1. Inhalable Dry Powder Aerosol Attenuated Live Virus Measles Vaccine
R. 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
1
Aktiv-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
ABSTRACT Freeze-fracture SEM of E-Z sub-micron
100000 5.0
measles virus encased in myo-inositol-
Needle-free aerosol delivery of dry powder vaccines may stabilized excipient microparticles. Particles
4.5
provide an effective and low-cost means of immunization1-5. formed at 50 - 60 °C from an aqueous solution
10000 4.0
Log CCID50 / 10 mg
Powder manufacturing with gentle, rapid drying by Carbon containing 11% total dissolved solids (50 g/L
3.5 Viral potency stability of
PFU / 10 mg
Dioxide Assisted Nebulization with a Bubble Dryer (CAN-BD) myo-inositol, 25 g/L gelatin, 16 g/L
1000 3.0 the reformulated E-Z
arginine-HCl, 1 g/L alanine, 2.1 g/L histidine,
2.5 live-attenuated measles
appears to be an attractive alternative to freeze drying of
3.5 g/L lactalbumin hydrolysate, 3 g/L tricine,
100 2.0 vaccine microparticles at
sensitive biologicals like live attenuated Edmonston-Zagreb 1.5 5 °C sealed in Aktiv-Dry
pH 6.5 - 7.0). Microparticles averaged 1 μm in
measles virus. Stability of measles virus in glassy, dry, diameter and encased spherical virus
10 1.0 PuffHaler blisters.
AD PFU 5 °C AD CCID50 5 °C
respirable microparticles made by CAN-BD with less than 1% particles averaged 120 nm.
0.5
water sealed in unidose aluminum foil blisters has been Mean viral potency of measles vaccine
1
0 4 8 12 16 20 24
0.0
demonstrated. The inhalable microparticles have a high fine powder samples was 4.6 log CCID50 / 10 mg. Incubation Time (Weeks)
particle fraction (>15% less than 3.1 microns aerodynamic SOME ADVANCES TO CELEBRATE
diameter), which is optimal for alveolar deposition. The dry SAFETY OF MYO-INOSITOL STABILIZER AND
REFORMULATED E-Z LIVE-VIRUS MEASLES VACCINE • Live-attenuated E-Z measles vaccine was reformulated as
powder aggregate, upon dispersion from an active dry
microparticles that rapidly dissolve in moist respiratory
powder inhaler, deposits in the moist respiratory tract where • Successfully completed in-life phase of GLP toxicology study in
Sprague-Dawley rats. Final report not yet available, but no deaths or mucosa and become the functional equivalent of the
the microparticles rapidly dissolve within minutes. Viral
serious adverse events from myo-inositol inhalation reported to date. wet-mist measles vaccine successfully administered to
replication has been demonstrated by RT-PCR, followed by a
• Inhalation of dry powder measles vaccine by macaques in a pilot more than 3 million children.
measles-specific immune response confirmed with plaque
immunogenicity study was well tolerated with no deaths or adverse • Dried and micronized stabilized live virus measles vaccine
reduction neutralization assays in rodent and non-human events reported. A robust measles-specific immune response was
primate models. To achieve a powder that was stable, easily without greater loss of viral activity than in the present
generated (see Rota et al. Poster 305, adjacent).
dispersed and rapidly reconstituted, myo-inositol was commercial lyophilization process.
• An inhalation toxicology study in additional Rhesus macaques is
substituted for sorbitol, historically used in lyophilized SIIL planned for next year. • Pressure release valves or rupturable membranes facilitate
vaccine. Dry powders have an advantage over lyophilized • A Phase I clinical trial is planned in 2010. dispersion and create stable aerosol clouds comparable to
vaccines for conventional subcutaneous injection or wet mist • Lam et al.6 have completed a Phase I clinical trial of orally ingested an FDA-approved active DPI.
aerosol delivery in that no water-for-injection, which must be myo-inositol powder with no SAEs reported following intake of up to • Greatly reduced cost and complexity of DPI‘s.
carried to the field in mass vaccination campaigns, is 18 g per day for one month; Phase II clinical trials are beginning in • Reduced water content of powders to < 1% and increased
required. Funded by FNIH Grant 1077. Canada.
fine particle fractions (FPF) to ~20% (w/w) FPF < 3.3 μm and
• Normal concentration of myo-inositol in human plasma is about 4-5
GRAND CHALLENGE 3: NEEDLE-FREE DELIVERY ~46% (w/w) FPF < 5.8 μm.
mg/liter.
What is specifically needed: • Intracellular concentration is 5-500x higher than in plasma. • Myo-inositol stabilized powders pass the WHO test for
• Well-formulated free flowing stable microparticles with less than 1% • Average dietary intake is 1 gram in the form of inositol hexaphosphate stability at 37 °C for one week with less than one log loss in
residual moisture, with high virus titers, rapidly dissolved, to replicate or myo-inositol in phospholipids. viral activity.
and create an immune response. • Human milk contains about 450 mg/liter of myo-inositol. • Generation of immune responses in Cotton rats and Rhesus
• Inexpensive unidose blister packs or capsules with overwraps to macaques.
protect vaccine from contamination, decomposing, reaction with water,
oxidants and UV, and to reduce vaccine wastage. (Presently 40% is • Development of myo-inositol as a stabilizing excipient; no
destroyed.)
