Saye Khoo
•www.hiv-druginteractions.org & www.hep-druginteractions.org
sponsorship from Janssen, ViiV, AbbVie, Merck, BMS, Gilead.
Editorial content remains independent.
•Research Grants: Merck, ViiV, Janssen, Novartis
•Speakers bureau: Merck, Janssen, AbbVie, ViiV, Gilead
•Travel grants: Gilead, ViiV,
Declaration of Interests
PK Challenges on Reservoirs
Cells (biology)
Location of Cells
(compartments)
Barrier to cure
Persistent inflammation
Low-level viraemia
Resistance
Transmission
HIV Drug
HIV
HIV
Reservoirs Consequences
Quality of Evidence for PK Reservoirs
Measure drug
concentrations in
cells/compartments
• direct observation
• target concentrations not defined
• protein binding, drug metabolism
• large inter-patient variability
• technical challenges
Comparison of viral
dynamics
• plasma-compartment VL discordance
• VL dynamics upon starting treatment
• significant inter-patient variability
• relevant to transmission
Comparison of drug
resistance profiles
• direct relevance
• track evolution of resistance
PK Compartments
CNS
PK, VL dynamics,
Resistance
Breast milk
PK, VL dynamics,
Resistance
Lungs
VL dynamics
GALT, lymph nodes
PK, VL dynamics
Generally concordant
nucleotide sequences
Genital tract
PK, VL dynamics,
Resistance
Jofesson et al PLoS Pathogens 2013; 9: e1003432
Placenta
PK
Are some ARVs more effective in the CNS than others ?
• Some [ARV] in CSF ≤ IC50-95
• CPE CSF HIV RNA and HIV genetic diversity
• Switch to CPE regimen – HIV CSF RNA, cognitive function
• Discordant resistance mutations
• Some ARVs neurotoxic in-vitro
ARGUMENTS FOR
Letendre et al. Arch Neurol 2008;65:65
Cysique et al. BMC Neurol 2011;11:148
Robertson et al. J Neurolvirol 2012;18:388
Peluso et al AIDS 2012;26:1765
Smit et al. J Virol 2004;78:10133
Heaton et al. Neurology 2010;14:536
Simioni et al. AIDS 2010;24:1243
Giancola et al. JAIDS 2006;41:332
Smurzynski et al. AIDS 2011;25:357
Ellis et al. Clin Infect Dis 2014; 58: 1015
Are some ARVs more effective in the CNS than others ?
• Some [ARV] in CSF ≤ IC50-95
• CPE CSF HIV RNA and HIV genetic diversity
• Switch to CPE regimen – HIV CSF RNA, cognitive function
• Discordant resistance mutations
• Some ARVs neurotoxic in-vitro
ARGUMENTS FOR
Letendre et al. Arch Neurol 2008;65:65
Cysique et al. BMC Neurol 2011;11:148
Robertson et al. J Neurolvirol 2012;18:388
Peluso et al AIDS 2012;26:1765
Smit et al. J Virol 2004;78:10133
Heaton et al. Neurology 2010;14:536
Simioni et al. AIDS 2010;24:1243
Giancola et al. JAIDS 2006;41:332
Smurzynski et al. AIDS 2011;25:357
MR spectroscopy (N-acetyl aspartate)
Schweinsburg. J Neurovirol 2005;11:356
Frontal white
matter 11%
Tovar-y-Romo et al. JPET 2012;343:696
• Dendritic spines of neurons
• Ca signalling
• Apoptosis & survival
8OH EFV is Directly Neurotoxic
Are some ARVs more effective in the CNS than others ?
