RESEARCH

Lymphatic Targeting of Tenofovir; Intracellular Pharmacokinetics and Viral

Dynamics

Arnold Fridland, Ph.D, William Lee, Ph.D.

Gilead Sciences, Inc.

2CONFIDENTIALRESEARCH

Tenofovir and Tenofovir DF (Viread)

Tenofovir

• intracellular t 1/2 ~ 50h• once daily dosing• good resistance profile

Viread

• human % F = 40• Approved 11/01

N

NN

N

NH2

O P O-

CH3

O

O-

N

NN

N

NH2

O P O

CH3

O

O

O O

O O

O

O

3CONFIDENTIALRESEARCH

Human PK after IV infusion (1.0 mg/kg) of Tenofovir and PO (300 mg) Tenofovir DF (Viread)

Time Post Dose (Hr)

0 6 1210

100

1000

5000

[Ten

ofo

vir]

(n

g/m

L)

0 12 24 36 4810

100

1000

PO

• No prodrug is observed in plasma following PO

F = 40%

IV

4CONFIDENTIALRESEARCH

Viread – What’s Not to Like?

• Good oral bioavailability > 40%• Excellent efficacy, Viread/3TC/EFV result in > 70%

patient below 50 copies/mL of HIV RNA @ wk 144• Safety comparable to placebo• Available as combination pill w/FTC• Leading selling NRTi

5CONFIDENTIALRESEARCH

Δ HIV RNA (day 7) vs Tenofovir Exposure After Tenofovir (iv) or Viread (po) in Patients

• Prodrug leads to higher intracellular levels of TPP

6CONFIDENTIALRESEARCH

Objectives for 2nd GenerationTenofovir Prodrug

• Increased tissue exposure/decreased renal excretion• Observable plasma levels of prodrug after p.o.• Increased stability in blood compartment• Preferential metabolism in lymphatic tissues

7CONFIDENTIALRESEARCH

In Vitro/In Vivo Criteria for Prodrug Selection

• EC50 ag HIV-1• MT-2 cell extracts• Plasma stability• PLCE

“Mono phenyl isopropylalaninyl mono

amidate - (R,S,S)” GS-7340

N

NN

N

NH2

O P

CH3

O

R3

HNOR2

R1

O

• Dog PK• PBMC levels• Tissue homogenates

N

NN

N

NH2

O P

CH3

OO

HNO

CH3

O

8CONFIDENTIALRESEARCH

Stereochemistry at Phosphorus Determines Metabolic Stability and Antiviral Activity

Tenofovir monoamidate

HIV EC50 (µM)

PLCE t1/2 (min)

MT-2 extract t1/2 (min)

Ala-Et Isomer A

0.005

2.9

2.9

Isomer B 0.05 8.0 151

Aba-Et Isomer A

0.004

3.9

49.2

Isomer B 0.03 11.3 > 1000

O P

O

NH-CH-COOR2

OPh

R1N

N

NH2

N

N

9CONFIDENTIALRESEARCH

In vitro Activity & Metabolic Stability (t1/2) of GS 7340

EC50 (M)

MT-2 Cell Extract (min)

Human Plasma (min)

Extract Plasma (min)

tenofovir 5 - - - tenofovir DF 0.05 70.7 0.41 172 GS 7340 0.005 28.3 90 0.3 GS 7485 (D-ala) 10 > 700 > 200 -

10CONFIDENTIALRESEARCH

HPLC Chromatograms after 1 hr Incubation of GS 7340 in Human Whole Blood

Plasma

PBMC

GS-7340GS-7161

PMPA

PMPA

7161

PMPAp

PMPApp

min

min

11CONFIDENTIALRESEARCH

Plasma and PBMC Profiles of GS 7340 and tenofovir after iv infusion (0.5 mg/kg) and po (4.8 mg/kg) in dogs

1

10

100

1000

10000

100000

0 5 10 15 20 25

Time (hr)

[ ] (

nM

) GS-7340 Plasma

PMPA Plasma

PMPA PBMC

Total PMPA PBMC

FPBMC = 15%

FPLASMA = 23 %

12CONFIDENTIALRESEARCH

Plasma and PBMC AUC0-24 After Tenofovir (s.c.) and Tenofovir Prodrugs (p.o.) in Dogs

1:>100

1:51:1.4

13CONFIDENTIALRESEARCH

Distribution in Dogs After Administration of a Single Oral Dose of [14C]-Tenofovir DF or [14C]-GS-7340

Tissue/Fluid Tenofovir DF GS-7340 Tissue Conc.Ratio of GS-7340

to TDF%

DoseConc.

(ug-eq/g)%

DoseConc.

(ug-eq/g)

Liver 12.40 38.30 16.45 52.94 1.4

Kidney 4.58 87.90 3.78 80.21 0.9

Lungs 0.03 0.53 0.34 4.33 8.2

Iliac Lymph Nodes 0.00 0.51 0.01 5.42 10.6

Axillary Lymph Nodes 0.00 0.37 0.01 5.54 14.8

Inguinal Lymph Nodes 0.00 0.28 0.00 4.12 15.0

Mesenteric Lymph Nodes 0.00 1.20 0.04 6.88 5.7

Thyroid Gland 0.00 0.30 0.00 4.78 15.8

Pituitary Gland 0.00 0.23 0.00 1.80 7.8

Salivary Gland (L+R) 0.00 0.45 0.03 5.54 12.3

Adrenal Gland 0.00 1.90 0.00 3.47 1.8

Spleen 0.00 0.63 0.17 8.13 12.8

Pancrease 0.00 0.57 0.01 3.51 6.2

14CONFIDENTIALRESEARCH

PK/PD Conclusions

• GS-7340-02 produces rapid achievement of high concentrations of GS-7340 in the plasma– Rapid distribution/elimination

• T1/2 ~ 20 - 40 minutes

• Sustained plasma concentrations of tenofovir– Lower tenofovir Cmax concentrations

– AUC ~ proportional to tenofovir dose

– Prolonged plasma T1/2 relative to TDF, greater accumulation to steady-state (150 mg 7340-02 300 mg TDF)

• Improved intracellular distribution of tenofovir to PBMCs– ~ 6 to 20-fold higher concentrations relative to TDF 300 mg

– No correlations between plasma or PBMC tenofovir exposure and antiviral activity