1. Epidemic and Pandemic Use of Antivirals Introduction
15 September 2008
Frederick G. Hayden, M.D.
University of Virginia Health System
Charlottesville, Virginia, USA

2. Antiviral Agents for Influenza

3. Antivirals for Influenza: Overview
Effective for prophylaxis and early therapy
Useful in outbreak control
Rapid antiviral effect
Differing anti-influenza spectra
Amantadine/rimantadine: influenza A only
Oseltamivir/zanamivir: influenza A + B
Favorable PK characteristics
Treatment 2X/d; prophylaxis 1X/d
Few drug interactions
Differences in dosing routes, PK, tolerance, efficacy, and resistance profiles

4. M2 Inhibitor Prophylaxis During Pandemic Influenza
Hayden. J Infect Dis 176:S56, 1997

5. Influenza Prevention In Households: PEP
Antiviral (Study)
No.
Contacts
(age)
Reduction in 2° influenza illness
Reduction in influenza infection
Oseltamivir (Welliver et al, 2000)
955
(13+ yr)
89%
49%
Oseltamivir* (Hayden et al, 2004)
792
(1+ yr)
73%
35%
(Index +)
Zanamivir* (Hayden et al, 2000)
837
(5+ yr)
79%
62%
Zanamivir
(Monto et al, 2002)
1,291
82%
59%
*Index case given treatment

6. Antiviral Treatment of Acute Influenza
Outcome
M2I
ZNV
OSEL
Symptom relief
Yes
Complications reduction ?
Decrease antibiotic use
28%
24-40%
Decrease hospitalization
~50%
Treatment of viral complications
Reduction in transmission
~30% No
? = No placebo-controlled study or not reported

7. Oseltamivir Treatment Effects in A(H5N1) Infection
Virus
Survivors/
Treated (%)
Untreated (%)
P-value
Presumed clade 1
45/82 (55%)
6/26 (23%)
0.006
Presumed clade 2
43/106 (41%)
1/30 (3%)
< 0.001
Total
88/188 (47%)
7/56 (12%)
Adapted from Writing Committee of Second WHO Consultation on Human H5 Infections. N Engl J Med 358: 261, 2008

8. Resistance to M2 Inhibitors (S31N) in Community Isolates of A/H3N2, 2000-07%
Bright et al. Lancet 2005, JAMA 2006; Klimov et al. CDC unpublished; Barr et al. Antiviral Res 2006; R Saito, Niigata Univ, unpublished

9. Oseltamivir Resistance in H1N1 (H274Y)
Rare in community H1N1 isolates,
0 to <1% in most surveys
2.2% in Japan in (but not in or early in )
High prevalence in Europe and globally,
Patients without known oseltamivir use or exposure to those on drug
Generally no obvious epidemiologic links
Household contacts, several apparent clusters
Typical influenza illness; some fatalities
Efficient person-to-person transmission
Revision of WHO Pandemic Preparedness Guidance
The working group process is under way to revise and update the pandemic preparedness guidance documents. The working group comprises experts, and representatives from Regional Offices and partner institution (ECDC, CDC, IFRC, UNICEF, UNSIC). It is anticipated that new guidance documents will be published by the end of 2008.

10. Oseltamivir Resistance in H1N1 Viruses
Region
Location
No. isolates tested
% resistant
AMRO+ (17%)
Canada
USA
508
1026
26%
12%
EURO+
(25%)
France
Norway
496
265
47%
67%
WPRO+
(5%)
China/HK
Japan
666
1652 3%
AFRO*
(85%)
South Africa*
Other*
107 20
100%
45%
Worldwide
4Q07- 1Q08
2Q08-20Aug08
7528
788
16%
31%
+4Q07- 1Q *2Q08-20Aug08
20 Aug 08

11. University of Virginia Health System Charlottesville, Virginia, USA
Antivirals for Seasonal, A(H5N1), and Pandemic Influenza: Efficacy, Resistance, and New Agents
15 September 2008
Frederick G. Hayden, M.D.
University of Virginia Health System
Charlottesville, Virginia, USA

