Mackenzie L. Cottrell, Katy L. Garrett, Cindi W. Emerson,

Amanda Schauer, Craig Sykes, Angela D.M. Kashuba

University of North CarolinaUNC Eshelman School of Pharmacy

Intracellular Metabolism of Tenofovir

Alafenamide in Cervical and Vaginal

Epithelial Cells

TAF Exhibits Favorable Pharmacology

Adapted from Gupta S. IAS 2015 Oral Abstract #TUAB0103.

HIV TARGET CELL

AMIDATE

ESTER

DIANION

GI TRACT

Tenofovir

alafenamide

(TAF)

Tenofovir

disoproxil

fumarate

(TDF)

Tenofovir

(TFV) Parent

Nucleotide

PLASMA

TAF25 mg

TDF 300 mg

TFV

TFV

TFV

TFV HIVTFVdpT1/2 = 0.4 min

†

T1/2 = 90 min†

TAF Achieves Higher TFVdp in PBMCs

Ruane P. CROI 2012 Oral Abstract #103.

TAF

8mg

TAF

25mg

TAF

40mg

TDF

300mg

TFVdp is Lower in FGT Tissues for TAF

Garrett K. CROI 2016 Oral Abstract #102LB.

Time (hr)

0 12 24 36 48 60 72

TF

V o

r T

FV

dp

(pM

)

103

104

105

●TFV

▼TFVdp

300mg TDF single dose

TFVdp is Lower in FGT Tissues for TAF

Garrett K. CROI 2016 Oral Abstract #102LB.

300mg TDF

Time (hr)

0 12 24 36 48 60 72

TF

V o

r T

FV

dp

(pM

)

103

104

105

300mg TDF single dose 25mg TAF single dose

●TFV

▼TFVdp

TFV 2-fold

TFVdp 1.3-fold

Mucosal Tissue Microanatomy

Adapted from Margolis L. 2006 Nature Reviews Microbiology.

Langerhans Cells

Dendritic Cells

Macrophages

T-Lymphocytes

Epithelial Cells

Mucosal Tissue Microanatomy

Adapted from Margolis L. 2006 Nature Reviews Microbiology.

Langerhans Cells

Dendritic Cells

Macrophages

T-Lymphocytes

Epithelial Cells

Whole blood Ovary Vagina Uterus

Cathepsin A Gene Expression

http://www.gtexportal.org/home/gene/CTSA

Lo

g10

RP

KM

Hypothesis: TFVdp will be

lower in epithelial cells dosed

with TAF vs TFV

In Vitro Approach

0.5 μΜ

PBMC

0.5 μM

Ect1

0.5 μM

VK2

10 μM

PBMC

10 μM

Ect1

10 μM

VK2

0.5 μΜ

PBMC

0.5 μM

Ect1

0.5 μM

VK2

10 μM

PBMC

10 μM

Ect1

10 μM

VK2

TAF TFV

3

hr

12

hr

24

hr

48

hr

72

hr

Cells: Freshly isolated PBMCs (single healthy volunteer*),

Ect1/E6E7 ectocervical and VK2/E6E7 vaginal cell lines (ATCC®)

Treat with:

Harvest at:

Count on: Muse™ Cell Analyzer

Lyse in: 70:30 methanol:water

*UNC Center for AIDS Research Sample Collection Protocol IRB 08-0047

Analytical Methods

LC-MS/MS TFVdp quantification: Calibrated range=0.02-500 ng/ml cell lysate

Statistical Analysis

‒ BLQ values imputed at ½ the LLOQ

‒ Median area under the concentration time curve (AUC) calculated by linear trapezoidal rule with sparse sampling function in WinNonlin v6.3

‒ Pearson’s correlation performed for dose-normalized log10 transformed matched samples with SAS v9.3

TFVdp Median (Min, Max) Concentration

vs Time: TAF (solid)

Time (hour)

0 12 24 36 48 60 72

TF

Vdp(f

mol/m

illio

n c

ells

)

100

101

102

103

104

105

106

PBMC

Time (hour)

0 12 24 36 48 60 72

TF

Vdp (

fmol/m

illio

n c

ells

)

100

101

102

103

104

105

106

PBMC

10μM Dosing 0.5μM Dosing

TFVdp Median (Min, Max) Concentration

vs Time: TAF (solid)

Time (hour)

