy that although a triple class 5-drug regimen resulted in a better suppressionn of the HIV-1 replication than a 3-drug regimen 1*2, HIV-1 replicationn was nevertheless not completely suppressed. Therefore new drugs orr new approaches with current drugs are necessary to further suppress this soo called residual replication 3. HIV-1 can only replicate in activated CD4+ T cellss 4, so strategies to limit the number of target cells might be an option. The usee of hydroxyurea, which reduces the availability of suitable target cells for HIVV by killing the dividing CD4+ T cells 5 and in vitro lowers endogenous deoxynucleosidee triphosphates 6, is such an approach. However, hydroxyurea cann result in serious toxicity and blunts the CD4+ T cell replenishment resultingg from effective antiretroviral therapy, and its effectiveness in HIV therapyy is disappointing 7.
Anotherr approach is the use of mycophenolate mofetil (MMF) in combination withh highly active antiretroviral therapy (HAART) 8 9. MMF is used to prevent organn rejection and to treat acute rejection after organ transplantation 10. It is aa prodrug that is rapidly converted into its active metabolite mycophenolic acid (MPA),, which selectively inhibits inosine 5'-monophosphate dehydrogenase (IMPDH)) conversion of inosine monophosphate (IMP) to xanthosine 5'-monophosphatee (XMP) in the de novo synthetic pathway of the purine guaninee 11,12. Blocking this enzyme in lymphocytes depletes guanosine triphosphatee (GTP) and deoxyguanosine triphosphate (dGTP) pools. This depletionn of dGTP pools inhibits the activation of T lymphocytes, thereby limitingg the availability of target cells for HIV. MPA also has a direct antiviral effectt on HIV itself, as dGTP is necessary for HIV replication 8'9.
Thee HIV reverse transcriptase inhibitor abacavir (ABC) is an analogue of guanosinee 13,14. Abacavir is phosphorylated to its active form carbovir triphosphatee (CBVTP), which competes with endogenous dGTP for incorporationn into the growing viral DNA chain. The MPA mediated depletion off endogenous dGTP may result in an altered competition between CBVTP andd dGTP, thereby increasing the antiretroviral efficacy of ABC 8. It has
recentlyy been demonstrated that MPA also synergistically enhances the activityy of didanosine and tenofovir15.
Iff indeed MMF is to have a role in the treatment of HIV, a greater degree of understandingg is required about interactions between MPA and antiretroviral drugs,, as no such data are available at this time. Knowledge of such drug interactionss is also important in case of organ transplantation in HIV patients onn antiretroviral therapy, which is no longer a novelty 16. We therefore studied thee pharmacokinetic interactions between MPA and HAART and studied the effectt of MPA on the intracellular dGTP pools in peripheral blood mononuclear cellss (PBMC) in vivo.
Methods s
Patients Patients
Nineteenn antiretroviral naive patients were recruited in this study. All patients startedd with a triple class antiretroviral drug regimen consisting of didanosine entericc coated 400 mg QD, ABC 300 mg BID, lamivudine (3TC) 150 mg BID, indinavirr (IDV) 800 mg BID, ritonavir 100 mg BID to boost IDV plasma concentrationss and nevirapine (NVP) 200 mg BID (first two weeks 200 mg QD).. The patients were stratified for type of HIV infection (acute or chronic infection)) and randomized to a group with MMF or another without MMF. The firstt three patients randomized to MMF started with 500 mg BID, and after 4 weekss the dose was increased to 1000 mg BID. Because the plasma MPA concentrationss were well above the minimum desired concentration of 0.14 ug/mll 17, the dose was decreased to 500 mg BID after 12-24 weeks of treatmentt and all further patients randomized to MMF in the study used 500 mgg BID.
Thee local Medical Ethics Committee approved the study and written informed consentt was obtained from all patients.
T-cellT-cell proliferative capacity
T-celll proliferative capacity was determined in a whole-blood lymphocyte culturee assay as described previously in detail 18. Briefly, cells in 10 uJ of
heparinizedd blood were stimulated with mAb to CD3 in the presence of CD28 mAb.. Proliferative responses were measured after 4 days of culture by means off incorporation of [3H]-thymidine, added 24 hours before harvest. All cultures weree done in triplicate. Reactivity was calculated back to counts per minute (c.p.m.)) in 103 CD3+ T cells and results are expressed as percentages of the mediann responses in healthy controls investigated simultaneously.
