copyright © 2007 merck & co., inc., whitehouse station, new jersey, usa all rights reserved...

Copyright © 2007 Merck & Co., Inc., Whitehouse Station, New Jersey, USA All rights Reserved

Resistance to the HIV-Integrase Inhibitor Raltegravir: Analysis of Protocol 005, a Phase 2 Study in

Patients with Triple-Class Resistant HIV-1 Infection

D.J. Hazuda, M. D. Miller, B. Y. Nguyen and J. Zhao for the P005 Study Team

Merck Research Labs, North Wales PA 19454


N N

HN

OOHO

HN

O

NN

O

F

• Integrase strand transfer inhibitor (IC50 ~ 10 nM)–hydroxypyrimidinone carboxamide

• Antiviral IC95 ~ 31 20 nM (50% NHS)• Active across diverse clinical isolates and HIV-2• Potent antiviral effect in treatment naïve patients

–Phase II 24 week data (Markowitz et. al. IAS 2006)• Robust activity in HIV-1 patients with triple class resistance

– Phase II 24 week data (Grinsztejn et al ICAAC 2006)– Phase III data (Cooper et al & Steigbigel et al CROI 2007)

MK-0518: Raltegravir

+ OBT§ GSS = Genotypic sensitivity score by Phenosense GTGrinsztejn et al. Lancet 2007; 369: 1261-69

PN005 Phase 2 Study in Treatment PN005 Phase 2 Study in Treatment Experienced Patients: Baseline CharacteristicsExperienced Patients: Baseline Characteristics

MK-0518*MK-0518* Placebo*Placebo*

200 mg200 mgN=43N=43

400 mg400 mgN=45N=45

600 mg600 mgN=45N=45 N=45N=45

Median Age (yrs)Median Age (yrs) 4343 4343 4444 4343

MaleMale 84%84% 89%89% 91%91% 89%89%

Mean Mean loglog1010HIV RNAHIV RNA 4.64.6 4.84.8 4.74.7 4.74.7

Mean CD4 Count Mean CD4 Count (/mm(/mm33)) 245245 221221 220220 274274

Median Years of Prior ARTsMedian Years of Prior ARTs 1010 1111 99 1010

OBT: Median # of ARTsOBT: Median # of ARTs 44 44 44 44

GSSGSS§§:: 0 to all ARTs0 to all ARTs 27 (63%)27 (63%) 38 (84%)38 (84%) 35 (78%)35 (78%) 28 (62%)28 (62%)

GSSGSS§§:: 0 to PI0 to PI

# pts with enfuvirtide as new # pts with enfuvirtide as new OBTOBT

38 (88%)38 (88%)

13 (30%)13 (30%)

45 (100%)45 (100%)

8 (18%)8 (18%)

41 (91%)41 (91%)

13 (29%)13 (29%)

39 (87%)39 (87%)

10 (22%)10 (22%)



Raltegravir Phase 2 PN005 Efficacy: Percent (95% CI) of Patients with Viral Load

< 400 copies/mL and <50 copies/mL

* BID + OBTNoncompleters = Failure approachGrinsztejn et al. Lancet 2007; 369: 1261-69

Number of Patients Number of Patients

0 2 4 8 12 16 24Week

-50

0

50

100

150

200

Cha

nge

in C

D4

cell

coun

t f

rom

bas

elin

e (c

ells

/L)

m518p5ms fig2v3 May 10, 2007

43 38 39 40 37 40 4145 45 44 43 42 44 4345 45 42 42 42 45 4245 41 44 41 40 45 43

Raltegravir 200mg* Raltegravir 400mg*Raltegravir 600mg* Placebo*

0 2 4 8 12 16 24Week

0

20

40

60

80

100

Pro

porti

on o

f pat

ient

s w

ith v

iral l

oad

<50

copi

es/m

L (%

)

43 43 43 43 43 43 4345 45 45 45 42 45 4545 45 45 45 45 45 4545 45 45 45 45 45 45

Raltegravir 200mg*Raltegravir 400mg*Raltegravir 600mg* Placebo*

0 2 4 8 12 16 24Week

0

20

40

60

80

100

P

ropo

rtion

of p

atie

nts

with

vira

l loa

d <4

00 c

opie

s/m

L (%

)

