Inhalable Dry Powder Aerosol Attenuated Live Virus Measles

Vaccine

R. Sievers1,3, S. Winston1, D. Griffin, P. Rota, B. Quinn1, J. Searles1, D. Krank1, P. Bhagwat1, P. Pathak1, L. Rebits1, R. Dhere2, V. Vaidya2, R. Muley2, J. Burger3, D. McAdams3, S. Cape1,3

K.Powell4 , C. Shermer4, L. Chan4 and K. Kisich5

1Aktiv-Dry LLC, 6060 Spine Rd., Boulder, CO USA, 2Serum Institute of India Ltd., Pune, Maharashtra, India,

3University of Colorado, Boulder, CO USA4BD Technologies, Research Triangle Park, NC, USA

5National Jewish Medical and Research Center, Denver, CO USA

GRAND CHALLENGE 3: NEEDLE-FREE DELIVERY

What is specifically needed:

• Well-formulated free flowing stable microparticles with less than 1% residual moisture, with high virus titers, rapidly dissolved, to replicate and create an immune response.

• Inexpensive unidose blister packs or capsules with overwraps to protect vaccine from contamination, decomposing, reaction with water, oxidants and UV, and to reduce vaccine wastage. (Presently 40% is destroyed.)

• Simple active dry powder inhalers that can disperse powder agglomerates to generate high emitted doses with high fine particle fractions.

ADVANTAGES OF AEROSOL DRY POWDER OVER LIQUID VACCINES

• Powders inherently more stable than liquids

• No line current or batteries required for aerosolization

• Lower risk of disease transmission

• Less vaccine wastage

• More difficult to contaminate

• No skin puncture or contact with blood

• No re-use of needles

• No need for water-for-injection (WFI)

NEEDLE-FREE DELIVERY VACCINE DEVELOPMENT

• Needle-free vaccination by inhalation of an aerosol of fine dry powder microparticles,~1-5 microns MMAD

• In-vivo rapid dissolution in moist respiratory mucosa

• Replication of live-attenuated measles vaccine virus

• Robust immune response demonstrated by plaque neutralization in two animal models: Rhesus macaques and Cotton rats; macaques will be challenged with wild-strain of measles virus one year after immunization.

• After demonstration of inhalation safety of the myo-inositol stabilizer in Sprague-Dawley rats and the E-Z live-virus vaccine in Rhesus macaques, an IND will be submitted for Phase I clinical trials in India.

PATH TO COMMERCIALIZATION

• CAN-BD is a closed, continuous process similar to spray-drying (but with a proprietary nebulizer), and is potentially faster and less expensive than freeze-drying.

• Demonstrated ability to manufacture sufficient materials for pre-clinical and early phase human clinical trials.

• Pilot scale GMP CAN-BD system has been designed, built, and installed at SIIL; scale-up to 400 million doses per year will be required for full-scale commercial production.

• Unidose packaging in blisters or capsules will prevent cross-contamination, circumventing vaccine wastage.

Particles formed at 50 - 60 °C from an aqueous solution containing 11% total dissolved solids (50 g/L myo-inositol, 25 g/L gelatin, 16 g/L arginine-HCl, 1 g/L alanine, 2.1 g/L histidine, 3.5 g/L lactalbumin hydrolysate, 3 g/L tricine, pH 6.5 - 7.0). Microparticles averaged 1 µm in diameter and encased spherical virus particles averaged 120 nm.

Mean viral potency of measles vaccine powder samples was 4.6 log CCID50 / 10 mg.

FREEZE-FRACTURE SEM OF E-Z SUB-MICRON MEASLES VIRUS ENCASED IN MYO-INOSITOL-

STABILIZED EXCIPIENT MICROPARTICLES

SAFETY OF MYO-INOSITOL STABILIZER AND REFORMULATED E-Z LIVE-VIRUS MEASLES

VACCINE• Successfully completed in-life phase of GLP toxicology

study in Sprague-Dawley rats. Final report not yet available, but no deaths or serious adverse events from myo-inositol inhalation reported to date.

• Inhalation of dry powder measles vaccine by macaques in a pilot immunogenicity study was well tolerated with no deaths or adverse events reported. A robust measles-specific immune response was generated.