12. Do We Need New Anti-Influenza Agents?
Antiviral resistance
Global spread of M2 inhibitor resistance in H3N2 > H1N1
New emergence of oseltamivir-resistant H1N1
Dual M2 and NAI resistance in IC hosts
Antiviral efficacy incomplete in H5N1 disease
Oseltamivir resistance emergence
Safety/efficacy ? in risk populations: infants < 1 yr, pregnancy, hospitalized, IC hosts
Lack of parenteral agents

13. Viral Loads and Antiviral Treatments in Immunocompromised Host with Fatal Oseltamivir-Resistant H1N1 Illness
67 yo male with CLL + recent chemoRx  neutropenia
Fever, cough, SOB  acute respiratory failure
Extubation 
Re-intubation 
Intubation 
 Oseltamivir resistance (H274Y) 
Amantadine resistance (L26F)
Van der vries et al. NEJM 359:1074, 2008

14. Medical Needs for Anti-Influenza Antivirals
Greater antiviral efficacy greater clinical benefit
Safety and efficacy in special risk populations: infants < 1 yr, pregnancy, hospitalized, immuno-compromised
Reliable drug delivery in seriously ill patients
Manage antiviral resistance
Combinations

15. Influenza Virus Replication and Sites for Antiviral Inhibition
De Clercq. Nature Reviews- Drug Discovery 5:1015, 2006

16. Investigational Anti-Influenza Agents
Neuraminidase (NA) inhibitors
- Zanamivir (IV), peramivir (IV/IM), A (oral)
Long-acting NA inhibitors (LANI)
CS8958/R (topical), Flunet (topical)
Conjugated sialidase- DAS181 (topical)
HA inhibitors- cyanovirin-N, sialyl-glycopolymer, arbidol
Polymerase inhibitors- ribavirin; viramidine; siRNA; T-705
Protease inhibitors- aprotinin
Biologics- antibodies, interferons

17. Investigational Agents in Clinical Development
Target
Sponsor
Route
Development phase
Zanamivir NA
GSK IV
Phase 1, 2a
Peramivir
Biocryst
IV, IM
Phase 2
CS8958
Sankyo, Biota
Topical
T-705
Polymerase
Toyama
Oral
DAS181
HA receptor
Nexbio
Phase 1

18. Neuraminidase Inhibitors
Peramivir

19. NA Inhibitor Resistance Profiles
NA mutation
NA type/ subtype
Susceptibility in the NAI assay (fold )
Oselt
Zana
Peram A
E119V
A/N2
R (>50->1000)
S (1)
S (1-2)
R292K
R (>1000)
S (4-25)
R (40-80)
S (8-13)
H274Y
A/N1
R (>700)
R (40-100)
S (3)
R152K B
R (>30-750)
R (10-100)
R (>400)
R (150)
Mishin et al. AAC 49:4516, 2005; Wetherall et al. AAC 41:742, 2003; Abed et al. Antiviral Res 77: 163, 2008

20. IV zanamivir 600 mg or placebo twice daily X 5 days starting 4 hr before virus inoculation
Highly protective against experimental infection (14% vs 100%), virus shedding (0 vs 100%), and illness

21. Pharmacokinetic Profiles of Intravenous and Intramuscular Peramivir
Linear PK; prolonged plasma T1/2elim (18 – 20 hr)
Kilpatrick JM, et al. Pharmacokinetics and Safety of Peramivir by Intramuscular Administration, Options for the Control of Influenza VI, Toronto, 2007

22. IM Peramivir: Time to Alleviation of Illness
J Alexander, BioCryst Pharmaceuticals, unpublished data

23. Long Acting Neuraminidase Inhibitors (LANI)- 2 Strategies
CS-8958
FLUNET R
Pro-drug
Clinical
Dimer
Preclinical

24. Yamashita et al. 43rd ICAAC, Abstract no. F-1830, 2003

25. Double-blinded trial found that inhaled CS-8958 administered once only was not statistically different than standard oseltamivir regimen.

