pharmacokinetics of plasma lamivudine(3tc), and its active...

Presented at the 12th Int. Workshop on Clin. Pharmacology of HIV Therapy, 13-15 April 2011, Miami, Fl, USA

Pharmacokinetics of plasma lamivudine (3TC), and its active intracellular anabolite 3TC‐triphosphate (3TC‐TP) over a 24 hour dosing interval following administration of 3TC 300 mg and 150 mg once daily (od) to HIV‐negative healthy volunteers.

The ENCORE 2 Study.

Else LJ1, Jackson A3, Puls R4, Hill A1, Fahey P4, Lin E4, Amara A2, Siccardi M1, Tjia J1, Emery S4, Khoo S1, Back DJ1 and Boffito M3

1 Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool, UK.2 NIHR Biomedical Research Centre, Royal Liverpool Hospital Trust, Liverpool, UK3 St Stephen’s Centre, Chelsea and Westminster Hospital, London, UK.4 National Centre in HIV Epidemiology and Clinical Research, University of New South Wales, Sydney, Australia.


• Lamivudine (3TC)‐ cytidine nucleoside analog converted intracellularly (3‐step phosphorylation) to active triphosphate form (3TC‐TP)‐ correlation between intracellular [3TC‐TP] exposure and efficacy1

• PD: no strong evidence for dose‐related differences in efficacy ‐ (300mg vs 150mg bd)2‐3 & (150mg bd vs. 300mg od)4‐ saturation of phosphorylation pathways at higher doses?

• PK: 3TC‐TP exposures equivalent when administered at 150mg bd & 300mg oddoses5

• OBJECTIVE (ENCORE 2): determine the pharmacokinetics, safety and tolerability of plasma 3TC and 3TC‐TP over a 24 hour dosing interval following administration of 300 mg and 150 mg once daily to HIV‐negative healthy volunteers in a randomised crossover design.

Background:Rationale

1Fletcher et al; AIDS 2000; 2Eron et al NEJM 1995; 3Barlett et al Ann Int Med 1996; 4DeJesus et al CID 2004; 5Yuen et al Antimicrob Agents Chemothr 2004


• open label, randomized, cross‐over pharmacokinetic (PK) study

• 24 healthy volunteers, randomised (1:1) into 2 groups

• Participant demographics, clinical data, concomitant medications, adverse events recorded

• plasma 3TC & intracellular 3TC‐TP PK sampling: Day 10 & Day 30 (*0, 1, 2, 4, 8, 12, 24 h)

3TC 150 mg OD 3TC 300 mg OD10 day washout

days1 10 20 30

PK sampling* PK sampling*

3TC 300 mg OD 3TC 150 mg OD10 day washout

PK sampling* PK sampling*

SCREENING

‐28 0

Randomized (1:1)

Arm 1:

Arm 2:

Background:ENCORE 2 Design


Bioanalytical Methods:Plasma 3TC quantification

Calibrators/QC: drug‐free plasma spiked with working 3TC solutions (range 5‐5000 ng/mL)

Clinical samples: ~ 1 mL plasma obtained (4 mL EDTA tube)

Sample pre‐treatment (all samples): 300 µL (plasma sample) + 300 µL formic acid (0.02%)IS (2‐chloroadenosine; 20 µL) added


Bioanalytical Methods:Plasma 3TC quantification

Calibrators/QC: drug‐free plasma spiked with working 3TC solutions (range 5‐5000 ng/mL)

Clinical samples: ~ 1 mL plasma obtained (4 mL EDTA tube)

Sample pre‐treatment (all samples): 300 µL (plasma sample) + 300 µL formic acid (0.02%)IS (2‐chloroadenosine; 20 µL) added

Analytes eluted via reverse phase (Synergi; polar C18) chromatography

Quantification performed via HPLC‐MS/MS(MRM; positive ionisation mode)

3TC extracted by solid phase extraction (SPE C18; bond‐eluent)‐ evaporated to dryness ‐ reconstituted in mobile phase (100 µL)


Bioanalytical Methods:Intracellular 3TC‐TP quantification

Calibrators/QC: drug‐free PBMCs (~4*106 cells) prepared via Ficoll gradient centrifugation

Clinical samples: PBMCs prepared via CPT tubes (cell count recorded)

Cells re‐suspended in 70% methanol & dispatched in 1mL fractions(stored at ‐80°C)

Calibrators/QC: PBMC samples spiked with working 3TC‐TP solutions (range 0.5‐100 ng/sample)