AA

43 43 43 43 43 43 4345 45 45 45 42 45 4545 45 45 45 45 45 4545 45 45 45 45 45 45

Raltegravir 200mg*Raltegravir 400mg* Raltegravir 600mg* Placebo*

0 2 4 8 12 16 24Week

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

Cha

nge

in v

iral l

oad

from

bas

elin

e (L

og10

cop

ies/

mL)

43 43 42 42 42 42 4145 45 44 45 42 45 4545 45 45 43 45 45 4445 45 45 45 43 45 45

Raltegravir 200mg* Raltegravir 400mg*Raltegravir 600mg*Placebo*

0 2 4 8 12 16 24Week

-50

0

50

100

150

200

Cha

nge

in C

D4

cell

coun

t f

rom

bas

elin

e (c

ells

/L)

m518p5ms fig2v3 May 10, 2007

43 38 39 40 37 40 4145 45 44 43 42 44 4345 45 42 42 42 45 4245 41 44 41 40 45 43

Raltegravir 200mg* Raltegravir 400mg*Raltegravir 600mg* Placebo*

0 2 4 8 12 16 24Week

0

20

40

60

80

100

Pro

porti

on o

f pat

ient

s w

ith v

iral l

oad

<50

copi

es/m

L (%

)

43 43 43 43 43 43 4345 45 45 45 42 45 4545 45 45 45 45 45 4545 45 45 45 45 45 45

Raltegravir 200mg*Raltegravir 400mg*Raltegravir 600mg* Placebo*

0 2 4 8 12 16 24Week

0

20

40

60

80

100

P

ropo

rtion

of p

atie

nts

with

vira

l loa

d <4

00 c

opie

s/m

L (%

)

AA

43 43 43 43 43 43 4345 45 45 45 42 45 4545 45 45 45 45 45 4545 45 45 45 45 45 45

Raltegravir 200mg*Raltegravir 400mg* Raltegravir 600mg* Placebo*

0 2 4 8 12 16 24Week

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

Cha

nge

in v

iral l

oad

from

bas

elin

e (L

og10

cop

ies/

mL)

43 43 42 42 42 42 4145 45 44 45 42 45 4545 45 45 43 45 45 4445 45 45 45 43 45 45

Raltegravir 200mg* Raltegravir 400mg*Raltegravir 600mg*Placebo*


Methods: PN005 Phase 2 Integrase Genotyping/Phenotyping Analysis

• Genotyping– population sequencing – first point identified as failure (non-responder and rebound)– compare sequence taken at baseline– cross sectional analysis – limited longitudinal studies (ongoing)

• Phenotyping– site directed mutants– limited data on patient derived sequences– single cycle infection assays with LTR- responsive reporter

cell lines


Analysis of raltegravir resistance in PN005: Phase 2 Study in patients w/triple class resistance:Genotype of first time point, 24 week results

• Virologic failure was observed in 38/133 (28.6%) patients on RAL

• Integrase mutations were observed in 35 of 38 patients failing RAL

• RAL failure was generally associated with either of two genetic pathways (N155 or Q148)

• Additional mutations were frequently observed with both pathways

• Q148 containing mutants were more common than N155

• Most common pattern Q148H/G140S

N155H pathwayTotal N = 14

Q148 pathwayTotal N = 20

N155H (n = 2) Q148H, G140S (N=13)

N155H, L74L/M (n = 1) Q148R (n = 1)

N155H, E92Q (n = 1) Q148R, G140S (n = 2)

N155H, T97A (n = 3) Q148K, E138K (n = 1)

N155H, Y143H (n = 1) Q148R, E138E/K (n = 1)

N155H, G163K (n = 1) Q148R, L74L/M, E138A (n = 1)

N155H, E92Q, T97A (n = 1) Q148H/R, G140S (n = 1)

N155H, V151I (n = 1)

N155H, G163G/R (n = 2*)

N155H, D232N (n = 1) Y143R (n = 1)


Profile of Patients in PN005 with virologic failure on raltegravir

• Similar number of patients with mutations at amino acids 148 or 155 across 200, 400, and 600 mg treatment groups

