an nih ip/cp for topical microbicides stability of mucosal cytokine profiles (proteins) and mucosal...
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an NIH IP/CP for Topical Microbicides
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
NIH IP/CP
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
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
McGowan et al JAIDS 2007
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
NIH IP/CP
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
NIH IP/CP
RM Phase 1 Trial Design
Randomization: 0.1% UC-781, 0.25% UC-781, or placebo
Visit 1 Visit 2 Visit 3 Visit 4 Visit 5 Visit 6
Screening Phone interview
Single-doseexam
Safety;Given 7 daily doses
7-day examBaseline
<4 wk 1 wk 1 wk~ 8 days
Week 0 Week 2 Week 5 Week 6 Week 8
flex flex flex
NIH IP/CP
Sample Collection & Processing
• Mucosal mononuclear cells (MMC) for FACS:• 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-1, IL-6, TNF, IFN-, MIP-1, 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
02
04
06
08
01
00
02
04
06
08
01
00
Group
% C
CR
5+
on
CD
4+
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
NIH IP/CP
MMC phenotypes by FACS: Do ‘double +s’ on CD4 change?
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
02
04
06
08
01
00
02
04
06
08
01
00
Group
% C
CR
5+
X4
+o
n C
D4
+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
NIH IP/CP
MMC phenotypes by FACS: Does activation on CD4 change?
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.
02
46
81
00
24
68
10
Group
DR
+C
D3
8+
CD
4+
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
Collection of rectal secretions for cytokines/chemokines
with surgical sponges
02
04
06
08
01
00
Boxplots of IL-1b
Group
IL-1
b
U19 (All) U19 (NON) U19 (Med) U19 (RESP)(n=27) (n=9) (n=9) (n=9)
V2 V3 V5
02
04
06
08
01
00
V2 V3 V5 V2 V3 V5 V2 V3 V5
02
04
06
08
01
00
02
04
06
08
01
00
Boxplots of IL-6
Group
IL-6
U19 (All) 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
Luminal cytokines by Luminex™: IL-1 or IL-6
No appreciable differences over time or within subgroups of IL-1 or IL-6.In general, IQR reasonably tight for whole group at baseline.
05
10
15
20
25
30
05
10
15
20
25
30
Boxplots of IL-12(p-40)
Group
IL-1
2(p-
40)
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 V50
50
10
01
50
20
00
50
10
01
50
20
0
Boxplots of Ing-gamma
Group
Ing
-ga
mm
a
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
Luminal cytokines by Luminex™: IL-12 or IFN-
No appreciable differences over time or within subgroups of IL-12 or IFN-.IQR reasonably tight for IL-12; broad for IFN-.
02
04
06
08
00
20
40
60
80
Boxplots of TNF-Alpha
Group
TN
F-A
lph
a
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 01
00
20
03
00
40
00
10
02
00
30
04
00
Boxplots of RANTES
Group
RA
NT
ES
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
Luminal cytokines by Luminex™: TNF- or RANTES
No appreciable differences over time/within subgroups of TNF- or RANTES.IQR reasonably tight for TNF-; broad for RANTES.
01
00
20
03
00
40
05
00
01
00
20
03
00
40
05
00
Boxplots of MIP-1 Alpha
Group
MIP
-1 A
lph
aU19 (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
Luminal cytokines by Luminex™: MIP-1
No appreciable differences over time or within subgroups of MIP-1-.In general, more variable IQR.
NIH IP/CP
“Blinded” Interpretations
• MMC phenotypes: Based on FACS data, there appears to be no difference among the all subjects in the 3 groups (placebo, high-does, 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
an NIH IP/CP for Topical Microbicides
•NIH NIAID U19 IP/CP #AI060614: “Microbicide Development Program”
Biosyn, Inc Anne Marie CornerLinda KnappLinda Kristekas
UCLAIan McGowan (U Pitt)Chomchay Siboliban Amy AdlerTerry SaundersElena KhanukhovaCharlie PriceJulie ElliottJohn BoscardinYing ZhaoDaniel ChoKaren AndrewsElizabeth JohnsonAlexis DominguezJulia Klein
NIHJim TurpinJeanna PiperCherylnn MathiasGrace Chow
ConsultantsAlex Carballo-DieguezAna Vetuneac
CONRADHenry GabelnickChristine MauckTim McCormickMarianne Callahan
VOLUNTEERS!
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