Structure of general safety issues observed in two animal models and
• Simple active dry powder inhalers that can disperse powder myo-inositol awaiting data analysis from formal GLP toxicology study of
agglomerates to generate high emitted doses with high fine particle inhaled myo-inositol.
fractions. REFERENCES
NEEDLE-FREE DELIVERY VACCINE DEVELOPMENT 1. R.E. Sievers, J.A. Best, and S.P. Cape, quot;Human-Powered Dry Powder
• Needle-free vaccination by inhalation of an aerosol of dry powder. Inhaler and Dry Powder Inhaler Compositionsquot;, US Patent Application
20080035143, February 14, 2008.
• Rapid dissolution in moist respiratory mucosa
2. R.E. Sievers, S.P. Sellers, and J.F. Carpenter, quot;Supercritical
• Replication of live-attenuated measles vaccine virus
fluid-assisted nebulization and bubble drying,quot; U.S. Patent 6,630,121;
• Robust immune response demonstrated by plaque neutralization in October 7, 2003.
two animal models: Rhesus macaques and Cotton rats; macaques will
be challenged with wild-strain of measles virus one year after 3. J.L. Burger, S.P. Cape, C.S. Braun, D.H. McAdams, J.A. Best, P.
immunization. Bhagwat, P. Pathak, L.G. Rebits, R.E. Sievers, quot;Stabilizing
Formulations for Inhalable Powders of Live-Attenuated Measle Virus
• After demonstration of safety of the myo-inositol stabilizer in
Vaccinequot;, J Aerosol Med Pulm Drug Deliv, 21 (1): 25-34, 2008.
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. 4. S.P. Cape, J.A. Villa, E.T.S. Huang, T-H Yang, J.F. Carpenter, R.E.
Sievers, quot;Preparation of Active Proteins, Vaccines and
Pharmaceuticals as Fine Powders using Supercritical or Near-Critical
ADVANTAGES OF AEROSOL DRY POWDER OVER
Fluidsquot;, Pharmaceutical Research, 25 (9):1967-1990, 2008.
LIQUID VACCINES SEM of Tsuno (rice-derived) myo-inositol (modified with amino acid) 5. R. E. Sievers, S. P. Cape, K. O. Kisich, D. J. Bennett, C. S. Braun, J. L.
• Powders inherently more stable than liquids microparticles produced by CAN-BD for inhalation safety study in rats. Burger, J. A. Best, D. H. McAdams, N. A. Wolters, B. P. Quinn, J. A.
• No line current or batteries required for aerosolization Searles, D. M. Krank, P. Pathak, P. A. Bhagwat, and L. G. Rebits,
• Lower risk of disease transmission quot;Challenges of Developing a Stable Dry Powder Live Viral Vaccinequot;,
• Less vaccine wastage Proceedings of the Respiratory Drug Delivery 2008, R.N. Dalby, P.R.
• More difficult to contaminate Byron, J. Peart, and J.D. Suman (eds.), May 11–15, Scottsdale, AZ
• No skin puncture or contact with blood (USA), pp. 281–290 (2008).
• No re-use of needles 6. S. Lam, A. McWilliams, J. leRiche, C. MacAulay, L. Wattenberg, E.
• No need for water-for-injection (WFI) Szabo, quot;A Phase I Study of myo-Inositol for Lung Cancer
Chemopreventionquot;, Cancer Epidemiol Biomarkers Prev, 15 (8):
PATH TO COMMERCIALIZATION 1526-1531, 2006.
• CAN-BD is a closed, continuous process similar to spray-drying (but
ACKNOWLEDGEMENTS
with a proprietary nebulizer), and is potentially faster and less
Funded in part by a grant from the Foundation for the National Institutes
expensive than freeze-drying.
of Health through the Grand Challenges in Global Health initiative. The
• Demonstrated ability to manufacture sufficient materials for authors thank B. Papahadjopoulos-Sternberg (Nano Analytical
pre-clinical and early phase human clinical trials. Laboratory), S. Godin (Bridge Laboratories), K. Powell (BD
• Pilot scale GMP CAN-BD system has been designed, built, and Technologies), C. Shermer (BD Technologies), L. Chan (BD
installed at SIIL (see photo at right); scale-up to 400 million doses per Technologies), N. Wolters, S. Buckingham, S. Evans, N. Breitnauer, J.
year will be required for full-scale commercial production. Carpenter, M. Hernandez and L. Lindsay for their technical support. We
• Unidose packaging in blisters or capsules will prevent GMP CAN-BD system installed and manufacturing microparticulate are also grateful to the 11 members of Aktiv-Dry’s Product Development
cross-contamination, circumventing vaccine wastage. live-virus measles vaccine at the Serum Institute of India in Pune Advisory Group for many helpful suggestions.