• Factors decreasing likelihood of developing mutations at a.a. 148 alone, 155 alone, either 148 or 155 (hazard ratio < 1):

– Lower viral load (≤ 100,000 copies/mL vs. > 100,000 copies/mL)– New use of enfuvirtide in OBT– PSS > 0

Baseline CharacteristicsPatients with virologic failure on RAL

vRNA > 100,00053%

vRNA < 100,00047%

CD4 <20058%

CD4 >20042%

GSS = 0 68%

GSS ≥ 1 32%

Seggevm

Could you turn the first three bullets into a two-column table?I'll attach an example next slide


Mutations observed in PN005 are similar to those selected with different integrase

inhibitors in cell culture

Q148

N155

G140

E138

Mg

E92

T97

Q148

N155

G140

E138

Mg

E92

T97

Catalytic Residues

Path 2Path 1

Catalytic ResiduesDiketoacidNaphthyridinePyrimidine


Effect of signature and secondary mutations on raltegravir resistance

0

100

200

300

400

500

600

Fold

-Cha

nge

IC50 Q148H

Q148H/G140SQ148KQ148K/E138AQ148K/G140AQ148K/E138A/G140AQ148RQ148R/G140S

0

10

20

30

40

50

60

70

Fold

-Cha

nge

IC50

N155HN155H/L74MN155H/T97AN155H/E92Q

0

100

200

300

400

500

600

Fold

-Cha

nge

IC50 Q148H

Q148H/G140SQ148KQ148K/E138AQ148K/G140AQ148K/E138A/G140AQ148RQ148R/G140S

0

10

20

30

40

50

60

70

Fold

-Cha

nge

IC50

N155HN155H/L74MN155H/T97AN155H/E92Q

Q148 Pathway N155 Pathway


Effect of signature and secondary mutations on infectivity

0102030405060708090

100

Infe

ctiv

ity

(% o

f wt)

Q148HQ148H/G140SQ148KQ148K/E138AQ148K/G140AQ148K/E138A/G140AQ148RQ148R/G140S

0102030405060708090

100

Infe

ctiv

ity

(% o

f wt)

Q148HQ148H/G140SQ148KQ148K/E138AQ148K/G140AQ148K/E138A/G140AQ148RQ148R/G140S

0

20

40

60

80

100

Infe

ctiv

ity

(% o

f wt) N155H

N155H/L74MN155H/T97AN155H/E92Q

0

20

40

60

80

100

Infe

ctiv

ity

(% o

f wt) N155H

N155H/L74MN155H/T97AN155H/E92Q

• Q148H/K/R and N155H single mutants display reduced infectivity.• Among all mutants examined, Q148H/G140S has highest replication capacity in both infectivity (shown) and multiple cycle replication assays (data not shown).

Q148 Pathway N155 Pathway


Summary of integrase genotyping and phenotyping results in PN005

• Signature mutations Q148H/R/K and N155H engender:– >10-fold reduced susceptibility to raltegravir– decreased viral replication capacity

• Secondary mutations were usually observed: – consistently enhanced the level of raltegravir resistance– sometimes improved viral replication capacity (subset of combinations)

• Q148-containing combinations displayed higher level resistance than N155H-containing combinations – Q148 mutations were more prevalent in PN005

• The G140S/Q148H variant was the most resistant and most fit variant– Resistance >100x; replication capacity near WT– Most common combination observed in PN005

Resistance is the primary driver for the acquisition of additional mutations, is there a pharmacologic threshold?


Longitudinal analysis of subjects with early N155H genotype: evidence of ongoing

evolution to acquire higher level resistance

Patient D

100

1000

10000

100000

1000000

-50 0 50 100 150 200 250 300 350Days in study

log[

vRNA

]

6

5

4

3

2

5.5

4.5

3.5

2.5

G140G/S, Q148Q/H, N155N/H (pop) Q148H and N155H unlinked (clonal)

400cp/ml

Q148H, G140S (pop)

Patient A

100

1000

10000

100000

1000000

-100 0 100 200 300 400 500 600 700

Days in study

log[

vRNA

]

N155H

Q148HG140SE138E/K

6

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2

5.5

4.5

3.5

2.5400cp/ml

Patient B

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1000000

-100 0 100 200 300 400 500 600 700

Days in study

log[

vRNA

]

