Transcript Document

Stability of Mucosal Cytokine Profiles (Proteins)
and Mucosal Mononuclear Lymphocyte
Phenotypes Following Rectal Administration of
UC-781 Microbicide Gel in a
Phase 1 Safety Assessment
(blinded data at 75% completion)
D Cho, I McGowan, J Elliott, G Cortina, A Dominguez, K Tanner,
EJ Johnson, T Saunders, A Adler, E Khanukhova, C Price, P Anton
UCLA, NIH, CONRAD
an NIH IP/CP for Topical Microbicides
Study Intent
• Safety assessment: using most detailed assays available
• WHAT are reasonable safety indices, outside of FDA requirement
• WHAT research assays may be important safety indices:
“immunotoxicity indices”
• HPTN 056 (McGowan PI) attempted to investigate this in 4 groups
of men (N=4/group) each seen every 2 weeks for 6 weeks to
evaluate reproducibility and stability of readouts.
 Groups: HIV- men, no hx of RAI
McGowan et al JAIDS 2007
HIV- men, + hx RAI
HIV+ men, undetectable PVL
HIV+ men, PVL >5000 copies/ml
 Indices: Histology (quantitative & qualitative), secreted Ig, cytokine
mRNA from tissue, MMC phenotypes
• GOALS: to reduce #, identify preferred sensitive assays
NIH IP/CP
Study Outline
• 75% Subjects studied thus far [19 men (70%), 8 women (30%)] with
all samples collected
• 3 collection timepoints (baseline, single dose/30 minutes, 7 days)
• 3 dosing groups: (i) placebo
(ii) high-dose UC-781 (0.25% gel)
(iii) low-dose UC-781 (0.1% gel)
NIH IP/CP
Trial Objectives and Indices
• Secondary Objectives: To determine whether use is
associated with rectal mucosal damage (immunotox):
Epithelial sloughing
Histopathology
Mucosal mononuclear cell phenotype (flow)
Mucosal cytokine mRNA (tissue)
Mucosal cytokines (secreted)
Mucosal immunoglobulins
Fecal calprotectin
Explants- susceptibility to HIV infection
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Trial Objectives and Indices
• Secondary Objectives: To determine whether use is
associated with rectal mucosal damage (immunotox):
Epithelial sloughing: No difference seen between subjects nor between visits
Histopathology: No difference seen between subjects nor between visits
Mucosal mononuclear cell phenotype (flow)
Mucosal cytokine mRNA (tissue): Batched for end-of-study analysis
Mucosal cytokines (secreted)
Mucosal immunoglobulins: No difference seen between subjects nor visits
Fecal calprotectin: No difference seen between subjects nor between visits
Explants- susceptibility to HIV infection: reported previously
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RM Phase 1 Trial Design
Randomization: 0.1% UC-781, 0.25% UC-781, or placebo
flex
<4 wk
Visit 1
flex
 1 wk
flex
 1 wk
Week 0
Week 2
Visit 2
Visit 3
Screening
Baseline
Single-dose
exam
~ 8 days
Week 5 Week 6
Visit 4
Visit 5
Safety;
Given
7 daily
doses
7-day
exam
Week 8
Visit 6
Phone
interview
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Sample Collection & Processing
•
Mucosal mononuclear cells (MMC) for FACS:
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•
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Mucosal biopsies collected pre routine, (large cup forceps) at 3 visits (Anton AIDS 2000)
V2 (Baseline) preceded at intervention; V3 (single dose) and V5 (7-day) post-intervention
MMC isolation and staining per routine (Shacklett J Immunol Meth 2003)
MMC stained with combination of: CD4-FITC, HLA-DR-FITC, CD38-PE, CCR5-PE,
CXCR4-APC)
• Rectal fluid collection for cytokines by Luminex™:
•
•
•
•
•
Surgical sponges inserted for 5 minutes to collect samples at 3 visits
V2 and V3 samples acquired before product introduced; data merged as baseline
V3 and V5 sample acquired after exposure
Rectal fluid eluted by centrifugation
Luminex™ multiplex bead array quantified: IL-1b, IL-6, TNFa, IFN-g, MIP-1a, IL-12,
RANTES (results: pg/ml)
NIH IP/CP
Data Presentation
• Blinded data presented with laboratory numbers
recoded to maintain blind…numerical sequence # are
the same as seen in the Explant presentation
• Data presented as box plot showing 25-75% range
(Interquartile range=IQR) with median identified;
‘whiskers’ reflect 1.5xIQR. “Outliers” are small circles
• Data presented as “grouped” at each visit
• Data next presented according to explant ‘responders’,
‘non-responders’ or ‘middle’
• When available, HPTN 056 data presented to give
context and evidence of reproducibility at baseline
NIH IP/CP
MMC phenotypes by FACS: Does CCR5 on CD4 change?
CCR5 expression on CD4+ MMC does not seem to change in group as a whole after
single or 7-day exposure; Subset analysis based on explant responders: no changes.
Mean similar to HPTN 056
U19 (Non-resp)
U19 (Med)
U19 (Responder)
(n=27)
(n=9)
(n=9)
(n=9)
V2 V3 V5
V2 V3 V5
V2 V3 V5
V2 V3 V5
HPTN056
(n=8,v=24)
60
40
20
0
% CCR5+on CD4+
80
100
U19 (All)
Group
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MMC phenotypes by FACS: Do ‘double +s’ on CD4 change?
