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

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