6

5

4

3

2

5.5

4.5

3.5

2.5

N155H

400cp/ml

N155H, L74L/M, T97A

+ DRV

Patient C

100

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1000000

-100 0 100 200 300 400 500 600 700

Days in study

log[

vRNA

]

6

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3

2

5.5

4.5

3.5

2.5

N155H

400cp/ml

N155H, Y143Y/C

+ ENF, DRV/r


The Q148 pathway may be preferred

• Combinations containing Q148 mutations were more frequent in PN005

• Resistance to raltegravir was generally higher with combinations containing Q148 mutations

• Patients who fail with Q148 combinations return to baseline vRNA– Some patients with N155H viruses appear to retain lower vRNA

set point, partial activity or fitness?

More longitudinal analysis is required


Mutations can confer cross resistance to structurally diverse integrase inhibitors

IC50

ratio

of m

utan

ts v

s. W

T H

IV-1

in

infe

ctiv

ity a

ssay

399 / 838 / 2194X

502XN

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HN

F

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N

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F

NN

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OHN

OH O

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Conclusions• Raltegravir failure <30% in triple class resistant patients in PN005

– GSS=0 in >68% of virologic failures• Raltegravir failure was associated with integrase mutations in two

distinct genetic pathways defined by two or more mutations including – A signature mutation at either Q148(H/R or K) or N155(H) and– one or more secondary mutations unique to each pathway

• Signature mutations at Q148 and N155 conferred >10X reduced susceptibility to raltegravir and resulted in decreased viral replication capacity

• Secondary mutations appear to serve primarily to enhance the level of resistance to raltegravir – Although mitigation of effects on replication capacity were observed for a

subset of combinations (eg. G140S/Q148H the most common variant), all combinations evaluated to date displayed increased resistance

– Despite >10X resistance, viruses with a single mutation at N155 evolved to acquire increased resistance suggesting a pharmacologic threshold

• Mutations at Q148 and N155 can confer cross resistance to structurally diverse integrase inhibitors


AcknowledgementsInvestigatorsInvestigatorsB. GrinsztejnB. Grinsztejn BrazilBrazilC. KatlamaC. Katlama FranceFrance J. GatellJ. Gatell SpainSpainA. LazzarinA. Lazzarin ItalyItalyD. VittecoqD. Vittecoq FranceFranceC. GonzalezC. GonzalezUSAUSAJ. SierraJ. Sierra Mexico MexicoG. CarosiG. Carosi ItalyItalyS. LittleS. Little USAUSAM. MoroniM. Moroni ItalyItalyJ. Rockstroh GermanyJ. Rockstroh GermanyM. KozalM. Kozal USA USAR. LiporaceR. Liporace USAUSAE. Jones-LopezE. Jones-Lopez USAUSAB. ClotetB. Clotet Spain SpainS. StaszewskiS. Staszewski

GermanyGermany

Merck Research LabsMerck Research LabsB-Y. NguyenB-Y. NguyenJ. ChenJ. ChenR. IsaacsR. IsaacsC. HarveyC. HarveyH. TepplerH. TepplerL. Wenning L. Wenning M. MillerM. MillerD. HazudaD. HazudaR. DanovichR. DanovichM. RowleyM. RowleyV. SummaV. SummaJ. VaccaJ. VaccaJ. WaiJ. Wai

D. McMahonD. McMahon USAUSAP. KumarP. Kumar USAUSAC. LeeC. Lee MalaysiaMalaysiaK. SquiresK. Squires USAUSAM. Opravil SwitzerlandM. Opravil SwitzerlandN. ClumeckN. Clumeck BelgiumBelgiumJ. LennoxJ. Lennox USAUSAJ. EronJ. Eron USAUSAJ. GallantJ. Gallant USAUSAM. NelsonM. Nelson UKUKS. BrownS. Brown USAUSAK. TashimaK. Tashima USAUSAV. SorianoV. Soriano SpainSpainC. CrumpackerC. Crumpacker USAUSAD. KuritzkesD. Kuritzkes USAUSA

All patients in Protocol 005All patients in Protocol 005

copyright © 2007 merck & co., inc., whitehouse station, new jersey, usa all rights reserved...

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