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: PIIan McGowan, MWRI/University of Pittsburgh: co-PI

Ian McGowan, MWRI/University of Pittsburgh: PI

NIH/NIAID: 2nd IPCP on Rectal Microbicides Developments (2009-2014)

NIH IP/CP

Outline

Intent

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)

context

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-of-range” 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. HIV-/RAI-II. HIV-/RAI+III. HIV+/RAI+: high PVLIV. 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(log10)

4.1 4.5 4.9 4.1 0.28 0.49 0.75

IFN-(log10)

2.7 3.2 3.3 3.0 0.29 0.50 0.74

IL-10(log10)

2.7 3.2 2.6 2.8 0.28 0.43 0.70

McGowan et al, HPTN 056 JAIDS 2007

ICC: Intra-class correlation >.75 shows strong stability

Colorectal (10cm)

Colonic (30cm)

Colorectal (higher than 10cm)

Upper intestinal tract (systemic delivery to targets)

Where to sample…”explants”

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

X

X

Colorectal: Biopsy location

NIH IP/CP

Colorectal Biopsy safety and location

NIH IP/CP

Success

Con

cent

rati

on

Ano-Rectal Distance

Anus

Failure

HIVHIV

Rectum Colon

MicrobicideMicrobicide

RM Clinical Relevance: Selection Site for Explants

Diagram courtesy: C Hendrix)

~10cm

+ effect?

~30cm

no effect?

Rectal Explant Model used in RM Clinical Trials:“ex vivo biopsy infection” experiments

Explant media soaked collagen rafts

b)1 cm2 raft placed atop the explant and inverted.

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 24-well plate containing 500ul of explant medium

Media 100% exchanged q 3 days for ~2 weeks (D1/D4/D7/D11/D14) Supernatant frozen for batched p-24 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

NIH IP/CP

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

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HEC Placebo UC781 0.10% UC781 0.25%

10cm

30cm

NIH IP/CP

(V2 = baseline) (V3 = single topical application UC781)

Confidence from RMP-01 UC781 RM trial (n=36; 3 arms)

in vivo exposed

ex vivo infected

Bx infection

data (V2 vs V3)

104 virus titer

NIH IP/CP

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HEC Placebo UC781 0.10% UC781 0.25%

10cm

30cm

Baseline variability

looks same….

but only 60%

infected with this

titer

(V2 = baseline) (V3 = single topical application UC781)

RMP-01 UC781 RM trial: now 102 virus titer

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

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

Baseline infectibility

NIH IP/CP

Use higher titer virus for explant infection studies

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

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 hyper-osmolar, vaginally formulated 1% tenofovir gel when

used rectally; in addition, aims were to investigate acceptability, mucosal injury and multi-compartment PK

Study Rationale

BaselineEvaluation

Open labelOral tenofovir

(N = 18)

Single rectal

tenofovir(N = 18)

2:1

7 DayRectal

tenofovir(N = 18)

2:1

Safety, PK / PD, acceptability

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)

Each participant completed 12 visits with 8 flexible sigmoidoscopies in 3.5 months → ~2300 bx>10,000 study samples

Clinical Unit

UCLA/MG Clin Lab

(Safety labs, HIV, RPRHSV 1 & 2)

Rectal Swabs

Blood Rectal Sponges

Stool Sample

Biopsies

UCLA/MGPlasma PBMC

MTNPK Lab

Magee NAATGC, CT

UCLA/MGCytokines

UCLA/MG

Genova (calprotectin)

Rectal Lavages

UCLA/MG(Sloughing Assay)

UCLA/MG

UCAL/MG (Explant Culture,

ImmunophenotypingPK processing

UCLAResearch Pathology

(Qualitative)

MTN Micro(rectal

microflora)

MTNPK Lab

VaginalSponge

UCLA/MG

MTNPK Lab

Magee-WRI(gram stain,pH)

