Transcript Document

DAIDS IPCP-HTM
NIH/NIAID: 1st IPCP on Rectal Microbicides Developments
(2004-2011)
Two Phase 1 Clinical Trials:
RMP-01: Topical UC781 (single & 7-day topical)
RMP-02 / MTN-006: Tenofovir (single oral, single & 7-day topical)
Peter Anton, UCLA: PI
Ian McGowan, MWRI/University of Pittsburgh: co-PI
NIH/NIAID: 2nd IPCP on Rectal Microbicides Developments
(2009-2014)
Ian McGowan, MWRI/University of Pittsburgh: PI
Outline
 Intent
context
 Assays for use in RM trials
 1st Phase 1 RM Clinical Trial utilizing assays:
RMP-01 (UC781)
 2nd Phase 1 RM Clinical trial:
RMP-02/MTN-006 (Tenofovir)
NIH IP/CP
Assay Optimization: selected for Phase 1
Assay factors addressed prior to trial (some still to address) :
 Apply to both vaginal and rectal samples
 Anticipate effect of standard clinical trial / home use such as
enemas/douches/gels (osm; pH; dilution effect)
 Sequence of sample collection: inherent confounder
 Multi-site trials: determine where samples (Flow, explants etc) collected
versus where/when processed (fresh, frozen, O/N, batched: 1 lab?)
 Semen/seminal fluid alter results/drug delivery? Same with sexual
activity / trauma
•
Relevance for interpreting “biopsy infection” experiments
 Compartment dilution effect on delivered drug concentrations?
•
Quantify [microbicide] likely exposed to tissue. Challenge much
greater in rectal than cervicovaginal explants
 NORMATIVE VALUES: to assess implications/actions of “out-ofrange” values (and then: clinical relevance)
NIH IP/CP
HPTN 056: Characterization of Baseline Mucosal Indices of Injury
and Inflammation in Men for Use in Rectal Microbicide Trials
Pre-Phase 1 rectal safety study:

normative ranges; inherent variability
Primary Objective

Determine variability of mucosal immunological, virological and
histopathological parameters in biopsies from 10cm & 30cm in the
recto-sigmoid colon in 4 defined study groups (n=16):
I.
II.
III.
IV.
HIV-/RAIHIV-/RAI+
HIV+/RAI+: high PVL
HIV+/RAI+: high PVL
Secondary Objective

Determine within group stability of defined measures over time (3
flexible sigs over 6 weeks) & biological variability between groups
Assays

Histopathology (qual/quant); flow cytometry, tissue cytokine mRNA,
rectal secreted Ig, tissue VL)
Total: 48 procedures; 1,440 biopsies
McGowan et al, HPTN 056 JAIDS 2007
HPTN-056: Data Analysis
 Data analyzed by group means
 Subject variability around means explored
 Definitions:
• Sig: within subject standard deviation
• Tau: between subject standard deviation
• Intra-class correlation (ICC) [ICC = Tau^2/ (Tau^2 + Sig^2)]
• ICC thresholds
– >0.75 shows strong stability
– >0.5 shows moderate stability
McGowan et al, HPTN 056 JAIDS 2007
Stability of Flow Cytometry Data
Mean
G1
G2
G3
G4
Sig
Tau
ICC
CD3%
68
66
74
70
5.5
9.8
0.76
CD4%
42
41
12
29
4.0
8.0
0.81
CD8%
29
28
62
45
5.2
11.9
0.84
CD31%
68
66
73
70
5.6
9.9
0.76
CD19%
32
30
26
37
7.4
8.3
0.56
ICC: Intra-class correlation >.75 shows strong stability
McGowan et al, HPTN 056 JAIDS 2007
Stability of Cytokine Data
Mean
G1
G2
G3
G4
Sig
Tau
ICC
RANTES 4.1
4.5
4.9
4.1
0.28
0.49
0.75
2.7
3.2
3.3
3.0
0.29
0.50
0.74
2.7
3.2
2.6
2.8
0.28
0.43
0.70
(log10)
IFN-g
(log10)
IL-10
(log10)
ICC: Intra-class correlation >.75 shows strong stability
McGowan et al, HPTN 056 JAIDS 2007
Where to sample…”explants”




Colorectal (10cm)
Colonic (30cm)
Colorectal (higher than 10cm)
Upper intestinal tract (systemic delivery to targets)
When to sample…explants
 Real-life factors: bowel movements, prep, pre/post sexual prep, menses
 Drug trial factors: how long post exposure, frequency, # samples
Safe to sample…?