26. T-705: Summary of Pre-Clinical Findings
Inhibitory for influenza A, B, C viruses: EC50s µg/ml
Active against flavi, arena, bunya, picorna, alpha, and paramyxo viruses
Cytotoxicity for mammalian cell > 1,000 µg/ml
Triphosphate is inhibitor of influenza RNA polymerase.
Active orally in murine models
6-fluoro-3-hydroxy-2-
pyrazinecarboxamide
Furuta et al. AAC 46:977, 2002; AAC 49:981, 2005

27. Mode of T-705 action (Hypothesis)
Nucleosidase
T-705
T-705 ribofuranose
Nucleoside- kinase
5’- Nucleotidase
Phosphoribosyl
- transferase
Weak inhibition of IMPDH
T-705RMP
Nucleotide- kinase
Nucleotidase
Potent inhibition of
influenza viral polymerase
T-705RTP
AAC 49:981 (2005)

28. T-705: In Vitro Activity vs Ribavirin
Compound
IC50(μM) ± SD
(A PR/8/34 H1N1 influenza)
CC50(μM) ± SD
(MDCK cells)
T-705
1.0 ± 0.9
> 6,370
Ribavirin
31.6 ± 9.2
94.3 ± 47.6
Furuta Y, Takahashi K, Kuno-Maekawa M, et al. Mechanism of action of T-705 against influenza virus. Antimicrob Agent Chemother. 2005;49: H1N1 cytotoxicity: p 982, T1
Furuta Y, et al. Antimicrob Agents Chemother. 2005;49:

29. Delay before starting treatment following viral exposure, hours
Effect of a T-705 Treatment in Mice Exposed to Lethal A/Duck/N/1525/81 (H5N1) Virus * * * * *
*P < .01 relative to control * * *
Survival (%)
Sidwell RW, Barnard DL, Day CW, et al. Efficacy of orally administered T-705 on lethal avian influenza A (H5N1) virus infections in mice. Antimicrob Agent Chemother. 2007;51: Treatment window: p 850, Fig 2.
Delay before starting treatment following viral exposure, hours
Sidwell RW, et al. Antimicrob Agent Chemother. 2007;51:

30. Pharmacokinetics of Oral T-705
Bioavailability > 97% in mouse
Rapid absorption in humans (Tmax < 1 hr)
Plasma T1/2elim from 1.3 to 3.9 hr
Mainly excreted as T-705M1 in urine
Single doses up to 1,600 mg or multiple up to 400 mg tid for 7 days well tolerated
Single oral doses of T-705 over a range of 30 to 1600 mg
Toyama Chemical Co, unpublished

31. Molecular Model of DAS181 (Fludase®)
Fusion construct with catalytic domain of A. viscosus sialidase and an epithelium-anchoring domain (human amphiregulin)
Active against both α2,6- and α2,3-linked sialic acid receptors
Malakhov et al. AAC 50:1470, 2006

32. Preclinical Features of DAS181
Inhibitory for range of influenza A and B viruses
In vitro EC90 values: 1-14 nM
Epithelial tag increases activity 5-30 fold
Pretreatment (24 hr) effective
Intranasal dosing shows
Prophylactic and therapeutic activities in mice
Antiviral effects with reduced inflammatory responses in ferrets
Malakhov MP, Aschenbrenner LM, Smee DF, et al. Sialidase fusion protein as a novel broad-spectrum inhibitor of influenza virus infection. Antimicrob Agent Chemother. 2006;50:
Malakhov et al. Antimicrob Agent Chemother 50:1470, 2006

33. Effect of DAS181 on S. pneumo Binding to Human Airway Epithelium (HAE) Cells
DAS181 treatment had no significant effect on adherence.
Nicholls et al. J Antimicrob Chemother 62:426, 2008

34. DAS181 Treatment in Mice with H5N1
Dose of 1 mg/kg/d for 7-8 d
Inoculum of 3 MLD50
Time-to-treatment effects on survival and lung titers on day 3 and 6.
Belser et al. JID 196:1439, 2007