0 12 24 36 48 60 72

TF

Vdp(f

mol/m

illio

n c

ells

)

100

101

102

103

104

105

106

Ect1

PBMCVK2

0.5μM Dosing

Time (hour)

0 12 24 36 48 60 72

TF

Vdp (

fmol/m

illio

n c

ells

)

100

101

102

103

104

105

106

Ect1

PBMCVK2

10μM Dosing

TFVdp Median (Min, Max) Concentration

vs Time: TAF (solid) & TFV (dashed)

TAF AUC0-72hr 192-1305 fold Higher compared to TFV

Time (hour)

0 12 24 36 48 60 72

TF

Vdp(f

mol/m

illio

n c

ells

)

100

101

102

103

104

105

106

Ect1

PBMCVK2

Time (hour)

0 12 24 36 48 60 72

TF

Vdp (

fmol/m

illio

n c

ells

)

100

101

102

103

104

105

106

Ect1

PBMCVK2

0.5μM Dosing 10μM Dosing

TFVdp AUC0-72hr

Cell Type DoseμM

TAFMean AUC (SE)

pmol*hr/million cells

TFVMean AUC (SE)

pmol*hr/million cells

PBMC0.5 2122 (209) 1.63 (0.173)

10 12646 (702) 33.5 (5.61)

Ect10.5 10037 (989) 26.7 (1.78)

10 210382 (18701) 469 (28.1)

VK20.5 3587 (826) 18.6 (1.60)

10 60604 (35752) 256 (11.1)

TAF: PBMC 1.7-16.6 fold Lower vs Epithelial

TFV: PBMC 7.6-16.4 fold Lower vs Epithelial

TFVdp Rate of Accumulation

0.5μM TAF 0.5μM TFV

Epithelial cells: 6

Times Faster

Epithelial cells: 17-30

Times Faster

Time (hour)

0 3 6 9 12

TF

Vd

p (

fmo

l/1

06

ce

lls)

0

20000

40000

60000

80000

100000

120000

140000

160000

PBMC

VK2

Ect1Rate=11204, r

2=0.97

Rate=10608, r2=0.94

Rate=1772, r2=0.75

Time (hour)

0 3 6 9 12

TF

Vd

p (

fmo

l/1

06 c

ells

)

0

100

200

300

400

500

600

PBMC

VK2

Ect1

Rate=23.61, r2=0.86

Rate=41.60, r2=0.92

Rate=1.383, r2=0.61

TFVdp Correlates Between Cell Types

Pearson’s r Ect1 VK2 PBMC

Ect1 1 0.71† 0.64*

VK2 0.90† 1 0.45*

PBMC 0.58* 0.42* 1

*p<0.05; †p<0.001

PBMC Log10

TFVdp (fmol/106 cells)

3.5 4.0 4.5 5.0 5.5

Ect1

Log

10 T

FV

dp (

fmo

l/1

06

ce

lls)

3.5

4.0

4.5

5.0

5.5

6.0

PBMC Log10

TFVdp (fmol/106 cells)

0.5 1.0 1.5 2.0 2.5 3.0 3.5

Ect1

Log

10 T

FV

dp (

fmol/10

6 c

ells

)

0.5

1.0

1.5

2.0

2.5

3.0

3.5

TFVTAF

Limitations

Static exposure over 72 hours

Immortalized cells exhibit altered

phenotypes − Ect1 TFVdp ~1.7-fold higher compared to

primary ectocervical cells1

1Shen Z. 2014 PlosOne.

Summary and Conclusions

TFVdp was HIGHER with matched TAF vs TFV dosing

− >100-fold AUC0-72hr in PBMCs, Ect1 and VK2 cells

TFVdp was HIGHER in epithelial cells vs PBMCs

− VK2 ≥ 1.7 fold AUC0-72hr

− Ect1 ≥ 4.7 fold AUC0-72hr

TFVdp was significantly correlated among cell types

− Ect1 vs PBMC TFVdp r2=0.58, p<0.05

− VK2 vs PBMCs TFVdp r2=0.42, p<0.05

Presence of epithelial cells within tissue homogenates does

not explain previous observations of low TFVdp

concentrations in tissue biopsies from women dosed with TAF.

This work was supported in part by the Centers for AIDS Research (Grant P30 AI50410)

and the John A. and Deborah S. McNeill Jr. Distinguished Professorship