• Some [ARV] in CSF ≤ IC50-95
• CPE CSF HIV RNA and HIV genetic diversity
• Switch to CPE regimen – HIV CSF RNA, cognitive function
• Discordant resistance mutations
• Some ARVs neurotoxic in-vitro
ARGUMENTS FOR
• CSF viral escape is uncommon
• Some observational studies show no association between CPE and cognitive function
(assay sensitivity, NPS tests, normative data)
• PK in CSF may not reflect PK in microglia or brain macrophages
ARGUMENTS AGAINST
Letendre et al. Arch Neurol 2008;65:65
Cysique et al. BMC Neurol 2011;11:148
Robertson et al. J Neurolvirol 2012;18:388
Peluso et al AIDS 2012;26:1765
Smit et al. J Virol 2004;78:10133
Heaton et al. Neurology 2010;14:536
Simioni et al. AIDS 2010;24:1243
Giancola et al. JAIDS 2006;41:332
Smurzynski et al. AIDS 2011;25:357
Nightingale et al. Lancet ID (in press)
ARV Penetration into Genital Tract
• Physiochemical Characteristics – QSAR predictions suggest Vd,
MRP1 substrate, MRP4 substrate drugs penetrate female genital tract
• Gender specific and drug-specific – rather than class-specific
• Viral dynamics generally concordant with exceptions
• PARTNERS suggests plasma VL is a good surrogate
• Insight from PreP pharmacokinetic studies - Rectal TFV / TFV-DP levels higher than vaginal - TFV-DP in VT: TFV gel >>> TDF (po), some rectal exposure also
Thompson et al. AIDS Res Hum Retrovirol 2014;29:1
Else et al. AVT 2011;16:1149
Hendrix et al PLoS One 2014; 8(1): e55013
Else et al. Antiviral Therapy 2011; 16:1149 ; Cohen, et al. Annals Int. Med, 2007
Breast Milk
• residual transmissions (3.3-5.6% in Kesho Bora trial)
• > three quarters vertically infected infants in Kisumu Breastfeeding Study (ZDV/3TC + NVP or NFV) had resistance to both NVP and 3TC
• in the BAN study (cART initiated post-partum) almost a third of HIV+ infants had multiclass resistance
Zeh et al. PLoS Med 2011;8:e1000430
Fogel et al. CID 2011;52:1069
Kesho Bora Study Group, Lancet ID 2011 Mar;11(3):171
van der Perre Science Trans Med 2012;4:
Tuaillon et al; J Immunol 2009
Valea et al Revrovirology 2011
Chasela NEJM 2010 ;362 :2271
Importance of cell-to-cell transmission in Breast Milk
• cell-to cell infection may be important route of early postpartum transmissions through breastfeeding
• Macrophages
• T & B cells more likely to be activated, memory
Tuaillon et al; J Immunol 2009
van der Perre Science Trans Med 2012;4:
Valea et al Revrovirology 2011;8:34
• CD4+ T cells freshly purified from BM and plasma
• T cells in BM more likely to be activated, memory cells
• Spontaneous HIV antigen-secreting T cells present in BM and plasma
• HIV detected in 4/7 aviremic, and 5/8 viraemic mothers
• ART does not completely suppress cell-associated viral replication
Importance of cell-to-cell transmission in Breast Milk
Breast Milk Transmissions – Phylogenetic analyses
• Nested within RCT of vitamin supplementation • Mothers were free to choose breastfeeding after counselling • ARVs not available during study • MTCT (N=61) – infant PCR- (6w) subsequently became PCR+
Koulinska et al. J AIDS 2006;41:93-99
Transplacental transfer of HIV drugs
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
Cord:maternal plasma
Amniotic:maternal plasma
10.7 2.8
Pinetti et al AVT 2010;15:127
Ivanovic Curr HIV Res 2009;7:620
Gavard. Am J Obstet Gynecol 2006;195:296
Bawdon. Obstet Gynecol 6:244, 1998
Sudharkaran Antimicrob Agents Chemother 2005;49:1023-8
Forestier Am J Obstet Gynecol 185:178, 2001
Ceddaldi P-F, et al: Am J Obstet Gynecol 2008;198:433. Mirochnick IWCPHT Sorrento 2010; Abst03
Moisan IWCPHT Sorrento 2010; Abst 01
Rati
o
HIV in the Lung
xx
• Acute (opportunistic & non-opportunistic) infections • Chronic Lung Disease – inflammation, emphysema, fibrosis • Increasing prevalence despite ART
• ? HIV related factors
Drug Targeting of Cellular Reservoirs
Ford et al. JAC 2004;54:982
CD4+ T cells – including latent infected resting memory T cells GALT Follicular Dendritic Cells Haemopoietic Stem Cells Monocyte-macrophages
Other cells ?
Buzon et al. Nat Med 2014;20:139
• Hierarchy of PI accumulation methodological differences
• Activated vs resting (ex-vivo) ZDV-TP, CBV-TP
ARV concentrations by compartment
relative to PBMC concentrations
Fletcher C V et al. PNAS 2014;111:2307-2312
Different patterns of HIV RNA decay
from FDCs in lymph nodes
Association between decay rate of
virions from FDC pool and
intracellular drug levels
ARV Concentrations in Lymphatic Tissue
• HIV+ patients starting ART (N=12; including EFV [6], ATVr [4], DRVr [2]) • Serial ileum/rectal/LN biopsies through M1-M6 • Flash-frozen tissue, minced and disaggregated • PBMCs and tissue extracts washed twice, lysed • LC-MS/MS analysis • Patterns of RNA decay in tissue: no change
initial decay, then plateau constant reduction
Macrophages
• A primary tissue reservoir of HIV
• Significant differences from T cells
• longer lived source of infection
• less prone to cytopathic effect
• terminally differentiated limited dNTP pools (22-320-fold [dNTP]
• ? Different TK activity (TK2 vs TK1 in lymphocytes)
• host restriction factors- SAMHD1, APOBEC3G, etc
Kumar et al. Mol Cell Ther 2014;2:10
Carter. Ann Rev Microbiol 2008;62:425
Gavegnano et al. Antivir Chem Chemother 2009;20:63
Perno et al. Antivir Res 2006;71:293
Aquaro et al. Antiviral Res 2002;55:209
Galvegano et al. Mol Biol Int 2012;625983
Gavegano et al. AAC 2013;57:1262
Aquaro et al. J Leukoc Biol 2006;80:1103
Could PK of ARVs differ in Macrophages ?