NIH IP/CP
Colorectal: Biopsy location
X
X
NIH IP/CP
Colorectal Biopsy safety and location
NIH IP/CP
RM Clinical Relevance:
Selection Site for Explants
Concentration
~10cm
Microbicide
~30cm
Success
Failure
HIV
Anus
+ effect?
Rectum
no effect?
Colon
Ano-Rectal Distance
Diagram courtesy: C Hendrix)
Rectal Explant Model used in RM Clinical Trials:
“ex vivo biopsy infection” experiments
HIV-1 infected
and washed
explants
a) Explants transferred by
transfer pipet to a dry
petri-dish, cut-side down
Mounted explants
transferred to well of 24well plate containing
500ul of explant medium
Explant media
soaked collagen
rafts
b)1 cm2 raft placed atop the
explant and inverted.
Media 100% exchanged q 3 days
for ~2 weeks (D1/D4/D7/D11/D14)
Supernatant frozen for batched p24 ELISA (= qPCR of HIVRNA)
Fletcher et al AIDS 2006
A PHASE 1 SAFETY AND ACCEPTABILITY
STUDY OF THE UC-781 MICROBICIDE GEL
APPLIED RECTALLY
IN HIV SERONEGATIVE ADULTS:
RMP-01
P Anton, T Saunders, A Adler, C Siboliban, E Khanukhova, C Price,
J Elliott, K Tanner, Ana Ventuneac, Alex Carballo-Dieguez, J Boscardin, Y Zhao,
W Cumberland, AM Corner, C Mauck, I McGowan
UCLA, NIH, CONRAD
submitted
Rectal Biopsy Infections ex vivo: RMP-01
 Samples acquired endoscopically (large-cup forceps): 14 biopsies at
each site (10cm and 30cm)
 NO DRUG ADDED TO EXPLANTS (except at V2): all drug applied in vivo
 To laboratory and set up within 2 hours
 HIV (pre-determined strain, titers) applied to explants, incubated 2 hrs
 Biopsies washed (>3-5 times), then mounted on gelfoam, placed in well
of 24-well plate; incubated for 12-14 days.
 Controls: media (uninfected control); UC781 at baseline visit only to
demonstrate in vitro efficacy
 Supernatants for p24 taken every 3 days (100%); each time point is
mean of 2 biopsies pooled; cumulative p24 graphed
NIH IP/CP
Confidence from RMP-01 UC781 RM trial (n=36; 3 arms)
(V2 = baseline) (V3 = single topical application UC781)
V2
UC781 0.10%
UC781 0.25%
22000
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
22000
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
V3
Cumulative P-24 at Day 14
(pg/ml)
in vivo
exposed
22000
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
Cumulative P-24 at Day 14
(pg/ml)
10cm
Cumulative P-24 at Day 14
(pg/ml)
HEC Placebo
V2
22000
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
V2
V3
Visit
22000
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
V2
V3
Visit
V3
Visit
Cumulative P-24 at Day 14
(pg/ml)
30cm
V2
V3
Visit
Cumulative P-24 at Day 14
(pg/ml)
ex vivo
infected
Cumulative P-24 at Day 14
(pg/ml)
Visit
Bx
infection
data
(V2 vs V3)
22000
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
104
virus titer
V2
V3
Visit
NIH IP/CP
RMP-01 UC781 RM trial: now 102 virus titer
6000
5000
4000
3000
2000
1000
0
V2
UC781 0.10%
UC781 0.25%
7000
7000
(ID=410)
6000
5000
4000
3000
2000
1000
0
V3
V2
6000
5000
4000
3000
2000
1000
0
V3
Visit
5000
Baseline
variability
looks
same….