PharmacokineticPharmacokinetic and intracellular triphosphate profiles
Afterr 8 weeks of therapy plasma pharmacokinetic profiles of MPA (the active metabolitee of MMF), ABC, IDV and NVP were determined during a dosing intervall in all patients. Heparinized blood was obtained at T=0,1, 2, 4, 6, 8,10 andd 12h after dosing. After centrifugation at 1500 g for 10 minutes, plasma wass collected and frozen until further analysis. On the same day at T=0, 2, 6 andd 12h after dosing, blood samples were taken in cell preparation tubes of 8 mll with sodium citrate (Beckton Dickinson's Vacutainer® CPT™, Beckton Dickinsonn and Company, Franklin Lakes, N.J., U.S.A.) and PBMCs were isolatedd for the measurement of the intracellular deoxycytidine triphosphate (dCTP)) and dGTP pools and triphosphate concentrations of 3TC (3TCTP) and ABCC (CBVTP). The CPT tubes were immediately centrifuged for 30 minutes at 15000 g and 21 C to obtain leukocyte rich plasma. Leukocyte rich plasma was centrifugedd for 10 minutes at 600 g and C to isolate the leukocytes. After discardingg the supernatant, the cell pellet was resuspended with 2.50 mL phosphatee buffered saline and (after removing 0.25 mL for a cell count) again centrifugedd for 10 minutes at 600 g and . After discarding the supernatant, thee remaining cell pellet was resuspended in 0.96 mL 50mM Tris-HCL, 1.44 mLL 100% methanol was added and the solution was stored overnight at . Thee next day the samples were centrifuged for 10 minutes at 600 g and C to gett rid of debris and the supernatant was dried by rotary evaporation and storedd at C until further analyses.
Assays Assays
Plasmaa concentrations of ABC and MPA were simultaneously measured with aa liquid chromatographic assay using dual spectrophotometric detection as
describedd earlier 19 which has been validated in the 0.05-2 pg/ml range for ABCC and in the 0.01-10 pg/ml range for MPA for 200 (Jl plasma samples. The lowerr limits of quantification (LLQ) are 0.05 and 0.01 ug/ml for ABC and MPA, respectivelyy with an intra- and interday variation of 6 and 1 % for ABC and 6 andd 5% for MPA.
Plasmaa concentrations of IDV were measured using liquid chromatography coupledd with electrospray tandem mass spectrometry (LC-MS/MS) 20 which hass been validated over a concentration of 0.01 to 10 ug/mL The intra- and interdayy variation is less than 10%, the (LLQ) is 0.01 ug/ml.
Plasmaa NVP concentrations were measured using high-performance liquid chromatographyy (HPLC) with ultraviolet detection. This assay is validated over thee range of 0.05-10 ug/ml using 250 uL of plasma, with an intra- and inter-dayy variation of less than 4.5% and a LLQ of 0.05 ug/ml21.
Analysiss of the intracellular endogenous and drug triphosphates were performedd using enzymatic assays as described earlier22. Intra- and inter-day variabilityy of the 3TC assay was 14 and 15% respectively. The LLQ was 0.05 pmoll for 3TCTP. Intra- and inter-assay variability of the endogenous deoxynucleosidee triphosphate assays were less than 10%. The LLQ of endogenouss triphosphates is 0.01 pmol.
StatisticalStatistical analysis
Dataa were analyzed using SPSS 11.0.1, (SPSS Inc. Chicago, Illinois). Pharmacokineticc parameters of MPA, ABC, IDV and NVP were calculated usingg non-compartmental analyses (WinNonLin, version 3, Pharsight, Mountainn View, U.S.A.). Baseline patient characteristics, the change in [3 H]-thymidinee incorporation and the influence of MPA on the pharmacokinetic parameterss of ABC, IDV and NVP and the intracellular triphosphate concentrationss were compared using the Mann-Whitney U-test. In this analysiss both dosages of MMF were taken together. Intracellular triphosphate concentrationss before and during HAART were compared using the Wilcoxon two-samplee test.
Results s
PatientPatient characteristics
Alll patients were males. Baseline characteristics are shown in table 1. After 8 weekss of therapy, the pharmacokinetic profiles of MPA, IDV, ABC, and NVP weree assessed in all patients. After lowering the dose of MMF, the pharmacokineticc profiles were repeated in patient 02, 03 and 04 after 32, 24 andd 24 weeks of therapy, respectively. In patients 01, 02, 03 and 04 intracellularr triphosphates were measured 48, 32, 24 and 24 weeks after startingg therapy. In all other patients this was done after 8 weeks of therapy. Theree were no differences between the patient groups with and without MMF withh respect to adherence or co-medication. There was also no difference in virologicall response between the two groups 23.