Ex-vivo / in-vitro studies of PK in macrophages
intracellular PK • Intracellular [ZDV- TP], [CBV- TP], [3TC- TP], [FTC-TP] : M<<< T cells • Intracellular [TFV/TFV-DP]: M T cells • Intracellular [RAL]: M << T cells (independent of activation)
Arner et al. JBC 1992;267:10968
Perno et al. AVT 2006;71:293
Aquaro et al. J Leukoc Biol 2006;80:1103
Gavegano et al. AAC 2013;57:1262
Aquaro et al. Antiviral Res 2002;55:209
Galvegano et al. Mol Biol Int 2012;625983
PD • In-vitro models suggest NRTIs more active in MM than CD4+ lymphocytes • ddNTP:dNTP ratios are higher • EC50 of NRTI/NNRTI : chronic >>> acutely infected M • RAL: poor potency in M • EC50 of PI : chronically infected M >> T cells, but still retained some
activity
Drug Metabolism within Compartments
CNS
CYP P450 (2B6, 3A4, 2D6)
UGTs
Transporters (ABCG2, ABCC5/10,
ABCB1, SLCO1A2
Breast milk
CYP P450 (1B1, 4Z)
may inhibit gut UGT1A1
Transporters (ABCG2, ABCC1/3/6,
SLC15A2, SLC29A1, SLCO2B1/3A1
Lungs
CYP P450 (1A1, 2C19)
Transporters (ABCC1/10,
ABCG2; SLCO3A,
SLC22A7, SLC15A2)
GALT, lymph nodes
Transporters (ABCB1,
ABCC1/2/10, ABCG2;
SLCO3A1, hENTs)
CYPs (3A4, 2B6)
Genital tract
Transporters (ABCG2,
ABCC1/10/12,; SLCO3A1)
Placenta
CYPs (1A1, 2E1, 3A4/5)
UGTs
transporters (ABCB1, ABCC1,
ABCG2; SLC22A3 [OCT3],
SLC29A1,2 [hENT1&2)
Brain CYP 2B6
Can we improve existing drugs?
• ‘Boost’ concentrations within compartments through transporter inhibition
• Development of pro-drugs – eg TAF
• Cell-targeting Nanoformulations – eg folate-coated nanoformulations
Tenofovir Alafenamide (GS7340)
• TAF : intra-PBMC TFV-DP (50x) , plasma TFV (90%) compared to TDF
• TFV in rectal secretions, TFV-DP following single dose of TAF 3 days before to macaques
• Despite tissue drug levels, did not protect macaques from rectal SHIV infection
García-Lerma J G et al. J. Virol. 2011;85:6610-6617
Can we improve existing drugs?
• ‘Boost’ concentrations within compartments through transporter inhibition
• Development of pro-drugs – eg TAF
• Cell-targeting Nanoformulations – eg folate-coated nanoformulations
Kanmogne et al. Int J Nanomed 2012; 7: 2373
Nano-RTV (orange-yellow) uptake by
human brain microvascular endothelial
cells cocultured with monocytes
Gautam et al. 2014 AAC 6th Oct
Cell-to-cell transmission FIB-SEM imaging reveals virological synapses
between infected and uninfected T cells.
Do T et al. J. Virol. 2014;88:10327-10339
• May be important in MTCT (breast milk transmission), CNS, GALT, sexual transmission
• Several orders of magnitude more efficient than cell-free diffusion
large MOI delivered through physical contact densely packed cells
• Can this evade ART ? NRTIs sensitivity than cell-free infection PIs and EFV retain activity in-vitro
Sigal et al. Nature 2011;477:95
Titanji et al. Retrovirol 2013;10:161
Agosto et al. PLoS Pathog 10(2): e1003982
Questions
Modern ART does not fully control HIV replication
• How much more can existing ARVs be enhanced to target reservoirs ? (and what are the implications for deintensification)
• Can existing ARV classes ever gain adequate control of residual replication ?
• What are the implications for mortality, morbidity, resistance and transmission ?
Acknowledgements
University of Liverpool David Back
Laura Else
Catriona Waitt
Andrew Owen
Marco Siccardi
Sam Nightingale
Anna Maria Geretti
Malawi-Liverpool-Wellcome Unit,
Blantyre Henry Mwandumba
Christine Kelly
St Stephens AIDS Trust Marta Boffito
Akil Jackson
Oxford University Clinical
Research Unit, Ho Chi Minh Estee Torok
.. and many others ....