johns hopkins school of medicine - hiv persistence during therapy · 2018. 9. 24. · •group 2a...
TRANSCRIPT
Lucio Gama, Ph.D. Department of Molecular and Comparative Pathobiology
Johns Hopkins School of Medicine
* Patent pending
Ingenol mebutate or Ingenol 3-angelate
Isolated from Euphorbia peplus (milkweed)
INGENOL
Euphorbia tirucalli
AVELOZ
Ingenol derivatives
• The sap of E. tirucali presented a mix of “ingenols” with various unstable radicals at C3.
• Solution: modify the molecule, keeping the core intact.
• Results:
– Ing A, B, and C
– Also named KyoII A, B, and C (KyoLab is the name of the company).
Dr. Luiz Pianowski
Ingenol A, B, and C
• Ing A:
– 3-trans-cinnamate
• Ingenol B:
– 3-caproyl or hexanoate
• Ing C:
– 3-dodecanoate
PMA
Ingenol
• Diterpene / Phorbol Ester (PMA, prostratin)
• PKC activator – Mimic diacylglicerol
• Efficiently activates the HIV-LTR in reporter cells
J-lat 10.6 Clone (48h)
0.01 0.10 0.50 1.000
25
50
75
100Ingenol A
Ingenol B
Ingenol C
Prostratin
PMA
TNF-α 10ng
[mM] Compounds
% G
FP
Dr. Celina Abreu
Ing B
• In CD4+ T cells: Upregulates CD38 & CD69. Slight upregulation of HLA-DR • In Mø: Does not upregulate CD80 or HLA-DR.
• Decreases HIV and SIV replication in MT-4 / CEMx174
• Downregulates CD4, CCR5, CXCR4
Ing B
• Upregulates in vitro expression of cytokines and PTEFb components.
Dr. Renato Aguiar
Ing B
• Does not promote cell proliferation.
Human PBMC
D1 D2 D30
5
10
15
20
25Ingenol B 10µM
IL-2 + PHA
Untreated
% o
f p
rolife
ratio
n
• Very well tolerated (orally) • Mice, rats, dogs
• Easy and cheap extraction
Rhesus macaque trial 1 – Proof of concept
Two SIVmac251-infected rhesus macaques (NOT SUPPRESSED) Escalating dosage ORAL
Off 1mg BID 2.5mg BID 5mg BID
Blood collection at day 0, 3, and 7
CBC, Chemistry, FACS, Viral load
Off
Each block: 7 days
Ingenol – Blood chemistry panel
Similar to our SIV-infected animals * Low Ca, BUN, high globulin
Within normal levels * Protein * Bilirubin * (low) AST
Dr. Pate
0 3 7 14 17 2128 31 350.5
2
8
32
128
512
Days post treatment / Ing B regimen
Chemistry Panel
ALT (mg/dL)
Ap (IU/L)
Creat (mg/dL)
1 mg BID 2.5 mg BID 5 mg BID
Ingenol – CD69 (activation)
Erin Shirk
0 3 7 14 17 2128 31 350
20
40
60
Days post treatment / Ing B regimen
% CD4+ T cells positive for CD69
1 mg BID 2.5 mg BID 5 mg BID
0 3 7 14 17 2128 31 3520
30
40
50
60
Days post treatment / Ing B regimen
% CD8+ T cells positive for CD69
1 mg BID 2.5 mg BID 5 mg BID
0 3 7 14 17 2128 31 350
50
100
Days post treatment / Ing B regimen
Monocytes positive for CD69
1 mg BID 2.5 mg BID 5 mg BID
Ingenol increases viral load
0.E+00
1.E+06
2.E+06
3.E+06
4.E+06
5.E+06
6.E+06
7.E+06
8.E+06
0 5 10 15 20 25 30 35 40
SIV
co
py
eq./
ml
4824
4886
1 mg BID 2.5 mg BID 5 mg BID
Ingenol increases virus diversity in
plasma
Day zero - before Ing B
Gabriel Gonsalves
Plasma RNA
Tissue DNA post necropsy Brain DNA
Gabriel Gonsalves
Tissue DNA post necropsy Brain DNA
Post treatment Ing B
Plasma RNA
Studies in cART treated macaques - 1
• Group 1 – Hopkins Model * 3 Pigtailed macaques / SIVDeltaB670 + SIV17E-Fr * TNV / PMPA - 30 mg/kg/day
* DNV - 480 mg/kg BID * RIV – 24 mg/kg BID * L-870812 - 10 mg/kg BID * EX VIVO EXPERIMENTS
Resting CD4+ T cells were isolated from PBMCs, split in two sets, and serially
diluted in duplicates. One set was kept as control while the other was treated
with Ingenol B 1 µM for 10 days
Ingenol B
Studies in cART treated macaques - 1
Animal 1 Animal 2
SIV
RN
A c
op
ies/
DN
A in
10
,00
0 c
ells
Resting CD4+ T cells were isolated from spleen biopsies and seeded on 24 well plates in
AZT containing media. Cells were kept untreated or treated with Ingenol 1 µM or PMA
10 ng/mL + ionomycin 1 µM for 18 hours. After treatment, cells were collected and viral
DNA and RNA were quantitated by qPCR. SIV RNA copy eq. was normalized by the
levels of SIV DNA measured in 10,000 cells.