Tablee 1 Patient characteristics
Numberr of patients Agee (years) [median (IQR)] Baselinee CD4+
T cells <x108
/l) [median (IQR)] Baselinee CD8+ T cells (x106/l) [median(IQR)] Baselinee plasma HIV-1 RNA (log copies/ml)
[mediann (IQR)] M M F --10 0 344 (28-40) 480(415-610) ) 1130(875-1240)* * 4.700 (4.26-5.79) M M FF + 9 9 355 (32-49) 4300 (320-500) 1410(905-2025) ) 4.63(3.12-5.46) ) P* * 0.50 0 0.26 6 0.14 4 0.55 5 #
Mann-Whitneyy test; 'Baseline CD4+
and CD8* T cells were not available for 1 patient. All patientss were male.
SideSide effects
Inn the group with MMF, one patient (11) discontinued MMF at week 16 at his ownn request. In the group without MMF, one patient (05) stopped ABC in weekk 7 because of a hypersensitivity reaction 24, two patients (07 and 09) switchedd from NVP to efavirenz because of a rash and elevated liver enzymes andd two patients (12 and 17) temporarily stopped therapy, after respectively 199 weeks and 4 days of therapy. These two patients restarted all medication exceptt NVP after 6 weeks (patient 12) and 4 days (patient 17). Overall, no moree side effects were seen in patients using MMF.
TT cell proliferative capacity
Thee median T cell proliferative capacity measured with [3H]-thymidine incorporationn was 8.0 % (interquartile range (IQR) 4.8 - 22.3) in the group of patientss using MMF compared with 64.0 % (IQR 28.8 - 76.0) in the group of patientss without MMF ( p=0.001, Mann-Whitney test). The T cell proliferative capacityy did not change after lowering the MMF dosage from 1000 mg BID to 5000 mg BID.
PharmacokineticsPharmacokinetics of MP A
Inn patients 02, 03 and 04, who were using MMF 1000 mg twice daily at that time,, the median MPA Cmin concentration was 1.20 ug/ml (IQR 0.44-1.45
ug/ml),, which is well above the minimum required concentration for lymphocytee inhibition 17. The median Cmaxwas 15.20 ug/ml (IQR 11.70-15.50
ug/ml)) and the median AUC was 46.71 ug.h/ml (IQR 25.29-55.34). To prevent sidee effects, we therefore lowered the dose to 500 mg BID after respectively 24,, 16 and 12 weeks of treatment. The pharmacokinetic profile was repeated forr these patients after using the lower dosage for at least 8 weeks. The mediann MPA Cmin concentration measured in all patients using 500 mg BID
(n=9)) was 0.39 ug/ml (IQR 0.37-0.66 ug/ml), which is above the minimum requiredd concentration 17(table 2, figure 1).
PharmacokineticsPharmacokinetics of antiretroviral drugs
Thee AUC and the clearance of ABC were comparable in the groups with and withoutt MMF, p=0.45 and p=0.95 respectively (Mann-Whitney test)(table 2, figuree 1). Likewise, the AUC and the clearance of IDV were not affected by MPA,, p=0.61 and p=0.09 respectively. The AUC of NVP was lower and the clearancee higher in the MMF group: p=0.07 and p=0.04 respectively (Mann-Whitneyy test) (table 2, figure 1).
IntracellularIntracellular triphosphates
Inn the first twelve patients, of which five also received MMF 500 mg BID, intracellularr triphosphates during therapy were measured. Unfortunately, CBVTPP could not be quantified in the cell extracts, due to assay interference.
