Neurokinin-1 Receptor Antagonist (Aprepitant) Inhibits Drug-Resistant HIV-1

Infection of Monocyte-Derived Macrophages in vitro

Xu Wang, Steven D. Douglas, Jian-Ping Lai, Pablo Tebas, Janet L. Lathey, and Wen-Zhe Ho

Research Aim

To investigate the role of Neurokinin-1 Receptor Antagonist (Aprepitant) in Drug-Resistant HIV-1 Infection.

Background

• Respite of the success of antiretroviral therapy in maintaining control of HIV viral replication, treatment failure occurs in more than 50% of the individuals on HAART therapy.

• Cellular targets are attractive because it is more difficult for HIV to develop resistance.

• We have shown that NK-1R antagonist (CP-96,345) inhibits HIV-1 infection of MDM in vitro by down regulating CCR5 expression. (PNAS, 98: 3970-3975,2001)

Aprepitant

Chemical Structure of Aprepitant

Aprepitant: FDA approved drug for the treatment of chemotherapy

induced nausea

Experimental DesignMonocyte/Macrophage from normal subjects

With or without the NK-1R antagonists

treat for 2 h

Different types of HIV strains infection

HIV p24 assay at day 0, 3,5 7 post-infection

HIV RT assay at day 4, 8,12 post-infection

Inhibition of HIV (Bal) Infection of MDM by NK-1R Antagonists (10-6 M)

0

5

10

15

20

25

30

Control

Aprepita

nt

CJ12,2

55

CP-96,

345

RP 67,5

80

L-733

060

HIV

RT

Act

ivit

y (1

03 c

pm

)

Effect of Aprepitant on HIV Infection

RT Activity (% of Contriol) R5 R5X4 X4

Dose Bal SF162 89.6 UG024

Aprepitant 10-6 M 17.2 14.3 36.9 89.1

Aprepitant 10-7 M 13.8 16.6 44.1 93.1

Aprepitant 10-8 M 57.6 60.1 52.4 89.9

Control 100.0 100 100.0 100

Virus

RT Activity (% of Contriol) R5 R5X4 X4

Dose Bal SF162 89.6 UG024

Aprepitant 10-6 M 17.2 14.3 36.9 89.1

Aprepitant 10-7 M 13.8 16.6 44.1 93.1

Aprepitant 10-8 M 57.6 60.1 52.4 89.9

Control 100.0 100 100.0 100

Virus

Dose-dependent Inhibition of Aprepitant on HIV Bal infection of MDM

0

50

100

150

0 200 400 600 800 1000

Aprepitant (nM)

HIV

RT

Act

ivit

y (

% o

f C

on

tro

l)

Effect of Aprepitant on AZT Resistant Virus Infection of MDM

0

4

8

12

16

20

24

HIV

P24

Lev

el (

x10

3 p

g/m

l) Aprepitant

Control

AZT Resistant HIV-1 (A012 G691-6)

Day 0 Day 3 Day 5 Day70

50

100

150

200

250

300

HIV

P2

4 L

ev

el (

pg

/ml)

Aprepitant

Control

AZT Resistant HIV-1 (A018 G901-6)

Day 0 Day 3 Day 5 Day7

A012 G691-6 and A018G901-6 were kind gifts from NIH AIDS Research and Reagent Program

Effect of Aprepitant on RT Inhibitor-Resistant HIV Infection of MDM

0

100

200

300

400

500

HIV

P24

Lev

el (p

g/m

l)

Aprepitant

Control

Day 0 Day 3 Day 5 Day7

RT Inhibitor-Resistance HIV-1 TC 60

B

0

5000

10000

15000

20000

25000

30000

HIV

P24

Lev

el (p

g/m

l)

Aprepitant

Control

Day 0 Day 3 Day 5 Day7

RT Inhibitor-Resistance HIV-1 TC49

A

TC49 and TC60 (R5 HIV strain) were kind gifts from Dr. Besty Johnston White and Dr. David Katzenstein (Stanford University)