Bioanalytical Methods:Intracellular 3TC‐TP quantification

Calibrators/QC: drug‐free PBMCs (~4*106 cells) prepared via Ficoll gradient centrifugation

Clinical samples: PBMCs prepared via CPT tubes (cell count recorded)

Cells re‐suspended in 70% methanol & dispatched in 1mL fractions(stored at ‐80°C)

Calibrators/QC: PBMC samples spiked with working 3TC‐TP solutions (range 0.5‐100 ng/sample)

Sample pre‐treatment (all samples): 1 mL ACN & IS (2‐chloroadenosine triphosphate; 20 µL) added samples centrifuged (13,000rpm), evaporated to dryness re‐suspended in mobile phase (100 uL)

Analytes eluted via reverse phase (Fortis C18) ion‐pair (N,N‐DMHA) chromatography

Quantification performed via HPLC‐MS/MS(MRM; negative ionisation mode)


3 4 5 6 7 8 9 10Time (min)

0

50

1000

50

1000

50

1000

50

1000

50

100

Rel

ativ

e A

bund

ance

0

50

1000

50

1000

50

100

NL: 3.83E3TIC F: - c ESI SRM ms2 465.973 [158.860-158.870, 368.098-368.108]


NL: 3.26E3TIC F: - c ESI SRM ms2 467.991 [159.053-159.063, 370.306-370.316] MS





TIC

dCTP

3TC-TP

3TC-TP (clinical sample: 12.2 * 106 cells)

dTTP

dATP

dGTP / ATP

2Cl-ATP (IS)

Chromatogram: 3TC‐TP, IS and endogenous dNTPs


• PK parameters (AUC24, C24, Cmax) calculated by non‐compartmental analysis (WinNonlin®), expressed as unadjusted geometric means (90%CI)

• Within‐subject changes expressed by geometric mean ratios (GMR, 90% CI; adjusted for study arm, period and intra‐individual variation)‐ Linear mixed effects modelling

• Results were considered statistically significant at <0.05 or if 90% CI of GMR ratios excluded 1 or 0

• Regimens were considered bioequivalent if 90% CI for the GMR fell within the acceptance range of 0.8‐1.25.

Methods:Pharmacokinetic and statistical analyses


Results:Baseline characteristics and lipids (screening and day 37‐44 follow up)

N (%) Median (range)

Sex, female 13 (54) ‐

Ethnicity‐Caucasian‐Black‐Oriental‐Other

16 (67)4 (17)1 (4)3 (13)

‐‐‐

Age, years 24 33.5 (27.0‐57.0)

Weight, kg 24 72.2 (48.8‐109.6)

BMI, kg/m2 24 23.7 (19.5‐34.7)

Total Cholesterol, mmol/L

‐ Screening 24 5.25 (3.7‐7.2)

‐ Follow up 24 4.80 (3.6‐7.1)

Triglycerides, mmol/L

‐ Screening 24 1.28 (0.53‐3.2)

‐ Follow up 24 0.87 (0.47‐2.4)

• No serious adverse events related to study medication were recorded


Results:Plasma 3TC pharmacokinetic data (geometric mean; 90% CI)

0

200

400

600

800

1,000

1,200

0 4 8 12 16 20 24

3TC

pla

sma

(ng/

mL)

Time Post Dose (h)

Phases 1&2 (300 & 150 mg)

300 mg; n=24

150 mg; n=24


Results:Plasma 3TC pharmacokinetic data (geometric mean; 90% CI)

0

200

400

600

800

1,000

1,200

0 4 8 12 16 20 24

3TC

pla

sma

(ng/

mL)

Time Post Dose (h)

Phases 1&2 (300 & 150 mg)

300 mg; n=24

150 mg; n=24

Cmax↓44%

AUC0‐12↓43%

C24 ↓37%


Results:Plasma 3TC pharmacokinetic parameters: unadjusted geometric means, full mixed model (fitting dose, arm, period with adjustment for cross‐over) and basic mixed model (fitting dose with adjustment for cross‐over).