Tablee 2 Pharmacokinetic parameters
MMFF 500 mg BID n=9 9 Cmaxx MPA (Mg/ml) [median (IQR)] 6.7 (4.90-12.85) Cmjnn MPA (Mg/ml) [median (IQR)] 0.39 (0.37-0.66)
AUGG MPA (ug.h/ml) [median (IQR)f 19.14 (16.33-33.51) Cl/FF MPA (Uh) [median (IQR)] 26.13 (15.80-30.64)
ABCC n=9 ABC n-10 (MMFF 500 mg BID) (No MMF) C ^^ ABC (Mg/ml) [median (IQR)] 1.98 (1.56-2.84) 1.86 (1.48-2.35) CminABCC (Mg/ml) [median (IQR)] <0.05* < 0.05+
AUCC ABC (ug.h/ml) [median (IQR)f 3.67 (3.10-6.13)) 3.25 (2.39-4.86) Cl/FF ABC (L/h) [median (tQR)] 96.23(78.17-148.17) 110.37(72.41-139.54)
IDVV n=8 IDVN=11 (MMFF 500 mg BID ) (No MMF)
Cmaxx IDV (ug/ml) [median (IQR)] 7.74(4.98-11.87) 9.32 (7.93-10.23) CminIDVV (Mg/ml) [median (IQR)] 0.17 (0.12-0.38) 0.34 (0.20-0.45)
AUCC IDV (|jg.h/ml) [median (IQR)f 33.63 (21.10-41.40) 34.63 (30.13-47.20) Cl/FF IDV (Uh) [median (IQR)] 24.04 (19.33-38.23) 23.11 (16.95-26.56)
NVPn=99 NVP N=8 (MMFF 500 mg BID) (No MMF) Cmaxx NVP (Mg/ml) [median (IQR)] 5.87(5.41-6.78) 7.49 (6.51-8.07) Cminn NVP (Mg/ml) [median (IQR)] 4.25 (3.67-4.74) 5.08 (3.76-5.98) AUCC NVP (|jg.h/ml) [median (IQR)f 61.63 (54.16-69.53) 70.90 (60.11-82.44) Cl/FF NVP (Uh) [median (IQR)] 3.25 (2.88-3.70) 2.83 (2.43-3.34)
"AUCC during a dosing interval (0-12 h); +ln all patients below the LLQ of 0.05 ug/ml; *ln one
patientt 0.06 ug/ml in the other patients below the LLQ of 0.05 ug/ml.
Theirr was no significant difference in dCTP, dGTP or 3TCTP pools or the 3TCTP/dCTPP ratio between the two groups at T=0 (p=0.15, 0.20, 0.88 and 0.20),, T=2 (p=0.54, 0.54, 0.66 and 0.43), T=6 (p=0.43, 0.34, 0.20 and 1.00) andd T=12h (p=0.27, 1.00, 0.64 and 0.27)(Mann-Whitney test)(figure 2). In 6 patientss without MMF, dCTP and dGTP concentrations were also assessed at thee day of starting antiretroviral therapy. There was no difference between
intracellularr dCTP or dGTP concentrations before and during HAART (p=0.12 andd p=0.46 respectively; Wilcoxon two-sample test). A baseline sample was onlyy available from one patient taking MMF. There was no difference between dCTPP or dGTP concentrations between baseline and week 8 in this patient.
Figuree 1 Pharmacokinetic profiles
r r
- —— MMF 500 mg BIO (n=9) MMFF 1000 mg BID (n=3) Minimumm desired concentrationn (0 14 ug/ml) —— No MMF {n=8) -- - MMF 500 mg BID (n=9) — -- MMF 1000 mg BID (n=3) 99 f s s -- No MMF (n=10) -- MMF 500 (n=9) - -- MMF 1000 (n=3) —— NoMMF(n=11) -- MMF 500 mg BID (n=8) — -- MMF 1000 mg BID (n=3)Mediann plasma concentrations during a dosing interval; bars represent inter-quartile ranges, a)) concentrations of mycophenolic acid (MPA); (minimum desired concentration of MPA: 0.14 Ug/ml).. b) concentrations of nevirapine (NVP) with or without MMF; c) abacavir (ABC) ; d) indinavirr (IDV).
Discussion n
Thee aim of our study was to investigate the effect of MMF therapy on the pharmacokineticss of a number of antiretroviral drugs, which is important to knoww in case MMF is used in HIV treatment or in case of an organ transplantationn in an HIV patient using antiretroviral drugs. We also wanted to assesss if MPA indeed lowers the intracellular dGTP pool in PBMCs, as in vitro dataa have suggested 12.
Figuree 2 Intracellular concentrations of dCTP, dGTP, 3TCTP and the ratio 3TCTP/dCT T MMFF -MMFF < baselinee 0 2 Time e 3TCTP P Ü Ü s s 1 1 s s « « b£ £ seline e -*-"*" " 0 0 „.. — * * 2 2 » f f 6 6 . . ** + I 2 h h jurs s M M F --MMFF + ff i. 7.5-- 5.0-- 2.5--00 0-b s s — seline e dGTP P MMF--M MMF--M F H H 3TCTP/dCTP P MMF--MMFF *
Horizontall line represents median triphosphate concentration or median ratio at baseline and duringg a twelve hour dosing interval after at least 8 weeks of therapy.