Urine

MageeNAAT GC/CT

MTNPK Lab

UCLA/MGBeta HcG &

U/A

Subject’s Samples Workflow: ‘chain of custody’

DAIDS IPCP-HTM

Primary Objective: Safety of 1% vaginally-formulated tenofovir gel, applied rectally Endpoint: ≥ Grade 2 AE

Secondary Objectives: Acceptability, Mucosal Immunotoxicity, PKEndpoints - Acceptability: - % of those at last visit liking product

- likely to use the candidate in the future if helpsEndpoints - Mucosal Injury: - fecal calprotectin

- rectal microflora- secreted rectal cytokines- rectal epithelial sloughing- rectal histology- rectal mucosal CD4+ T cell

phenotype/activationEndpoints - 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

Study Endpoints

All Adverse Events

n

All Adverse Events GI System Events

Subjects Events Subjects Events

ORAL SD 18 15 37 7 15

RECTAL SDPlacebo

6 5 13 3 9

RECTAL SDTenofovir

12 8 17 6 10

RECTAL 7-DPlacebo

6 5 9 2 6

RECTAL 7-DTenofovir

12 12 47 12 37

Safety

p=0.10 p=0.002 p=0.001 p=0.002

RECTAL 7-DTenofovir

12 2 5 2 5

Clinical Grade 3 Events

Acceptability

75% likely to use TFV in the future

if they felt it might be helpful, despite relatively more dislike and

discomfort.

DAIDS IPCP-HTM

Mucosal Injury Indicessingle ORAL

(compared to baseline)

single RECTAL

(between groups)

7-day RECTAL

(between groups)

fecal calprotectin no difference no difference no difference

rectal microflora ND ND no difference

secreted rectal cytokines IL-1b no difference no difference p=0.01 IL-6 no difference no difference no difference

IL-12 no difference no difference no difference

INF no difference no difference no difference

TNFa no difference no difference p=0.03 MIP-1a no difference no difference no difference

RANTES no difference no difference no difference

epithelial sloughing no difference no difference no difference

histology no difference no difference no difference

mucosal lymphocytes CD3, CD4or CD8 on CD45 no difference no difference no difference

CD38 or CD38 RFI on CD4 no difference no difference no difference

CCR5 or CCR5 RFI on CD4 no difference no difference no difference

HLA-DR on CD4 no difference no difference no difference

HLA-DR & CD38 on CD4 no difference no difference no difference

ND=not done

Mucosal Index

With RECTAL dosing: plasma TFV Cmax & AUC: 2% of ORAL ‘7-day’ RECTAL dosing: no TFV accumulation in plasma

PK: TFV Exposure in Plasma

7/18 10/12 12/12

PK: TFV-DP in Rectal Tissue(30 minutes post dose)

Single ORAL: (i) TFV DP Tmax ≥ 24h post-dose. (ii) At 10 days, TFV DP detectable in 55% subjects

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

ANCOVA p=0.005

(HIV-1BaL TCID50=104 at ~10 cm)

effect size: 0.80

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

• 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

0 1 2 3 40

5000

10000

15000

r2 = 0.33P = 0.0011

Oral Dose Single Rectal Dose Multiple Rectal Dose

Log10[Tissue TFV-DP ]fmol/mg

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24 (p

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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

Support from MTN: funded by NIAID (5U01AI068633), NICHD, NIMH, all of NIH.

U19 Integrated Preclinical-Clinical Program for HIV Topical Microbicides (IPCP-HTM) grant funded by DAIDS, NIAID, NIH grant (AI060614)

DAIDS IPCP-HTM

Participants

Study Teams at each site:UCLAMWRI, University of Pittsburgh

NIH/DAIDS MTN Network Support MTN Microbiology Laboratory MTN Clinical Pharmacology Analytical Lab at JHU CONRAD Gilead Alpha StatConsult LLC

DAIDS IPCP-HTM

QUESTIONS ?