Full mixed model Basic mixed model

Dose Arm Period Dose

Dose (OD) Parameter Geometric mean(90% CI) GMR (90% CI) P value P value P value GMR (90% CI)

150 mg AUC0‐24ng.h/mL

4773 (4408, 5169)0.57 (0.55, 0.60) <0.001 0.002 0.277 0.57 (0.56, 0.59)

300 mg 8354 (7609, 9172)

150 mg C24ng/mL

38.1 (34.0, 42.7)0.63 (0.59, 0.67) <0.001 0.105 0.955 0.63 (0.59, 0.67)

300 mg 60.8 (53.4, 69.2)

150 mg Cmaxng/mL

757 (688, 833)0.56 (0.53, 0.60) <0.001 0.019 0.558 0.56 (0.53, 0.60)

300 mg 1344 (1247, 1448)







4773 (4408, 5169)0.57 (0.55, 0.60) <0.001 0.002 0.277 0.57 (0.56, 0.59)

300 mg 8354 (7609, 9172)

150 mg C24ng/mL

38.1 (34.0, 42.7)0.63 (0.59, 0.67) <0.001 0.105 0.955 0.63 (0.59, 0.67)

300 mg 60.8 (53.4, 69.2)

150 mg Cmaxng/mL

757 (688, 833)0.56 (0.53, 0.60) <0.001 0.019 0.558 0.56 (0.53, 0.60)

300 mg 1344 (1247, 1448)

• ~43% reduction in 3TC plasma exposure with 150 mg od regimen (50% dose reduction)

• None of the 3TC plasma PK parameters meet the criteria for bioequivalence







4773 (4408, 5169)0.57 (0.55, 0.60) <0.001 0.002 0.277 0.57 (0.56, 0.59)

300 mg 8354 (7609, 9172)

150 mg C24ng/mL

38.1 (34.0, 42.7)0.63 (0.59, 0.67) <0.001 0.105 0.955 0.63 (0.59, 0.67)

300 mg 60.8 (53.4, 69.2)

150 mg Cmaxng/mL

757 (688, 833)0.56 (0.53, 0.60) <0.001 0.019 0.558 0.56 (0.53, 0.60)

300 mg 1344 (1247, 1448)

• ~43% reduction in 3TC plasma exposure with 150 mg od regimen (50% dose reduction)

• None of the 3TC plasma PK parameters meet the criteria for bioequivalence• Significant differences between study arms (Arm 1 > Arm 2)


Results:Intracellular 3TC‐TP pharmacokinetic data (geometric mean; 90% CI)

0.00

1.00

2.00

3.00

4.00

5.00

0 4 8 12 16 20 24

300 mg; n=24

150 mg; n=24

3TC

-TP

intr

acel

lula

r (pm

ol/1

06ce

lls)

Time Post Dose (h)

Phases 1&2 (300 & 150 mg)


Results:Intracellular 3TC‐TP pharmacokinetic data (geometric mean; 90% CI)

0.00

1.00

2.00

3.00

4.00

5.00

0 4 8 12 16 20 24

300 mg; n=24

150 mg; n=24

3TC

-TP

intr

acel

lula

r (pm

ol/1

06ce

lls)

Time Post Dose (h)

Phases 1&2 (300 & 150 mg)

Cmax↓30%

AUC0‐12↓27%

C24 ↓18%


Results:Intracellular 3TC‐TP pharmacokinetic parameters: unadjusted geometric means, full mixed model (fitting dose, arm, period with adjustment for cross‐over) and basic mixed model (fitting dose with adjustment for cross‐over).




150 mg AUC0‐24pmol.h/106 cells

44.0 (38.0‐51.0)0.73 (0.64, 0.83) <0.001 0.637 0.008 0.74 (0.64, 0.86)

300 mg 59.5 (51.8‐68.3)

150 mg C24pmol/106 cells

1.23 (1.00, 1.52)0.82 (0.68, 0.99) 0.083 0.404 0.246 0.83 (0.68, 1.01)

300 mg 1.49 (1.19, 1.86)

150 mg Cmaxpmol/106 cells

2.95 (2.47, 3.51)0.70 (0.61, 0.82) <0.001 0.981 0.008 0.72 (0.61, 0.85)

300 mg 4.10 (3.59, 4.69)







44.0 (38.0‐51.0)0.73 (0.64, 0.83) <0.001 0.637 0.008 0.74 (0.64, 0.86)

300 mg 59.5 (51.8‐68.3)


1.23 (1.00, 1.52)0.82 (0.68, 0.99) 0.083 0.404 0.246 0.83 (0.68, 1.01)

300 mg 1.49 (1.19, 1.86)


2.95 (2.47, 3.51)0.70 (0.61, 0.82) <0.001 0.981 0.008 0.72 (0.61, 0.85)

300 mg 4.10 (3.59, 4.69)

• ~27% reduction in intracellular 3TC‐TP exposure with 150 mg od regimen (50% dose reduction)

• None of the 3TC‐TP intracellular PK parameters meet the criteria for bioequivalence