35. Literature review for reports of using convalescent blood products in treating pneumonia patients hospitalized in
8 studies; total of 336 treated patients
1,219 controls received supportive care
None blinded, randomized, or placebo-controlled
Overall mortality reduced from 37% to 16% (difference 21%; 95% CI, 15% - 27%).
Benefit if treated < 4 days after pneumonia Dx

36. Therapeutic Effect of Human Monoclonal Antibodies to A/Vietnam/1203/04 in Mice
Enhanced survival with treatment start delayed up to 72 hr post-infection.
Simmons et al. PLoS Med 4:e178, 2007

37. Potential Role of Combination Antiviral Therapy in Influenza Treatment
Combinations evaluated in animal models
Amantadine + interferon
M2 inhibitors + ribavirin
M2 inhibitors + oseltamivir
Oseltamivir + ribavirin
Combinations evaluated in humans
Oral rimantadine + nebulized zanamivir
Future considerations
Dual NAIs
Triple therapy: M2 inhibitor + ribavirin (or other transcriptase inhibitor) + IFN-α or NAI
Inclusion of other novel agents
Ong AK, Hayden FG. John F. Enders Lecture 2006: antivirals for influenza. J Infect Dis. 2007:196: p 186, col 1 para 3 to p 186, col 3, para 1.
Hayden FG. Antivirals for influenza: historical perspectives and lessons learned. Antivir Res. 2006;71: antiviral combinations: p 375 col 2 para 2.
Ong and Hayden. J Infect Dis 196:181, 2007; Hayden FG. Antivir Res 71:372, 2006

38. Zanamivir + Rimantadine
CASG* Trial of Nebulized Zanamivir + Rimantadine in Hospitalized Adults
Measure
Zanamivir + Rimantadine
Rimantadine alone
P value
No or mild cough,
day 3
15/16 (94%)
11/20 (55%)
.01
Days of hospitalization
4.7 ± 2.3
5.2 ± 2.3
.52
Frequency of rimantadine resistance 3 ND
Ison MG, Gnann JW Jr, Nagy-Agren S, Treannor J, Paya C, Steigbigel R, Elliott M, Weiss HL, Hayden FG; NIAID Collaborative Antiviral Study Group. Safety and efficacy of nebulized zanamivir in hospitalized patients with serious influenza. Antivir Ther. 2003;8: page 189, T5.
No or mild cough = 13% + 81% (Z+R), 20% + 35% (R only)
M2 inhibitor resistance: page 187 col 2 para 3, but isolates were only available for 21 patients total.
*CASG = Collaborative Antiviral Study Group.
Ison et al. Antiviral Ther. 2003;8:

39. Survival of mice inoculated with rg VN-1203/04 –Amantadine susceptible
Single-drug therapy
Combination therapy
AM 30 + OS 10
AM 30
AM 15 + OS 10
Survival distribution function
Survival distribution function
AM 15
OS 10
AS 1.5
Control
OS 1
Control
Days after inoculation
Days after inoculation
Ilyushina et al. Antiviral Therapy 12;363, 2007

40. Comparison of monotherapy with i. p
Comparison of monotherapy with i.p. zanamivir (ZNV), celecoxib, mesalazine, or gemfibrizol to triple regimen of ZNV + celecoxib + mesalazine in mice
High inoculum of A/Vietnam/1194/04 (103 LD50)
Therapy initiated at 48 hrs post-inoculation
No survival benefit of early therapy (4 hrs) with single agents except ZNV
Zheng et al. PNAS, on line 6/2008

41. Antiviral + Immunomodulator Therapy for H5N1 in Mice
 survival with ZNV + celecoxib + mesalazine
2/8 surviving mice in triple therapy group had detectable titers at day 21.