4000
3000
2000
1000
0
V3
V2
V3
but only
60%
infected
with this
titer
Visit
7000
7000
Cumulative P-24 at Day 14
(pg/ml)
7000
V2
6000
Visit
Cumulative P-24 at Day 14
(pg/ml)
Cumulative P-24 at Day 14
(pg/ml)
Visit
30cm
Cumulative P-24 at Day 14
(pg/ml)
7000
Cumulative P-24 at Day 14
(pg/ml)
10cm
Cumulative P-24 at Day 14
(pg/ml)
HEC Placebo
6000
5000
4000
3000
2000
1000
0
V2
6000
5000
4000
3000
2000
1000
V3
Visit
0
V2
V is it
V3
(V2 = baseline) (V3 = single topical application UC781)
NIH IP/CP
Lessons learned thus far for these types of trials:
Do infectibility results from 30cm and 10cm differ: NO
Need infectibility of ‘all’ baseline sample explants: YES
Baseline infectibility
Compare 10cm vs. 30cm for all subjects at baseline (V2)
Paired t-test
102 viral dose
0.8600
104 viral dose
0.5228
No difference seen when using 36 subjects’ baseline data:
“OK” to use 1 site in next trial
Use higher titer virus for explant infection studies
NIH IP/CP
DAIDS IPCP-HTM
RMP-02/MTN-006: A Phase 1, Placebo-Controlled Trial
of Rectally Applied 1%Vaginal Tenofovir Gel with
Comparison to Oral Tenofovir Disoproxil Fumarate
P Anton1, R Cranston3, A Carballo-Dieguez4, A Kashuba5,
E Khanukhova1, J Elliott1, L Janocko6,8, N Richardson-Harman7,
W Cumberland9, C Mauck10, C Hendrix2,8, I McGowan6,8
UCLA: Dept. of Medicine1 & School of Public Health9, Johns Hopkins University2,
University of Pittsburgh: Dept. of Medicine3 & Magee-Womens Research Institute6,
Columbia University4, UNC CFAR & School of Pharmacy5, Alpha StatConsult LLC7,
MTN8, CONRAD10
Presented: CROI 2011, Boston, MA
Study Rationale
 Rectal intercourse is commonly practiced by men and women
 HIV transmission during receptive anal intercourse (RAI) is
significantly greater per sexual act
 CAPRISA 004 demonstrated 39% reduction in HIV infection
using 1% tenofovir gel formulated for vaginal use
 Given the prevalence of RAI and the success of this agent,
this Phase 1 trial aimed to evaluate safety of the hyperosmolar, vaginally formulated 1% tenofovir gel when used
rectally; in addition, aims were to investigate acceptability,
mucosal injury and multi-compartment PK
RMP-02/MTN-006 Study Design
 3 stage trial at 2 sites (UCLA/MWRI): open label TDF (oral) followed by 2:1
randomization of tenofovir: HEC placebo (for single rectal topical dose; 7-day rectal
topical dosing)
 Each dosing stage with 2 weeks of sampling (then: 2 weeks wash-out/healing)