44.0 (38.0‐51.0)0.73 (0.64, 0.83) <0.001 0.637 0.008 0.74 (0.64, 0.86)

300 mg 59.5 (51.8‐68.3)


1.23 (1.00, 1.52)0.82 (0.68, 0.99) 0.083 0.404 0.246 0.83 (0.68, 1.01)

300 mg 1.49 (1.19, 1.86)


2.95 (2.47, 3.51)0.70 (0.61, 0.82) <0.001 0.981 0.008 0.72 (0.61, 0.85)

300 mg 4.10 (3.59, 4.69)

• ~27% reduction in intracellular 3TC‐TP exposure with 150 mg od regimen (50% dose reduction)

• None of the 3TC‐TP intracellular PK parameters meet the criteria for bioequivalence• Significant effect of study period (Day 10 vs. Day 30)


Results:Intracellular 3TC‐TP pharmacokinetic parameters (AUC0‐24): unadjusted geometric means, mixed model (stratified by Study Arm).

Mixed model

Period

Dose (OD) Dose (OD) Parameter Geometric mean(90% CI) GMR (90% CI) P value

Arm 1150 mg AUC0‐24

pmol.h/106 cells47.2 (38.6, 57.8)

0.90 (0.75, 1.09) 0.374300 mg 52.3 (41.1, 66.5)


pmol.h/106 cells40.5 (31.8, 51.5)

1.71 (1.41, 2.07) <0.001300 mg 69.2 (63.2, 75.8)



Mixed model

Period



pmol.h/106 cells47.2 (38.6, 57.8)

0.90 (0.75, 1.09) 0.374300 mg 52.3 (41.1, 66.5)


pmol.h/106 cells40.5 (31.8, 51.5)

1.71 (1.41, 2.07) <0.001300 mg 69.2 (63.2, 75.8)

• Arm 1: (300 mg → 150 mg) no significant differences



Mixed model

Period



pmol.h/106 cells47.2 (38.6, 57.8)

0.90 (0.75, 1.09) 0.374300 mg 52.3 (41.1, 66.5)


pmol.h/106 cells40.5 (31.8, 51.5)

1.71 (1.41, 2.07) <0.001300 mg 69.2 (63.2, 75.8)

• Arm 1: (300 mg → 150 mg) no significant differences • Arm 2: (150 mg → 300 mg) highly significant differences



Mixed model

Period



pmol.h/106 cells47.2 (38.6, 57.8)

0.90 (0.75, 1.09) 0.374300 mg 52.3 (41.1, 66.5)


pmol.h/106 cells40.5 (31.8, 51.5)

1.71 (1.41, 2.07) <0.001300 mg 69.2 (63.2, 75.8)

• Arm 1: (300 mg → 150 mg) no significant differences • Arm 2: (150 mg → 300 mg) highly significant differences

• Consistent with a combined DOSE and PERIOD effect


Results:Geometric mean 3TC‐TP (AUC0‐24): stratified by dose and study arm

Stratified by Arm:

‐ Arm 1: reduction in dose cancels the effect of period

‐ Arm 2: ↑ due to combined dose/period effects

3TC

-TP

(pm

ol.h

/106

cells

)

Stratified by dose: ‐ clear dose and period effect

‐ ~40% increase attributable to dose

‐ ~30% increase attributable to period

0

10

20

30

40

50

60

70

80

300 mg 150 mg 150 mg 300 mg

ARM 1 ARM 2

0

10

20

30

40

50

60

70

80

Day 10 Day 30 Day 10 Day 30

150 mg 300 mg


Conclusions:

• Mean plasma 3TC concentrations:‐ significantly higher for the 300 mg vs. 150 mg dose‐ significant differences between study arms

• Mean intracellular 3TC‐TP concentrations:‐ significantly higher for the 300 mg vs. 150 mg dose‐ significantly higher at Day 30 vs. Day 10 (period effect)‐ no prospect of an interaction/order effect

• For key plasma/intracellular pharmacokinetic parameters: 3TC at 150 mg is not bioequivalent to the standard regimen of 300 mg od

‐ indicating that saturation of cytosine phosphorylation pathways is not achieved at a dose of 150 mg od.


Acknowledgments:

Funder: Bill and Melinda Gates Foundation Principal Contact: Dr. Stephen Becker

Sponsor: National Centre in HIV Epidemiology and Clinical Research (NCHECR) University of New South Wales, Sydney, Australia

Principal Investigator: Dr. Marta BoffitoSt Stephen’s Centre Chelsea and Westminster Hospital, London

pharmacokinetics of plasma lamivudine(3tc), and its active...

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