FAPESP WEEK WASHINGTON, DC

PROF. JORGE KALIL October 24th – 26th, 2011 AGENDA

 BUTANTAN - SCIENCE IS THE BASIS

 BUTANTAN – SOME PROJECTS

Instituto Butantan | 1 BUTANTAN – A PUBLIC INSTITUTION OF THE STATE GOVERNMENT OF

 In 1901 Butantan was established to produce serum against the  Vital , the first director, investigated against bites

 Currently, Butantan is the main public producer of , antivenoms, antitoxins in  Fully dedicated to develop scientific research and production of immunebiological products for public

Instituto Butantan | 2 BRAZIL HAS BECOME AN INTERNATIONAL REFERENCE IN IMMUNIZATION

Strategy Brazil decided in the mid 80's to become self-sufficient in vaccines and immunization programs

Decision

This was a State decision rather than a government decision

Why

Too important to depend on availability and pricing

Instituto Butantan | 3 NATIONAL IMMUNIZATION PROGRAM (PNI)

PNI – Founded in 1973

PNI – General Information of Brazil (2010) . Smallpox Eradication . Poliomyelitis . Measles (autocne transmission) . 193 MM inhabitants

. 43 types of immunobiological

 26 Vaccines  13 sera from animal . Neonatal  4 sera from human Under . Accidental tetanus Control . . 77% produced in Brazil

. . ~ 300 MM doses of vaccines per year . Pertussis . 30 K vaccination rooms . Hepatite B . Expansion of national self-sufficiency . Influenza . Pneumococcus

Source: SVS/Ministry of Health, National Immunizatation Program, Brazil, 2010. Instituto Butantan | 4 NATIONAL SUPPLIERS OF VACCINES FOR THE MINISTRY OF HEALTH

‘Market Share’ per Suppliers(1) Products (2010)

FUNED 3% FAP 4% DTP DT dT

Butantan Biomanguinhos 51% 42% Anti B

Note: Part of Butantan’s production was sent to other Institutes, Influenza such as Biomanguinhos. Not computed in the analysis (Flu)

1Source: Ministry of Health, 2010 Instituto Butantan | 5 NATIONAL SUPPLIERS OF ANTIVENOMS AND ANTITOXINS FOR THE MINISTRY OF HEALTH

‘Market Share’ per Suppliers(1) Products (2010)

Snakes AV

IVB 25%

Butantan AV AV Caterpillar AV 56% FUNED 19%

Tetanus AT Diphitheria AT Anti Rabies

Note: Part of Butantan’s production was sent to other AV Institutes, such as FUNED and CPPI. Not computed in the analysis AB & E

1Source: Ministry of Health, 2010 Instituto Butantan | 6 EXPORTING PRODUCTS

Instituto Butantan | 7 VACCINES WITH EXTERNAL COOPERATION

Cooperation Projects NIH-PATH Rotavirus (pentavalent) NIH-DVI Dengue (tetravalent) Sabin Institute - George Washington University Necator - Schistosoma

Children’s Hospital Harvard - PATH Pneumococcus (cellular) Infectious Diseases Research Institute Leishmaniosis (for dogs) Ludwig Institute for Cancer Research Adjuvant for ovarian cancer BR Foods Lung Surfactant Recombinant OncoBCG for bladder Universidade de São Paulo – Medical School cancer Institut Pasteur – / Novartis - Siena / Recombinant BCG-Pertussis Albert Einstein College of Medicine

Instituto Butantan | 8 INDEXED SCIENTIFIC PUBLICATIONS

PAPERS PUBLISHED AND CITED

2500 250 sum of times cited (TOTAL) Papers Published 2000 Poly. (sum of times cited (TOTAL)) 200 Expon. (Papers Published)

1500 150

1000 100

500 50

0 0 1980 1985 1990 1995 2000 2005 2010

Instituto Butantan | 9 SCREENING OF BIOACTIVE COMPONENTS OF POISONOUS ANIMALS

Venom Composition Species Pharmacological activities (Transcriptomics and Proteomics)

 ANALGESICS

 ANTI- INFLAMMATORY

 ANTICOAGULANTS

 ANTITUMOR

 ACTION IN THE NERVOUS SYSTEM

 ANTIVENINS

 ANTIHIPERTENSIVE

Instituto Butantan | 10 PATENTS

Derived from animal secretions (32)

LOPAP (4 PATENTS APPLICATIONS) • Prothrombin activator, • defibrinogenating/ antithrombotic agent • kit for diagnosis of dysprothrombinemias • anti-apoptotic activity

TICKS’ SALIVARY GLANDS • A new anticoagulant and anti-tumoral agent • Microarray analysis

Lonomia obliqua

Amblyomma cajennense

Instituto Butantan | 11 PATENTS

Biotechnology (16)

