pursuing-new-avenues-in-the-anit-influenza-therapy-stephen-ludwig

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					Institute of
Molecular Virology
Westfälische-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
   Increasing incidence of resistance to
    clinically approved classes of drugs
Urgent need for novel antiviral agents against
               influenza viruses


- Wide availability

- Broad activity

- Low cost

- No resistance
                             Novel viral targets
Viral Polymerase
   Favipiravir, T-705 (Toyama Chemical Co., Ltd.)
                          Clincial Trail Phase 2->3

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


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

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

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

Taken from: Watanabe et al. (2010) Cell Host Microbe, 7, 427-439
                                    Cellular targets
B) Inhibitors of cellular factors that regulate the virus life cycle

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




                                             DAS181                   Budding
                       Adsorption



                                                                 Packaging
             Entry                      Posttranslational         Actin
                                        Processing                Rab 11
             Endocytosis                   Translation
             Rabs, V-type ATPases
             PKC
              Fusion and
              Release
                                                                 RNP-
                                                      mRNA
                                                                 Export
                                                      vRNA (-)   CRM1
                                                                 Hsc70
                           Import
                                                      cRNA (+)   NUP153
                         Importins
                         NUPs
                                          Cellular targets
                 Viral penetration of cellular barriers is controlled
                          by cellular signaling cascades



                                                                            Budding
                                 Adsorption
RTKs (e.g. EGFR)
Eierhoff et al. (2010)
PLoS Pathog (in press)
                                                                     Packaging
                         PI3K                   Posttranslational     Raf/MEK/ERK
                                                Processing            Pleschka et al. (2001) Nat Cell Biol.
                         Entry
                         Endocytosis             Translation          IKK/NF-kB
                                                                      Wurzer et al. (2004) Cell Microbiol.
                                                                      Wurzer et al. (2003) EMBO J.

                          Fusion and
                          Release
                                                                     RNP-
                                                          mRNA
                                                                     Export
                                                          vRNA (-)

                                       Import
                                                          cRNA (+)
 NF-kB inhibition efficiently blocks viral RNP export
                       0h       2h      4h    8h   10h

          DAPI
          NF-kB Inh.   -    -        + anti-NP - + - + merge
                                        - +
               -M
            -JNK1
- INH
             -NS1

            -ERK2

              -NP

            -ERK2
+ INH
             -PB1

            -ERK2
The NF-kB inhibitor SC75741 blocks replication of influenza viruses
                                                             A/FPV/Bratislava/79 (H7N7)

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




                                                                               Hours post infection




                                                          A/Thailand/KAN-1/2004 (H5N1)
                                                         1000
                         % der unbehandelten Kontrolle




                                                          100

                                                           10
                                  Virustiter




                                                            1

                                                           0,1

                                                          0,01

                                                         0,001
                                                                 DMSO    KH1      DMSO      KH1       DMSO KH1 5µM
                                                                  0,1%   1µM      0,25%    2,5µM       0,5%
SC75741 shows no tendency to induce resistant virus variants




                                                                                         Control
                                                                                             SC75741
                                                                                              Oseltamivir




 Progeny virus titer supon repeated passaging in the presence and absence of the drugs
Therapeutic treatment of H5N1 infected mice with SC75741

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

                                                 twice daily 7,5mg/Kg i.p.
                                                                                                                                    once daily 15 mg/Kg i.p.
                                       1.2                                                                                1.2

                                                                                                                                                     SC75741
                   100
                     1.0                                                                                  1001
                                                                              SC75741
                  Wahrscheinlichkeit




                                                                                                     Wahrscheinlichkeit
                                       0.8                                                                                0.8




                                                                                             % Survival
     % Survival




                                       0.6                                                                                0.6
                                 50                                                                                       50
                                       0.4                               Placebo                                          0.4                           Placebo

                                       0.2                                                                                0.2


                                       0.0
                                                                                                                           0
                                                                                                                           0
                                       0                                                                                        0           5          10         15
                                             0          5                10             15
                                                                                                                                             Tage/Dauer
                                                            Tage/Dauer                                                                    Days after infection
                                                     Days after infection




                  SC75741 shows a significant (p = 0.05) anti H5N1 activity, when
                            treatment starting 4 days after infection
                                                                      SC75741 results in reduced
                                                                 cytokine/chemokine expression in vivo


                                                          IL6                                                                     IP-10              Cytokine/Chemokine specific
                                                                                                                                                      real time RT-PCR
Comparative delta CT value relative to control




                                                                         Comparative delta CT value relative to control
                                                 120                                                                      120
                                                                                                                                                          Primer: Qiagen RT-PCR
                                                 100                                                                      100
                                                                                                                                                          RNA isolated from the lung 48h
                                                 80                                                                       80
                                                                                                                                                           p.i.
                                                                                                                                                          SC75741 15mg/kg
                   (100%)




                                                                                            (100%)
                                                 60                                                                       60
                                                                                                                                                          Mallard/Bavaria/2005 (H5N1)
                                                 40                                                                       40


                                                 20                                                                       20


                                                  0                                                                        0
                                                       Control     SC75741                                                      Control   SC75741




                                                 SC75741 leads to a reduced transcription of IL-6 and IP-10 in H5N1
                                                                          infected mice
Targeting signal transduction pathways as an antiviral approach


     -broad activity

     -no emergence of resistent variants detectable

     -can be done by using existing drugs

     -NF-kB or MAPK blockers have indirect beneficial effects
      due to their immunemodulatory function
Traditional Medicine
      4.2 Improve Clinical Management of Patients


       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.
       .....
               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 roots
Tsai et al. (P-450) Platform to screen Chinese herbal medicines
Ehrhardt 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 activity

                              Viral titers
   100000
                                                      CYSTUS052 is very rich in highly
         10000                                        polymeric polyphenols
x102PFU/0.5ml




                1000
                                                      Acts antiviral by inhibiting binding of virus
                100
                                                      particles to cells
                 10

                  1                                   Does not interfere with cell viablity,
                 0,1
                                                      metabolism, intracellular signaling or
                       8h          24h          36h   binding of cytokines to cellular receptors
                                                      No pharmacological effects
                            50µg/ml CYSTUS052
                            25µg/ml CYSTUS052
                            untreated
                                                      Does not induce resistant virus variants



                                                                           Ehrhardt et al. 2007, Antiviral Res.
                                                                           Droebner et al. 2007, Antiviral Res.
                                                                           Kalus et al. 2009, Antiviral Res.
CYSTUS052 protects mice against
 H7N7 influenza virus infection



                                        Bodyweight
              110
                                                                               CYSTUS052




              100
weight in %




              90                                                               Control




              80
                    1   2   3   4   5    6   7   8   9   10 11 12 13 14 15
                                             days p.i.


                                    Survival Control: 4/10
                                     CYSTUS052: 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 range
of 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

				
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