• An inhalation toxicology study in additional Rhesus macaques is planned for next year.

• A Phase I clinical trial is planned in 2010.

SAFETY… (Continued)

• Lam et al.* have completed a Phase I clinical trial of orally ingested myo-inositol powder with no SAEs reported following intake of up to 18 g per day for one month; Phase II clinical trials are beginning in Canada.

• Normal concentration of myo-inositol in human plasma is about 4-5 mg/liter.

• Intracellular concentration is 5-500x higher than in plasma.

• Average dietary intake is 1 gram in the form of inositol hexaphosphate or myo-inositol in phospholipids.

• Human milk contains about 450 mg/liter of myo-inositol.*S. Lam, A. McWilliams, J. leRiche, C. MacAulay, L. Wattenberg, E. Szabo, "A Phase I Study of myo-Inositol for Lung

Cancer Chemoprevention", Cancer Epidemiol Biomarkers Prev, 15 (8): 1526-1531, 2006.

Structure of myo-inositol

SEM of Tsuno (rice-derived) myo-inositol (modified with amino acid) microparticles produced by CAN-BD for inhalation safety study in rats

5 August 2008 FNIH Grant 1077 10

Modifications to CAN-BD for Processing Larger Volumes of Vaccine Fluids

• Used to produce batches of powder (6 to 8 grams): Solovent and PuffHaler stability studies 2nd round of Cotton rat study Monkey immunogenicity study

GMP CAN-BD system installed and manufacturing microparticulate live-virus measles vaccine at the Serum Institute of India in Pune

MYO-INOSITOL BASED FORMULATION STABILITY IN AKTIV-DRY PUFFHALER™ BLISTERS

Viral potency stability of the reformulated E-Z live-attenuated measles vaccine microparticles at 5 °C sealed in Aktiv-Dry PuffHaler blisters.

1

10

100

1000

10000

100000

0 4 8 12 16 20 24Incubation Time (Weeks)

PFU / 10 mg

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

Log CCID50 / 10 mgAD PFU 5 °C AD CCID50 5 °C

Requirements– Deliver microparticles to subjects from 9 months to 40 years of age with active

dry powder inhaler devices using air pressurization, then depressurization» B-D’s Solovent® and Aktiv-Dry’s PuffHaler® DPIs are both designed

specifically to disperse the powder- with only muscle power- into the necessary size range to coat the entire respiratory tract

– Micoparticles with Mass Median Aerodynamic Diameter ~3 m, with a high fine particle fraction to encase and preserve virus nanoparticles

» ACI testing of both the Solovent and PuffHaler has shown dispersive performance similar to an FDA-approved DPI

– Readily deliverable and acceptable in developing countries with minimal caregiver training

» Both systems can be used without line-power or batteries, and no water for reconstitution is required

Active Dry Powder Inhaler Specifications

The Devices

The PuffHaler (L) and Solovent (R) inhalers configured for delivery through a mask.

SOME ADVANCES TO CELEBRATE

• Live-attenuated E-Z measles vaccine was reformulated as microparticles that rapidly dissolve in moist respiratory mucosa and become the functional equivalent of the wet-mist measles vaccine successfully administered to more than 3 million children.

• Dried and micronized stabilized live virus measles vaccine without greater loss of viral activity than in the present commercial lyophilization process.

• Pressure release valves or rupturable membranes facilitate dispersion and create stable aerosol clouds comparable to an FDA-approved active DPI.

• Greatly reduced cost and complexity of DPI‘s.

SOME ADVANCES TO CELEBRATE (Continued)

• Reduced water content of powders to < 1% and increased fine particle fractions (FPF) to ~20% (w/w) FPF < 3.3 µm and ~46% (w/w) FPF < 5.8 µm.

• Myo-inositol stabilized powders pass the WHO test for stability at 37 °C for one week with less than one log loss in viral activity.

• Generation of immune responses in Cotton rats and Rhesus macaques.

• Development of myo-inositol as a stabilizing excipient; no general safety issues observed in two animal models and awaiting data analysis from formal GLP toxicology study of inhaled myo-inositol.