U19 (All)
U19 (Non-resp)
U19 (Med)
U19 (Responder)
HPTN056
(n=27)
(n=9)
(n=9)
(n=9)
(n=8,v=24)
V2 V3 V5
V2 V3 V5
V2 V3 V5
V2 V3 V5
60
40
20
0
% CCR5+ X4+on CD4+
80
100
CCR5/CXCR4 dual expression on CD4+ MMC does not seem to change in group as a
whole after single or 7-day exposure; Subset analysis based on explant responders: no
changes. Mean similar to HPTN 056
Group
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MMC phenotypes by FACS: Does activation on CD4 change?
U19 (All)
U19 (Non-resp)
U19 (Med)
U19 (Responder)
HPTN056
(n=27)
(n=9)
(n=9)
(n=9)
(n=8,v=24)
V2 V3 V5
V2 V3 V5
V2 V3 V5
V2 V3 V5
6
4
2
0
DR+CD38+ CD4+
8
10
While trends in CD38 and HLA-DR on CD4 MMC may be suggested, there is no difference
over time compared to baseline changes. HPN 056 data not immediately available.
Group
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Collection of rectal secretions
for cytokines/chemokines
with surgical sponges
Luminal cytokines by Luminex™: IL-1b or IL-6
No appreciable differences over time or within subgroups of IL-1b or IL-6.
In general, IQR reasonably tight for whole group at baseline.
100
U19 (All)
Boxplots of IL-6
U19 (NON) U19 (Med) U19 (RESP)
(n=9)
(n=9)
(n=9)
V2 V3 V5
V2 V3 V5
V2 V3 V5
V2 V3 V5
U19 (NON) U19 (Med) U19 (RESP)
(n=27)
(n=9)
(n=9)
(n=9)
V2 V3 V5
V2 V3 V5
V2 V3 V5
V2 V3 V5
0
20
40
IL-6
60
60
40
20
0
IL-1b
U19 (All)
80
(n=27)
80
100
Boxplots of IL-1b
Group
Group
Luminal cytokines by Luminex™: IL-12 or IFN-g
No appreciable differences over time or within subgroups of IL-12 or IFN-g.
IQR reasonably tight for IL-12; broad for IFN-g.
U19 (Low) U19 (Med) U19 (High)
(n=27)
(n=9)
(n=9)
(n=9)
V2 V3 V5
V2 V3 V5
V2 V3 V5
V2 V3 V5
U19 (All)
200
U19 (All)
Boxplots of Ing-gamma
U19 (Low) U19 (Med) U19 (High)
(n=27)
(n=9)
(n=9)
(n=9)
V2 V3 V5
V2 V3 V5
V2 V3 V5
V2 V3 V5
150
50
100
Ing-gamma
20
15
10
0
5
0
IL-12(p-40)
25
30
Boxplots of IL-12(p-40)
Group
Group
Luminal cytokines by Luminex™: TNF-a or RANTES
No appreciable differences over time/within subgroups of TNF-a or RANTES.
IQR reasonably tight for TNF-a; broad for RANTES.
U19 (All)
U19 (Low) U19 (Med) U19 (High)
(n=27)
(n=9)
(n=9)
(n=9)
V2 V3 V5
V2 V3 V5
V2 V3 V5
V2 V3 V5
400
U19 (All)
Boxplots of RANTES
U19 (Low) U19 (Med) U19 (High)
(n=27)
(n=9)
(n=9)
(n=9)
V2 V3 V5
V2 V3 V5
V2 V3 V5
V2 V3 V5
300
Group
0
100
200
RANTES
40
20
0
TNF-Alpha
60
80
Boxplots of TNF-Alpha
Group
Luminal cytokines by Luminex™: MIP-1a
No appreciable differences over time or within subgroups of MIP-1-a.
In general, more variable IQR.
U19 (All)
U19 (Low) U19 (Med) U19 (High)
(n=27)
(n=9)
(n=9)
(n=9)
V2 V3 V5
V2 V3 V5
V2 V3 V5
V2 V3 V5
300
200
100
0
MIP-1 Alpha
400
500
Boxplots of MIP-1 Alpha
Group
“Blinded” Interpretations
• MMC phenotypes: Based on FACS data, there appears to be no
difference among the all subjects in the 3 groups (placebo, highdoes, low-dose) over all visits on co-receptor expression or
activation status.
• Rectal fluid cytokines: Using Luminex™ data from rectal sponges
eluates, there does not appear to be any trend/significant differences
among 7 measured cytokines/chemokines after single or 7 day
exposure.
• Based on these still blinded data, if these assays (MMC phenotype and
‘secreted’ cytokines) are relevant and sensitive enough to assess
early/mild immunoreactive changes to topical UC-781 exposure, we are
not detecting such changes
NIH IP/CP
•NIH NIAID U19 IP/CP #AI060614: “Microbicide Development Program”
Biosyn, Inc
Anne Marie Corner
Linda Knapp
Linda Kristekas
CONRAD
Henry Gabelnick
Christine Mauck
Tim McCormick
Marianne Callahan
UCLA
Ian McGowan (U Pitt)
Chomchay Siboliban
Amy Adler
Terry Saunders
Elena Khanukhova
Charlie Price
Julie Elliott
John Boscardin
Ying Zhao
Daniel Cho
Karen Andrews
Elizabeth Johnson
Alexis Dominguez
Julia Klein
NIH
Jim Turpin
Jeanna Piper
Cherylnn Mathias
Grace Chow
Consultants
Alex Carballo-Dieguez
Ana Vetuneac
VOLUNTEERS!
an NIH IP/CP for Topical Microbicides