New developments in Pertussis Vaccines with Appropriate technologies

Whole Cell Pertussis Vaccine

Low LPS content- Whole Cell Pertussis less reactogenic – low cost

Infants B. pertussis WCP WCPLow fermentation Tangential Organic Patent filtration extraction Clinical Trials IMMUNOGENIC COMPLEX FORMED BY VACCINAL 2007 ANTIGENS ENCAPSULATED BY NANOSTRUCTURED Adolescents MPLA Acellular MESOPOROUS SILICA Adjuvant Neonates Pertussis Patent CRISTALOGRAPHY Vaccine Clinical Trials planned 2009 LABORATORY Recombinant BCG-Pertussis Patent Immunization at birth Patent Under GMP production

Instituto Butantan | 12 RESEARCH & DEVELOPMENT LABORATORIES

. ~21 scientific labs

. ~180 Researchers  85% are PhD

. 1 Center  Multiple laboratories

. 1 Hospital (10 hospital beds)

. 1 Central Animal Facility

. Training programs (PAP)

. Graduate studies in Toxicology

. Masters and PhDs Instituto Butantan | 13 INDUSTRIAL COMPLEX

. 7 Main Industrial Plants (Buildings)  Anaerobic vaccines (tetanus and botulinic ) and Anatoxin Purification  Biological control  Aerobic Vaccine (Diphtheria and Pertussis)  Hepatitis  Influenza  Rabies  Blood Products (under construction)  Control, Serums, Formulation and Filling

. 6 Pilot Plants  Dengue / Rotavírus (Under Construction)  Recombinant (BCG)  Monoclonal Antibodies  Influenza  Blood Products

Instituto Butantan | 14 AGENDA

 BUTANTAN - SCIENCE IS THE BASE

 BUTANTAN – SOME PROJECTS

Instituto Butantan | 15 Presentation and discussion of some Projects

Area Projects 1 Vaccines for immunepreventable . Rotavirus (pentavalent) diseases . Dengue (tetravalent)

Research and improvement . Adjuvant BpMPLA . Lung Surfactant . Silica nanostructure mesoporous – vaccine antigens encapsulated . Monoclonal antibodies (anti-human CD3) . Clotalfina (analgesic product) . Amblyomin-X (anti cancer) . Lopap (antithrombotic agent) 1 Not exhaustive

Instituto Butantan | 16 Rotavirus Vaccine - Overview

Product – Pentavelent Rotavirus Vaccine Butantan –

. Composition . Challenges:

– Attenutated – To perform Phase II and III - non-inferiority study – Sorotypes: G1, G2, G3, G4 e G9 – To find funding for: . Technology of Production  Clinical Trial and laboratory assay – Phase II / III – Cell substrate: Vero cells

– Reassortment – Human/bovine . Objective: – Nº lots produced: 09 (6.324 doses) – Pentavalent low cost vaccine . Phase of Development – Phase I: 2010  Results: safe and immunogenic – Phase II: 2012

• Partnership – NIH / PATH / BNDES Instituto Butantan | 17

Dengue Vaccine - Overview

Product – Tetravalent Butantan –

. Composition . Challenges:

– Attenuated virus – To speed up Phase I, II, and III – (to avoid – Sorotypes: DEN1, DEN2, DEN3, DEN4 non-inferiority study)

. Technology of Production – To find funding for:  Clinical Trial and Laboratory assay - Phase II – Cell substrate: Vero cells and III – Recombinant DNA technology  Equipment – Chimeric  Plant – Nº lots produced: 06 (12.640 doses)  Maintenance of “The Global Solutions for Infectious Disease” support . Phase of Development – To define target population for immunization – Phase I: 2011/2012 – Production capacity x national and international demand

• Partnership . Objective: – NIH / DVI / BNDES / FAPESP – Tetravalent low cost vaccine

Instituto Butantan | 18

Dengue Vaccine - technical and scientific aspects Attenuation strategies for Dengue virus and Pre-Clinical studies

Attenuation strategies for Dengue virus Pre-Clinical Studies vaccine candidates

 30 nucleotide deletion in DENV 3’ UTR = 30  SCID-HuH-7 mice mutation  The candidate vaccine were attenuated,  rDEN1 30 and rDEN4 30 replicating to a peak titer from 100.9 up to 102.4 PFU/mL  The D30 mutation partially attenuated DEN2 and did not attenuated DEN3 for rhesus monkeys  Rhesus macaques

1.0 1.3  Antigenic chimeras - rDEN4 30 background  Low/undetectable levels of viremia in (10 to 10 PFU/mL) . rDEN2/4 30 – infectious in monkeys and  Immunogenic humans

. rDEN3/4 30 – infectious in monkeys and  Mosquitoes poorly infectious in humans  Vaccine viruses restricted ability to infect the midgut  DEN3/4 30 and to cause a disseminated (Ae. Aegypti - Toxorhynchites splendens)

 Additional 3’-UTR deletion mutations  Ae. albopictus fed on viremic subjects - vaccine virus was not recovered from any mosquitoes . rDEN3 30/31 – infectious in monkeys and humans

Instituto Butantan | 19 Adjuvant – Monophosphoryl lipid A (BpMPLA)

Product - Monophosphoryl lipid A (BpMPLA) Butantan –

. Composition . Challenges: – BpMPLA derived from LPS of B.pertussis – Scale-up

. Production Technology . Objectives: – Purification of B.pertussis followed by LPS – To optimize immunolgical response of pre- hydrolises exisiting and new vaccines . Phase of Development – To increase production capacity