Ingenol B 1µM
Studies in cART treated macaques - 1
Studies in cART treated macaques - 2
• Group 2A – Bioqual – 4 rhesus macaques / SIVmac251
• TNV / PMPA – 20 mg/kg/day • FTC – 50 mg/kg/day • RAL – 50 mg/kg BID
• 2 animals received ingenol 0.4 mg/kg/day
• 2 weeks / 1 week wash off / 2 weeks • 2 control animals
Studies in cART treated macaques - 2
Viral Load
0 8 14 21 28 35 42 49 56 63 66 70 72 74 78 84 92 94 96 99101
102
103
104
105
106
107
12079
12099
12106
12110
Ingenol withdraw + Ing2
Ing treated
days post cART
co
pie
s / m
L
+ Ing B + Ing B
CD4 T cells (HLADR)
8 14 28 42 56 70 74 78 84 92 94 99
0
2
4
6
8
10
12079
12099
12106
12110
Ingenol
Ing treated
withdraw + Ing2
days post cART
% H
LA
DR
+
Studies in cART treated macaques - 2
Cell associated SIV RNA
49 56 63 66 70 72 74 78 84 92 94 96 99100
101
102
103
12079
12099
12106
12110Ing treated
Ingenol withdraw + Ing2
days post cART
co
pie
s/1
0ˆ6
ce
lls
Studies in cART treated macaques - 2
Cell-associated HIV RNA – Siliciano’s Lab
�PH-011 �PH-014
DMSO 1 1
Ingenol B (1 uM) 203.7138135 466.8767709
PMA + Ionomycin 409.9110298 1798.585465
1
10
100
1000
10000
Fold
Ch
ange
Patient 1
Greg Laird
Meanwhile At Hopkins…
Cell-associated HIV RNA – Siliciano’s Lab
Greg Laird
0
5
10
15
20
25
30
35
40
45
Untreated �Ingenol 1nM �Ingenol 10nMIngenol 100nM
�Bryostatin 10nMPMA/Ionomycin
Fold
ch
ange
re
lati
ve t
o u
ntr
eat
ed
Patient 2
Fold change Rep 1 Fold change Rep 2 Average Fold Change
Studies in cART treated macaques - 3
• Group 2B – Bioqual – 2 rhesus macaques / SIVmac251
• TNV / PMPA – 20 mg/kg/day • FTC – 50 mg/kg/day • RAL – 50 mg/kg BID
• 2 animals received ingenol 0.4 mg/kg/day for 30 days
Tests in SIV-infected cART-treated macaques
cART-suppressed macaques
0 50 100 150101
102
103
104
105
106
107
days post treatment
SIV
co
py e
q/m
L p
lasm
a
* * *
cART + Ing B Removal of
cART and Ing B
1 3 50
2
4
6
week post Ing B treatment
% H
LA
-DR
+
CD4 T cells + for HLA-DRcART-suppressed macaques
0 50 100 150101
102
103
104
105
106
107
days post treatment
SIV
co
py e
q/m
L p
lasm
a
+ ING B
No ING B / No cART
* * *
1 3 50
5
10
15
20
25
week post Ing B treatment
% H
LA
-DR
+
CD8 T cells + for HLA-DR
1 3 5-20
0
20
40
60
80
100
week post Ing B treatment
% C
D6
9+
CD4 T cells + for CD69
1 3 50
20
40
60
80
week post Ing B treatment
% C
D6
9+
CD8 T cells + for CD69
SIV DNA per 10^6 cells
0 50 100 15010
100
1000
10000
days post treatment
SIV
DN
A c
op
ies /
10
^6
PB
MC
s
PK Studies
* In rats: 10 µg/kg i.v. – in macaque: 5 µg/kg i.v. * In rats: 1 mg/kg p.o. – in macaque: 0.5 mg/kg p.o. (2.5 mg total)
Start: 100,000 pg/mL
Ingenol in uninfected macaques
0
100
200
300
400
500
600
700
800
900
0 200 400 600
Minutes post administration
MCP1 in CSF (pg/mL)
PO Ingenol
IV Ingenol
0
50
100
150
200
250
300
350
400
0 200 400 600
Minutes post administration
IL6 in CSF (pg/mL)
PO Ingenol
IV Ingenol
Cytokines (ELISA)
0
50
100
150
200
0 100 200 300 400
Minutes post administration
MCP1 levels in Plasma (pg/mL)
PO Ingenol
IV Ingenol
0
2
4
6
8
10
12
14
0 200 400 600
Minutes post administration
IL6 levels in plasma (pg/mL)
PO Ingenol
IV Ingenol
Conclusions and Perspectives
• Ing B is a potent PKC activator and it is well tolerated when given to mammals orally.
• It is a potential drug for the reactivation of latent reservoirs • Evaluate bioavailability and pharmacokinetics
• Detection in tissues (Dr. Angela Kashuba) • Radiolabeled compound (Aurigon – Germany)
As a proof of concept: PKC activators are not Satan * Molecule modifications * Tools to understand pharmacophysiology
The Retrovirus Lab Johns Hopkins School of Medicine
Janice Clements
Chris Zink
Joe Mankowski
David Graham
Ken Witwer
Kelly Pate
Zhaohao Liao
Brandon Bullock
Suzanne Queen
Erin Shirk
Ming Li
Sarah Price
Liz Engle
Lakesha Johnson
Melissa McAlexander
Alexey Lyashkov
Ceereena Ubaida Mohien
Kelly Meulendyke
Claudia Avalos
Julia Russell
Julia Drewes
Jeanne Sisk
Josh Croteau
Ravi Tharakan
Adero Francis
Sarah Beck
Kathleen Kelly Brennan
UFRJ and Kyolab (Brasil)
Amilcar Tanuri Luiz Pianowiski Renato Aguiar
Celina Monteiro Abreu Gabriel Gonsalves Rodrigo Delvechio
Siliciano’s Lab
Greg Laird Korin Bullen
Bioqual
Mark Lewis
Acknowledgments