Thee first 3 patients (patient 02, 03 and 04) using MMF started with 1000 mg BID.. Because these first three patients had high MPA plasma concentration andd because the lymphocyte proliferation was completely blocked, the dose wass lowered to 500 mg BID in these and all new patients to prevent side effects.. It has been demonstrated that with a dose of 500 mg BID the inhibitionn of the lymphocyte proliferation is only slightly lower compared with a 10000 mg BID regimen 25, and when the lymphocyte proliferation is fully inhibitedd a further increase of the MMF dosage does not have any beneficial effectt 17. MPA has antiretroviral activity at lower concentrations of MPA than necessaryy for inhibition of lymphocyte proliferation 815. After dose reduction thee lymphocyte proliferation remained completely blocked.
Theree were no differences in the pharmacokinetics of ABC and IDV between thee 2 groups. In the group using MMF the NVP clearance was slightly but significantlyy higher than in the group without MMF. The primary routes of NVP biotransformationn and elimination include CYP metabolism by CYP3A4 and
CYP2B66 isoenzymes, glucuronide conjugation and urinary excretion of the glucuronidee metabolite 26 . Following oral administration MMF is rapidly and completelyy absorbed and de-esterified to MPA. MPA is almost completely glucuronidatedd to its stable phenolic glucuronide metabolite (MPAG) which is excretedd in urine 27. Both NVP28 and MPA27 undergo enterohepatic recycling. Itt is possible that there is interference of MPA with the enterohepatic recycling off NVP. This could be due to competition for transporters necessary for excretionn in bile. Another possibility might be that MPA has a local immunosuppressivee effect in the gut resulting in a change in normal gut flora, whichh could result in a change of the enterohepatic recycling of NVP. If indeed NVPP concentrations are lowered because of alterations of the enterohepatic recycling,, this could mean that the use of antibiotics could also influence NVP concentrationss by affecting the normal gut flora necessary for the enterohepaticc recycling. Because MPA undergoes enterohepatic recycling a secondd peak in plasma MPA concentration would have been expected. The absencee of this peak in our patients might be caused by interference of the antiretrovirall drugs with the excretion of MPA into bile. This effect on MPA has beenn reported for cyclosporine A 29.
Inn the first 12 patients intracellular dCTP, dGTP and 3TCTP pools were measured.. In 6 of these patients, who were all without MMF, samples before thee start of treatment were available. Both dCTP and dGTP concentrations did nott change during treatment, which can be explained by the fact that both dCTPP and dGTP concentrations are not influenced by 3TC and ABC 30. We hadd a sample before treatment of only one patient using MMF. During therapyy the intracellular concentrations of dCTP did not differ between patientss with and without MMF, as was expected because the dCTP synthesis iss not directly affected by MPA. As a consequence the ratio 3TCTP/dCTP was comparablee in both groups. The relative high concentration of 3TCTP leads to aa preference for 3TCTP incorporation during HIV replication, thereby inhibiting replication.. Because MPA inhibits dGTP production, we expected a lower dGTPP concentration in patients treated with MMF. Although lymphocyte proliferationn was completely blocked in patients using MMF, dGTP pools were
nott significantly different in patients with or without MMF. This is substantially inn accordance with a recent small study, in which a consistent decrease of dGTPP was not demonstrated in each of the 5 patients receiving MMF 31. Also inn patients using hydroxyurea in combination with antiretroviral therapy, the expectedd decrease of endogenous triphosphates, particularly dATP, predicted fromm in vitro studies 32 was not seen in vivo 33. One possible explanation could bee the variability of triphosphorylation in vivo. It is also possible that proliferationn is more sensitive than dGTP measurement. Another explanation mightt be that indeed dGTP is lowered by MPA after a first dosing, but that cellss in vivo develop a compensation mechanism probably by the salvage pathway.. However this would not explain the decreased lymphocyte activity thatt was observed throughout the study. CBVTP and dGTP have similar affinityy for HIV reverse transcriptase 34, so it is unlikely that these findings are causedd by an accumulation of dGTP in the presence of CBVTP. Assessment off dGTP pools shortly after the start of MMF, as compared to prolonged use of MMF,, might elucidate this issue further.
Althoughh this was a small study, we demonstrated that MPA alters the pharmacokineticss of NVP but has no effect on ABC and IDV. Furthermore, despitee effectively blocking lymphocyte proliferation, MPA did not decrease intracellularr dGTP pools at the time points tested in this study. Clearly, these resultss warrant further investigations on the supposed working mechanism of MPA. .
Acknowledgement t
WeWe thank Marijke Roos, Sanquin Research and Landsteiner Laboratory, Universityy of Amsterdam, Amsterdam, the Netherlands for measuring lymphocytee proliferation. This study was financially supported by a private foundationn that wishes not to be named.
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