 Product: Tenofovir Disoproxil Fumarate (TDF) 300mg tablet, Tenofovir 1% gel
(vaginal formulation/applicator) or HEC placebo gel
 8.5 months; 3.5 months/participant
 all 18 enrolled → completed. 100% retention (78% male; 22% female)
Safety, PK / PD, acceptability
Baseline
Evaluation
Open label
Oral tenofovir
(N = 18)
Single
rectal
tenofovir
(N = 18)
2:1
7 Day
Rectal
tenofovir
(N = 18)
2:1
Each participant completed 12 visits with 8 flexible sigmoidoscopies in 3.5 months → ~2300 bx
>10,000 study samples
DAIDS IPCP-HTM
Subject’s Samples Workflow: ‘chain of custody’
Clinical Unit
Blood
UCLA/MG
Clin Lab
(Safety labs,
HIV, RPR
HSV 1 & 2)
Urine
Magee
NAAT
GC/CT
UCLA/MG
Plasma
PBMC
Vaginal
Sponge
Rectal
Swabs
UCLA/MG
Cytokines
UCLA/MG
UCLA/MG
Beta HcG &
U/A
Rectal
Sponges
Magee NAAT
Stool
Sample
Rectal
Lavages
UCLA/MG
UCLA/MG
Biopsies
UCLA/MG
(Sloughing Assay)
MTN
PK Lab
GC, CT
MTN Micro
(rectal
microflora)
MTN
PK Lab
Magee-WRI
(gram stain,pH)
MTN
PK Lab
Genova
UCAL/MG
(calprotectin)
(Explant Culture,
Immunophenotyping
PK processing
MTN
PK Lab
UCLA
Research
Pathology
(Qualitative)
Study Endpoints
Primary Objective: Safety of 1% vaginally-formulated tenofovir gel, applied rectally
Endpoint: ≥ Grade 2 AE
Secondary Objectives: Acceptability, Mucosal Immunotoxicity, PK
Endpoints - Acceptability:
- % of those at last visit liking product
- likely to use the candidate in the future if helps
Endpoints - Mucosal Injury: - fecal calprotectin
- rectal microflora
- secreted rectal cytokines
- rectal epithelial sloughing
- rectal histology
- rectal mucosal CD4+ T cell phenotype/activation
Endpoints - PK: Tenofovir/diphosphate concentrations (9-10 compartments):
• Blood: plasma, PBMC, PBMC subsets (CD4+/CD4-)
• Fluids: rectal fluid, vaginal fluid (sponges)
• Tissue: Whole biopsy homogenates, isolated mucosal mononuclear cells
(MMC) & CD4+/CD- subsets
Exploratory Objective: ex vivo infectibility of in vivo exposed rectal tissue biopsies
Endpoint: cumulative HIV-1 p24 levels in colorectal explant supernatants at 14d
Safety
All Adverse Events
All Adverse
Events
n
ORAL SD
RECTAL SD
Placebo
RECTAL SD
Tenofovir
RECTAL 7-D
Placebo
RECTAL 7-D
Tenofovir
GI System Events
Subjects
Events
Subjects
Events
18
15
37
7
15
6
5
13
3
9
12
8
17
6
10
6
5
9
2
6
12
12
47
12
37
p=0.10
p=0.002
p=0.001
5
2
p=0.002
Clinical Grade 3 Events
RECTAL 7-D
Tenofovir
12
2
5
Acceptability
75% likely to use
TFV in the future
if they felt it
might be helpful,
despite relatively
more dislike and
discomfort.