42. High throughput identification of host genes important for influenza replication by Drosophila RNAi screen
HEK293
COX6A1 : cyt-c dep transport in mitochondria-->PB2; PB1-F2?
ATP6V0D1: ATPase subunit --> endocytosis M2?
NXF1: mRNA exporter-->export unspliced mRNAs NS1?
Functional studies
Steps of virus multiplication?
Contribution to host range?
Hao et al 2008 Nature

43. Influenza Antivirals: Future Directions
Goal: Rapid inhibition of influenza viral replication at all affected sites
Near-term: parenteral NAIs
IV zanamivir or IV/IM peramivir
Next: antiviral combinations
NAI plus M2 inhibitors, polymerase inhibitor (T-705 or ribavirin), or neutralizing antibodies
Longer-term:
Antivirals with immunomodulators
Host function-targeted agents

44. Forthcoming Book from ASM Press
Third edition
Updates 2002 version
Available first quarter 2009
Cover art obtained from:

45. Back-up Slides

46. Pre-clinical assessment of arbidol toxicity and antiviral activity
Ethyl-6-bromo-4-[(dimethylamino)-methyl]-5-hydroxy-1-methyl-2-[(phenylthio)methyl]-indole-3-carboxylate hydrochloride monohydrate
Previous reports of activity for influenza, hepatitis B and C viruses
Shi et al. Arch Virol 152:1447, 2007

47. Influenza testing by CPE inhibition in MDCK cells
Arbidol causes overt cytotoxicity at >16 ug/ml
Broad spectrum; narrow therapeutic index
Shi et al. Arch Virol 152:1447, 2007

48. In Vivo Activity of Arbidol
Murine model of A/PR/8/34(H1N1)
Drugs by oral gavage X 6 d starting 24 hr pre-virus
Up to ~3 log10  lung virus titers
LD50 of 314 mg/kg/d for arbidol
Narrow TI
Shi et al. Arch Virol 152:1447, 2007

49. Kinetic analysis of NA (sialidase) activity
Whole virus suspensions of isolates from and prior seasons
Vm (reflecting enzyme activity) similar in susceptible and resistant isolates from but both ~3X  than in earlier H1N1 viruses
Km (reflects substrate affinity) ~2X  in susceptible H1N1 from than earlier; intermediate for oseltamivir-resistant isolates
Rameix-Welti et al. PLoS Pathogens 4:e , 2008

50. Growth of H1N1 Viruses from 2007-8 in MDCK SIAT-1 Cells
Replication of oseltamivir-resistant H1N1 (H274Y) isolates not impaired in vitro compared to susceptible H1N1 viruses from or earlier. S R S R
Rameix-Welti et al. PLoS Pathogens 4:e , 2008

51. NA Gene Phylogeny
NA substitutions found in majority of H1N1 from include 3 near catalytic site (222, 249, 344).
 NA affinity for substrate and NAIs may have altered HA-NA balance to  fitness ?
Rameix-Welti et al. PLoS Pathogens 4:e , 2008

52. Clinical Experience Suggests No Role for Corticosteroids in A(H5N1) Treatment
Survival
Steroid Rx
No steroids
P-value
Hanoia
12/29 (41%)
29/38 (76%)
0.008
Published casesb
3/19 (16%)
10/15 (66%)
0.007
Vietnam
a Cao T, Liem NT. N Engl J Med 2008; 358: 261
b Emerg Infect Dis 2005; 11: 201; N Engl J Med 2004; 350: 1179;
N Engl J Med 2006; 355:

53. Convalescent Plasma Therapy in H5N1 Disease
Case report of 31 yo male who presented with 4 day Hx of fever, cough, and sputum
CXR on day 6 showed LLL pneumonia
Tracheal aspirate + H5N1 by RT-PCR and culture
Oseltamivir 150 mg bid started day 9 of illness but progressive bilateral pneumonia
Convalescent plasma infusions from H5 survivor (200 ml X 3) on days 12-13
Plasma neutralizing ab titer of 1:80
Hospital discharge on day 30
Zhou et al. NEJM 357:1450, 2007

54. Convalescent Plasma Therapy in H5N1 Disease
Relative contributions of exogenous plasma, endogenous immune responses, and oseltamivir ?
Zhou et al. NEJM 357:1450, 2007

55. H5N1 hyperinduces COX-2 and proinflam-matory cytokine RNAs in macrophages but not type 2 alveolar epi cells, compared to H1N1.
COX-2 expressed in epithelial cells of autopsy lung tissue of H5N1 patients