– Scale-up BpMPLA – Clinical trial  Pandemic H1N1 + BpMPLA – Pre-clinical  Human rabies – Animal Study  Dog Leishimania – In development  Hepatites B + BpMPLA  Seazonal Influenza + BpMPLA

Instituto Butantan | 21 Adjuvant – Monophosphoryl lipid A (BpMPLA)

Pathogen-Associated Molecular Patterns (PAMPs) and Danger- Associated Molecular Patterns are potential new adjuvants

Monophosphoril Lipid A (MPLA) derived from Bordetella pertussis as a new vaccine adjuvant

Instituto Butantan | 22 Lung Surfactant

Product – Surfactant Butantan –

. Composition . Challenges:

– Origin porcine – To test a new process of production (ultracentrifugation) and formulation – 2nd – Phospholipids + Proteins SP-B and SP-C generation of surfactant

. Production Technology

– Porcine pulmonary extract adsorbed on a cellulose derivative followed by organic . Objectives: extraction, solvent evaporation, lyophilize drying and final formulation – To make available a low cost surfactant to Brazilian Public Maternities . Phase of Development

– Licensed in Brazil in 2009

Instituto Butantan | 23 Lung Surfactant - technical and scientific aspects

Alveolus

Normal

Sindrome

Respiratory

Surfactant Distress

Lung Parenchyma Instituto Butantan | 24 Silica (SBA-15) Immunogenic complex formed by vaccinal antigens encapsulated by nanostructured mesoporous silica

Features  The SBA-15 possesses hexagonal porous uniformity (3.1 – 6.5 nm)  Thermal and hydrothermal stability  Exhibits potential applications for selective adsorption and catalysis

Instituto Butantan | 25 Adjuvant effect of SBA-15

Instituto Butantan | 26 Modulation of antibody response - Silica

10 μg/animal de BSA:250 μg/animal de SBA-15 ou Al(OH)3

IM VO

Source: Carvalho et al. 2010. Instituto Butantan | 27 Monoclonal Antibodies Anti-human CD3

AcMo Murine AcMo Quimeric AcMo Humanized

. Crohn's disease . Encephalomyocarditis autoimmune POTENTIAL . Psoriatic arthritis CLINICAL USES . Diabetes Type I

. Lupus erythematosus anti-human CD3 . Control of acute rejection in kidney transplant . Prevention of rejection in transplanted pancreatic islet . Steroid-refractory acute GVHD

Instituto Butantan | 28 Clotalphine Potent analgesic drugs from animal and

Crotalphine Molecular mechanisms involved in the analgesic action

Venom of Activation of CB2 cannabinoid receptors

release of endogenous Potent analgesic opioids long-lasting analgesic action (2-5 days) Activation of peripheral and opioid receptors

Crotalus intracellular signaling pathways activation Recent studies show involvement of PKC Activation of NO-cGMP-PKG which activates MAPKs and opening of potassium (Mechanism that may contribute to its long-lasting channels analgesic action)

Instituto Butantan | 29 Amblyomin-X ® An anti-cancer from Amblyomma cajannense

Experiments were performed in C57BL/6J mice

Amblyomin-X ® A Amblyomma cajennense

Scale-up Production

Tumor implants - Suspensions of B16F10 (2.5 x 104) cells subcutaneously injected

Kunitz type Inhibitor

. Partnership – União Química / BNDES / FAPESP

. Phase of Development – Pre-clinical and clinical studies Experimental model: Macroscopic aspect of nude dorsal Mia-PaCa-2 and SKMel-28 tumors ® = Patents (INPI, PCT)

Source: Chudzinski-Tavassi et al. Toxicon 2010 Instituto Butantan | 30 Lopap ® – Patents Application A prothombin activator from Lonomia obliqua

1- INPI (PI0200269-8, Brazil, 2002) Prothombin Activator Chudzinski-Tavassi AM, Reis CV. Native prothrombin activator. WO03/070746 / AU2003208190 / CAN2,471,410 / EP1482969 / JP2003- 569653 / MX04007344 / US10/501,238 2- INPI (PI0403882-7, Brazil, 2004). Antithrombotic Chudzinski-Tavassi AM, Reis CV, Ramos CR, Ho PL. Recombinant molecule Agent and its use as defibrinogenating/ antithrombotic agent and a kit for diagnosis of dysprothrombinemias PCT/BR2005/000171 Lopap Anti apoptotic 3- INPI (PI0504199-6, Brazil, 2005). activity Reis CV, Maria DA, Falsi M, Chudzinski-Tavassi AM. Anti-apoptotic activity of CNF 021.01 and pharmaceutical formulations

4- USPTO Peptide, compositions, and uses thereof. Chudzinski-Tavassi AM, USPTO Carrijo-Carvalho LC, Ventura JS, inventors. US patent application. 2009.

L. obliqua

Instituto Butantan | 31 Av. Vital Brasil, 1500 - Butantã São Paulo – SP Zip Code - 05503-900 (+ 55 11) 3726.7222 www.butantan.gov.br

Instituto Butantan | 32