Institute of Molecular VirologyWestfälische-Wilhelms-University Münster

Pursuing New Avenues in Anti-Influenza Therapy

Stephan Ludwig

Options for the Control of Influenza VII, Hongkong, Sep, 7, 2010

Outline

Introduction

Viral targets

Cellular/host targets- Immunemodulators- Factors directly regulating viral functions

Traditional Medicine

Urgent need for novel antiviral agents against

influenza viruses

- Wide availability

- Broad activity

- Low cost

- No resistance

Increasing incidence of resistance to clinically approved classes of drugs

Novel viral targets

Viral Polymerase

Favipiravir, T-705 (Toyama Chemical Co., Ltd.)Clincial Trail Phase 2->3

FLU-PHARM - New drugs targeting influenza virus polymeraseCoordinator: Stephen Cusack, EMBL (France)14 Partners from 7 European Countries

FLUCURE - Development of novel antiviral drugs against InfluenzaCoordinator: Heather Marshall-Heyman, VIRONOVA AB (Sweden)9 Partners from 7 European Countries

Viral Nonstructural Protein 1JJ3297 and other blockers of interferon antagonistic NS1 function, Basu et al. (P-460), (Basu et al. (2009) J Virol. 83:1881-91)

Viral NucleoproteinNucleozin triggers aggregation of NP and inhibits nuclear accumulation(Kao et al. (2010) Nat Biotech 28: 600-5)

Cellular/Host targets

A) Modulators or inducers of the immune response

B) Inhibitors of cellular factors or pathways that regulate the virus life cycle

Cellular targets

A) Modulators or inducers of the immune response

- IFN inducing agents, e.g. ASN2 (Ortigoza et al., O-869)

- Low dose interferon treatment for prophylaxis (Bennet et al., O-822)

- Protease-activated receptor (PAR) agonists act antiviral through induction of IFN gamma (Khoufache et al., P-446)

- anti-influenza activity of PS-341 (Velcade) through induction of an antiviral state (Dudek et al. (2010) J Virol. 84:9439-51)

- COX-2 inhibitors down modulate hyperinduction of immune responses (Lee et al., O-868) (Zheng et al. PNAS (2008) 105: 8091-6)

- Use of existing immunmodulatory drugs (Statins, Glycyrrhizin, Glitazones) (Fedson, P-447, Korossy-Horwood et al. P-458, Allevea et al. P-459)

Taken from: Watanabe et al. (2010) Cell Host Microbe, 7, 427-439

Cellular targetsB) Inhibitors of cellular factors that regulate the virus life cycle

Cell 139, 1243–1254

Nature 454, 890–893

Nature 463, 813–817

Nature 463, 818–822

Cell 139, 1255–1267

Virology 387, 473–481

Adsorption

Endocytosis

Fusion andRelease

vRNA (-)

cRNA (+)

mRNA

Import

Translation

PosttranslationalProcessing

RNP-Export

Budding

PackagingEntry

Cellular targetsB) Inhibitors of cellular factors that regulate the virus life cycle

DAS181

ImportinsNUPs

Rabs, V-type ATPasesPKC

CRM1Hsc70NUP153

ActinRab 11

Hillaire et al. (P-452) Collectin pSP-DNicol et al. (P-449) FLUPEP

Adsorption

Endocytosis

Fusion andRelease

vRNA (-)

cRNA (+)

mRNA

Import

Translation

PosttranslationalProcessing

RNP-Export

Budding

Packaging

Cellular targetsViral penetration of cellular barriers is controlled

by cellular signaling cascades

Entry

PI3K

RTKs (e.g. EGFR)Eierhoff et al. (2010)PLoS Pathog (in press)

Raf/MEK/ERKPleschka et al. (2001) Nat Cell Biol.

IKK/NF-kBWurzer et al. (2004) Cell Microbiol.Wurzer et al. (2003) EMBO J.

NF-B Inh.

-M

-PB1

-NP

-ERK2

-ERK2

- - + - - -+ + +

-JNK1

-NS1

-ERK2

0h 2h 4h 8h 10h

NF-B inhibition efficiently blocks viral RNP export

DAPI anti-NP merge

- INH

+ INH

The NF-B inhibitor SC75741 blocks replication of influenza viruses

A/FPV/Bratislava/79 (H7N7)

0,001

0,01

0,1

1

10

100

1000

DMSO0,1%

KH1 1µM

DMSO0,25%

KH12,5µM

DMSO0,5%

KH1 5µM

Vir

ustit

er

% d

er u

nbeh

ande

lten

Kon

trol

le

A/Thailand/KAN-1/2004 (H5N1)

Hours post infection

Ehrhardt et al, P- 457Reiling et al, P-453

SC75741 shows no tendency to induce resistant virus variants

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8

Infektionsrunde

rela

tiver

Vir

usti

ter

[%]

Kontrolle

Progeny virus titer supon repeated passaging in the presence and absence of the drugs

Control

SC75741

Oseltamivir

Therapeutic treatment of H5N1 infected mice with SC75741

SC75741 shows a significant (p = 0.05) anti H5N1 activity, when

treatment starting 4 days after infection

SC75741 shows a significant (p = 0.05) anti H5N1 activity, when

treatment starting 4 days after infection

twice daily 7,5mg/Kg i.p.