Mucosal Injury Indices
single
ORAL
single
RECTAL
7-day
RECTAL
(compared
to baseline)
(between
groups)
(between
groups)
no difference
no difference
no difference
ND
ND
no difference
no difference
no difference
p=0.01
no difference
no difference
no difference
no difference
no difference
no difference
no difference
no difference
no difference
no difference
no difference
no difference
no difference
no difference
no difference
epithelial sloughing
histology
mucosal lymphocytes
no difference
no difference
no difference
no difference
no difference
no difference
CD3, CD4or CD8 on CD45
CD38 or CD38 RFI on CD4
CCR5 or CCR5 RFI on CD4
HLA-DR on CD4
HLA-DR & CD38 on CD4
ND=not done
no difference
no difference
no difference
no difference
no difference
no difference
no difference
no difference
no difference
no difference
no difference
no difference
no difference
no difference
no difference
Mucosal Index
fecal calprotectin
rectal microflora
secreted rectal cytokines
IL-1b
IL-6
IL-12
INFg
TNFa
MIP-1a
RANTES
p=0.03
no difference
no difference
DAIDS IPCP-HTM
PK: TFV
Exposure
in Plasma
Tenofovir
Plasma Concentration
median (IQR)
1000
Tenofovir Concentration (ng/mL)
Oral Dose
Single Rectal Dose
Multiple Rectal Dose
100
10
1
0.1
0.01
0
4
8
12
16
20
24
Time Post-Dose (h)
 With RECTAL dosing: plasma TFV Cmax & AUC: 2% of ORAL
 ‘7-day’ RECTAL dosing: no TFV accumulation in plasma
PK:TFVTFV-DP
in Comparison
Rectal~30min
Tissue
DP Homogenate
Post-Dose
(30 minutes post dose)
105
TVF DP Tissue Homogenate
Concentration (fmol/mg)
7/18
10/12
12/12
104
103
102
101
100
Oral Dose
Single Rectal Dose
Multiple Rectal Dose
Single ORAL: (i)
TFV DP Tmax ≥ 24h7/18
post-dose. (ii) At 10/12
10 days, TFV DP detectable
in 55% subjects
N detectable
12/12
Single RECTAL dose: (i) Tissue TFV DP Cmax was 112x > single ORAL and AUCall was 1.5x > single
ORAL. (ii) At 10 days, TFV DP detectable in 80%
‘7-day’ RECTAL dosing: Tissue TFV DP accumulated: Cmax was 5x > single RECTAL dose
Biopsy Infection ex vivo of 7-day RECTAL dosing in vivo:
Significant suppression seen
(HIV-1BaL TCID50=104 at ~10 cm)
effect size: 0.80
ANCOVA p=0.005
Single ORAL dosing: no significant changes (p=0.65)
Single RECTAL dosing: no significant changes (p=0.12)
Dose-Response Relationship:
rectal tissue TFV DP with rectal biopsy infectibility
Oral Dose
Single Rectal Dose
Multiple Rectal Dose
Cumulative p24 (pg/mL)
15000
r2 = 0.33
P = 0.0011
10000
5000
0
0
1
2
3
Log10 [Tissue TFV-DP ]fmol/mg
4
• Virus inhibition correlated with increasing tissue TFV-DP, even with small study n
• Feasible to assess both dose and response following in vivo exposure to drug
CONCLUSIONS
 The vaginal formulation of 1% TFV gel was sub-optimal for clinical
safety and acceptability when rectally applied.
 Despite extensive mucosal indices of injury, none were seen with
product use.
 Consistent with other studies, the systemic absorption of
rectally-delivered TFV was ~2% of oral dose.
 Rectal dosing was associated with 100x more active TFV-DP in the
target mucosa than oral dose.
 Rectal tissue biopsy infection ex vivo was significantly reduced
compared to control; may be a potential biomarker of efficacy.
 PK/PD: Dose/response correlations were evaluable and significant
in this intensive small trial.
Acknowledgments
 U19 Integrated Preclinical-Clinical Program for HIV Topical Microbicides (IPCP-HTM)
grant funded by DAIDS, NIAID, NIH grant (AI060614)
 Participants
 Study Teams at each site:
UCLA
MWRI, University of Pittsburgh
 NIH/DAIDS
 MTN Network Support
 MTN Microbiology Laboratory
 MTN Clinical Pharmacology Analytical Lab at JHU
 CONRAD
 Gilead
 Alpha StatConsult LLC
 Support from MTN: funded by NIAID (5U01AI068633), NICHD, NIMH, all of NIH.
DAIDS IPCP-HTM
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