Inhalable Dry Powder Aerosol Attenuated Live Virus Measles VaccineR. Sievers1,3, S. Winston1, 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

1Aktiv-Dry LLC, 6060 Spine Rd., Boulder, CO 80301 USA, 2Serum Institute of India Ltd., Pune, Maharashtra, India 411028, 3University of Colorado, Boulder, CO 80309-0214, USA

ABSTRACTNeedle-free aerosol delivery of dry powder vaccines may provide an effective and low-cost means of immunization1-5. Powder manufacturing with gentle, rapid drying by Carbon Dioxide Assisted Nebulization with a Bubble Dryer (CAN-BD) appears to be an attractive alternative to freeze drying of sensitive biologicals like live attenuated Edmonston-Zagreb measles virus. Stability of measles virus in glassy, dry, respirable microparticles made by CAN-BD with less than 1% water sealed in unidose aluminum foil blisters has been demonstrated. The inhalable microparticles have a high fine particle fraction (>15% less than 3.1 microns aerodynamic diameter), which is optimal for alveolar deposition. The dry powder aggregate, upon dispersion from an active dry powder inhaler, deposits in the moist respiratory tract where the microparticles rapidly dissolve within minutes. Viral replication has been demonstrated by RT-PCR, followed by a measles-specific immune response confirmed with plaque reduction neutralization assays in rodent and non-human primate models. To achieve a powder that was stable, easily dispersed and rapidly reconstituted, myo-inositol was substituted for sorbitol, historically used in lyophilized SIIL vaccine. Dry powders have an advantage over lyophilized vaccines for conventional subcutaneous injection or wet mist aerosol delivery in that no water-for-injection, which must be carried to the field in mass vaccination campaigns, is required. Funded by FNIH Grant 1077.

Freeze-fracture SEM of E-Z sub-micron measles virus encased in myo-inositol-stabilized excipient microparticles. 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.

NEEDLE-FREE DELIVERY VACCINE DEVELOPMENT• Needle-free vaccination by inhalation of an aerosol of dry powder.• 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 safety of the myo-inositol stabilizer in Sprague-Dawley rats and the inhaled E-Z live-virus vaccine in Rhesus macaques, an IND will be submitted for Phase I clinical trials in India.

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)

GRAND CHALLENGE 3: NEEDLE-FREE DELIVERYWhat 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.

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

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

ACKNOWLEDGEMENTSFunded in part by a grant from the Foundation for the National Institutes of Health through the Grand Challenges in Global Health initiative. The authors thank B. Papahadjopoulos-Sternberg (Nano Analytical Laboratory), S. Godin (Bridge Laboratories), K. Powell (BD Technologies), C. Shermer (BD Technologies), L. Chan (BD Technologies), N. Wolters, S. Buckingham, S. Evans, N. Breitnauer, J. Carpenter, M. Hernandez and L. Lindsay for their technical support. We are also grateful to the 11 members of Aktiv-Dry’s Product Development Advisory Group for many helpful suggestions.

REFERENCES1. R.E. Sievers, J.A. Best, and S.P. Cape, "Human-Powered Dry Powder

Inhaler and Dry Powder Inhaler Compositions", US Patent Application 20080035143, February 14, 2008.

2. R.E. Sievers, S.P. Sellers, and J.F. Carpenter, "Supercritical fluid-assisted nebulization and bubble drying," U.S. Patent 6,630,121; October 7, 2003.

3. J.L. Burger, S.P. Cape, C.S. Braun, D.H. McAdams, J.A. Best, P. Bhagwat, P. Pathak, L.G. Rebits, R.E. Sievers, "Stabilizing Formulations for Inhalable Powders of Live-Attenuated Measle Virus Vaccine", J Aerosol Med Pulm Drug Deliv, 21 (1): 25-34, 2008.

4. S.P. Cape, J.A. Villa, E.T.S. Huang, T-H Yang, J.F. Carpenter, R.E. Sievers, "Preparation of Active Proteins, Vaccines and Pharmaceuticals as Fine Powders using Supercritical or Near-Critical Fluids", Pharmaceutical Research, 25 (9):1967-1990, 2008.

5. R. E. Sievers, S. P. Cape, K. O. Kisich, D. J. Bennett, C. S. Braun, J. L. Burger, J. A. Best, D. H. McAdams, N. A. Wolters, B. P. Quinn, J. A. Searles, D. M. Krank, P. Pathak, P. A. Bhagwat, and L. G. Rebits, "Challenges of Developing a Stable Dry Powder Live Viral Vaccine", Proceedings of the Respiratory Drug Delivery 2008, R.N. Dalby, P.R. Byron, J. Peart, and J.D. Suman (eds.), May 11–15, Scottsdale, AZ (USA), pp. 281–290 (2008).

6. 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.

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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.

• 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.

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 (see photo at right); 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.

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 (see Rota et al. Poster 305, adjacent).

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

• A Phase I clinical trial is planned in 2010.

• Lam et al.6 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.

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

Structure of myo-inositol