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

Antiviral Agents for Influenza

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

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

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

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

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

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 CDC unpublished; Barr et al. Antiviral Res 2006; R Saito, Niigata Univ, unpublished

Oseltamivir Resistance in H1N1 (H274Y) Rare in community H1N1 isolates, 1996-2007 0 to <1% in most surveys 2.2% in Japan in 2005-6 (but not in 2006-7 or early in 2007-8) High prevalence in Europe and globally, 2007-8 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.

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- 1Q08 *2Q08-20Aug08 http://www.who.int/csr/disease/influenza/h1n1_table/en/index.html; 20 Aug 08

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

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

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

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

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

Investigational Anti-Influenza Agents Neuraminidase (NA) inhibitors - Zanamivir (IV), peramivir (IV/IM), A-315675 (oral) Long-acting NA inhibitors (LANI) CS8958/R-118958 (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

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

Neuraminidase Inhibitors Peramivir

NA Inhibitor Resistance Profiles NA mutation NA type/ subtype Susceptibility in the NAI assay (fold ) Oselt Zana Peram A-315675 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

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

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

IM Peramivir: Time to Alleviation of Illness J Alexander, BioCryst Pharmaceuticals, unpublished data

Long Acting Neuraminidase Inhibitors (LANI)- 2 Strategies CS-8958 FLUNET R-125489 Pro-drug Clinical Dimer Preclinical

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

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

T-705: Summary of Pre-Clinical Findings Inhibitory for influenza A, B, C viruses: EC50s 0.01 - 0.5 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

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)

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;49:981-986. H1N1 cytotoxicity: p 982, T1 Furuta Y, et al. Antimicrob Agents Chemother. 2005;49:981-986.

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;51:845-851. Treatment window: p 850, Fig 2. Delay before starting treatment following viral exposure, hours Sidwell RW, et al. Antimicrob Agent Chemother. 2007;51:845-851.

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

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

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;50:1470-1479. Malakhov et al. Antimicrob Agent Chemother 50:1470, 2006

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

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

Literature review for reports of using convalescent blood products in treating pneumonia patients hospitalized in 1918-20 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

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

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. 2007:196:181-190. 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;71:372-378. antiviral combinations: p 375 col 2 para 2. Ong and Hayden. J Infect Dis 196:181, 2007; Hayden FG. Antivir Res 71:372, 2006

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;8:183-190. 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;8:183-190.

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

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

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.

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

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

Forthcoming Book from ASM Press Third edition Updates 2002 version Available first quarter 2009 Cover art obtained from: http://estore.asm.org/viewItemDetails.asp?ItemID=311

Back-up Slides

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

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

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

Kinetic analysis of NA (sialidase) activity Whole virus suspensions of isolates from 2007-8 and prior seasons Vm (reflecting enzyme activity) similar in susceptible and resistant isolates from 2007-8 but both ~3X  than in earlier H1N1 viruses Km (reflects substrate affinity) ~2X  in susceptible H1N1 from 2007-8 than earlier; intermediate for oseltamivir-resistant isolates Rameix-Welti et al. PLoS Pathogens 4:e1000103, 2008

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 2007-8 or earlier. S R S R Rameix-Welti et al. PLoS Pathogens 4:e1000103, 2008

NA Gene Phylogeny NA substitutions found in majority of H1N1 from 2007-8 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:e1000103, 2008

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: 2186-94.

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

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

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