Effect of Aprepitant on HIV co-receptor(CCR5) Expression in MDM

0

20

40

60

80

100

120

140

CC

R5

mR

NA

Lev

el (

% o

f C

on

tro

l)

Aprepitant Control

Exp1 Exp 2 Exp 3 Exp 4

0

20

40

60

80

100

120

140

CD

4+C

CC

R5+

cel

ls (

% o

f C

on

tro

l) Aprepitant Control

Exp1 Exp 2 Exp 3 Exp 4

Enhancing Effect of Aprepitant on anti-HIV Drug-mediated HIV Inhibition in MDM

0

5

10

15

20

25

HIV

RT

(1

03 c

pm

)

Aprepitant 10-6M

AZT 10-11M

Efavirenz 10-10M

Indinavir10-15M

----

+

---

-+

--

+

+

--

--+

-

+

-+

-

---+

+

--+

Summary 1

• Among the NK-1R antagonists examined

Aprepitant had a better inhibitory effect on

HIV infection of MDM.

• Aprepitant, in a dose-dependent manner,

inhibits HIV R5 strain infection, partially

inhibited R5X4 strain infection, and had little

effect on HIV X4 strain infection.

Summary 2

• Aprepitant potently inhibited the AZT-

resistant strains (A018 G901-6 and A012

G691-6) infection of MDM.

• Aprepitant also suppressed RT inhibitor

resistant strains (TC 49 and TC 60) infection

of MDM.

Summary 3

• Aprepitant down-regulated CCR5, the co-

receptor for HIV entry into MDM.

• Aprepitant enhanced the anti-HIV activity of

antiretrovirals (AZT, efavirenz, and

indinavir) in MDM.

Conclusion

Our data suggest that NK-1R antagonists merit further investigation as potential HIV therapeutic agents

• This investigation was supported by MH 076388 and MH 049981 from NIH

Acknowledgments

• Jian-Ping Lai• Li Song• Wenzhe Ho

• Pablo Tebas

Seracare BioServices Janet L. Lathey

• Donald E. Campbell • Eric Riedel• Steven D. Douglas

NK-1R Antagonists: Anti-HIVMechanisms

Project 1PI: Wenzhe Ho

NK-1R Antagonist

Anti-HIV Mechanisms

PBMC from Normal Subjects Drug ResistantSynergistic Studies

PBMC from HIV-infected Subjects

HIV Activation SP Induction

Microglia

HIV Replication Cytokine Production

Neuronal Cells (NT-2N)

HIV gp120 HIV Tat

Schematic Diagram of Project 1

HIV Replication (Direct Effect) CCR5 (Indirect Effect) SP (Indirect Effect)

Aims 1 & 2 Aims 2 & 3 Aim 4 Aim 4

Aim 1a: To determine whether the NK-1Rantagonists inhibit HIV infection of PBMC

M-, T-, and dual tropic HIV strains

HIV-1 p24 assay at day 7 post-infection (Core B)

With or without the NK-1R antagonists

PBMC from normal subjects

Cytotoxicity assay

Table 1 – Drug Resistant Isolates to be used

Isolates Biotype Resistance

139-1 SI (R5X4) AZT

HIV-174V/MT2 X4 DdI, ddC

HIV-1 LAI-M184V

SI 3TC

N119 SI (X4) Nevirapine

J302 SI Saquinavir

PBMC from Normal Subjects

Antiretroviral drugs (RT and protease inhibitors)

and/or

HIV p24 (Core B)

M- and T-tropic strains

w or w/o NK-1R antagonists

w or w/o NK-1R antagonists

Drug resistant strains

Aim 1b – To determine the effect of NK-1R antagonists on drug-resistant isolates and the synergistic effect of the antagonists

Table 2 – Antiretroviral Drugs to be Used

Drug Classification

ZidovudineDDC

EfavirenzLopinavirRitonavir

T20PA-457

RT Inhibitor (NRTI)RT Inhibitor (NRTI)

RT Inhibitor (NNRTI)Protease InhibitorProtease InhibitorFusion Inhibitor

Maturation Inhibitor