SC75741

Placebo

Days after infection

100

50

0

% S

urv

iva

l

once daily 15 mg/Kg i.p.

SC75741

Placebo

Days after infection

100

50

0

% S

urv

iva

l

A/Mallard/Bavaria/2005 (H5N1), induces severe disease in mice without adaptation (LD50: 8x101)

SC75741 results in reduced cytokine/chemokine expression in vivo

SC75741 leads to a reduced transcription of IL-6 and IP-10 in H5N1

infected mice

SC75741 leads to a reduced transcription of IL-6 and IP-10 in H5N1

infected mice

IP-10

0

20

40

60

80

100

120

Control SC75741Co

mp

arat

ive

del

ta C

T v

alu

e re

lati

ve t

o c

on

tro

l (1

00%

)

IL6

0

20

40

60

80

100

120

Control SC75741Co

mp

arat

ive

del

ta C

T v

alu

e re

lati

ve t

o c

on

tro

l (1

00%

)

Cytokine/Chemokine specific real time RT-PCR

Primer: Qiagen RT-PCR

RNA isolated from the lung 48h p.i.

SC75741 15mg/kg

Mallard/Bavaria/2005 (H5N1)

Targeting signal transduction pathways as an antiviral approach

-broad activity

-no emergence of resistent variants detectable

-can be done by using existing drugs

-NF-B or MAPK blockers have indirect beneficial effects due to their immunemodulatory function

Expansion and optimization of the current repertoire of antiviral drugs and development of clinical research to assess efficacy of putative adjuvant treatment modalities such as immunomodulators, passive immunotherapy and traditional medicine that are suitable for use in under-resourced areas would be most beneficial.

Research Recommendations: .....4.2.4 Develop novel and effective treatment strategies including adjunctive treatments (e.g. immunomodulators, immunoglobulin, natural products) that are applicable in low resource settings and easy to administer......

4.2 Improve Clinical Management of Patients

Traditional Medicine

Traditional Herbal Medicine

Numerous reports of the anti-influenza activity of medicinal plant extracts and plant products

Korrossy-Horwood et al. (P-458) Glycyrrhizin from licorice rootsTsai et al. (P-450) Platform to screen Chinese herbal medicinesEhrhardt et al. (O-871) Cystus052, a polypenol-rich extract from pink rockrose

Plant polyphenols possess ant-influenza activity:

Anti-influenza activity of resveratrol from red grapes:Improved survival and reduced lung titers in infected mice(Palamara et al. J.Infect. Dis.191,1719–1729)

Epigallocatechin-3-gallate and theaflavindigallate from green tea:Unspecific binding of the HA and agglutination of virus particles (Nakayama et al.; Antiviral Res. 21,289–299)

CYSTUS052 possess anti-influenza activityCYSTUS052 possess anti-influenza activity

0,1

1

10

100

1000

10000

100000

8h 24h 36h

x102 P

FU

/0.5

ml

untreated25µg/ml CYSTUS05250µg/ml CYSTUS052

CYSTUS052 is very rich in highly polymeric polyphenols

Acts antiviral by inhibiting binding of virusparticles to cells

Does not interfere with cell viablity, metabolism, intracellular signaling orbinding of cytokines to cellular receptorsNo pharmacological effects

Does not induce resistant virus variants

Viral titers

Ehrhardt et al. 2007, Antiviral Res.Droebner et al. 2007, Antiviral Res.Kalus et al. 2009, Antiviral Res.

80

90

100

110

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

days p.i.

wei

gh

t in

%Bodyweight

Control

CYSTUS052

CYSTUS052 protects mice against CYSTUS052 protects mice against H7N7 influenza virus infection H7N7 influenza virus infection

Survival Control: 4/10Survival Control: 4/10CYSTUS052: 10/10CYSTUS052: 10/10

(O-871)

Novel Antiviral Approaches -Perspectives

-Numerous promising approaches under investigation, but still in an early stage

-Inhibitors of cellular targets may be best suited to cover a broad rangeof viruses (also newly emerging strains) and to prevent emergence of resistant variants

- Inhibitors that possess dual action (immunemodulation and direct antiviral activity) might be of major advantage

- Use of existing drugs against cellular targets

- Drug combinations should be considered

- Medicinal products from traditional medicine should be given more attention to meet WHO recommendations for low-resource settings and to provide safe options for prophylactics