MEDICAL AEROSOLS TO IMPROVE GLOBAL HEALTH

R.E. Sievers1,2, S. Winston, S.P. Cape1,2, D. Bennett1, J.L. Burger2, D.H. McAdams2, B.P. Quinn1, J.A. Searles1, D.M. Krank1, P. Pathak1, P. A. Bhagwat1, L.G. Rebits1, K.O. Kisich3, R. Dhere4, P. Kulkarni4, V. Vaidya4, R. Muley4, K. Powell5, C. Shermer5, L. Chan5, D. Griffin6 , W-H. Lin6 , P. Rota7, M. Papania7

1Aktiv-Dry LLC, 6060 Spine Road, Boulder, CO, USA 2Center for Pharmaceutical Biotechnology, Department of Chemistry and

Biochemistry, and CIRES, University of Colorado, Boulder, CO, USA 3National Jewish Medical and Research Center, Denver, CO, USA 4Serum Institute of India, Ltd., Pune, India5BD Technologies, Research Triangle Park, NC, USA6Johns Hopkins School of Public Health, Baltimore, MD, USA 7Centers for Disease Control and Prevention, Atlanta, GA, USA

A lecture to honor Dr. Fred Fehsenfeld for all he has done

for my group and for CIRESSept.24, 2008

Fehsenfeld and Sievers Group Collaborations

Over 3 decades, Fred Fehsenfeld and I have mentored together a number of students and post-docs:

Students:Susan Wells Buhr Jim RobertsTom Ryerson Michael PhillipsMartin Buhr Mark BollingerSteve Montzka Ric Hutte*Eric Williams

In addition, we have had several collaborators at both NOAA and CU with whom we have co-authored papers: Collaborators:

Paul GoldanRichard Norton Dan AlbrittonMichael Trainer David FaheyDavid Parrish Bill KusterBob Barkley* Barbara Watkins MilesRobert Meglen John Birks

Every generation may believe that it faces unprecedented and “the Worst” challenges:

Consider the 20th and 21st Centuries

• World War I, the “War to end All Wars.”• Global Influenza Pandemic 1918•The Great Depression of 1929•The Dust Bowl and collapse of farming• Racial and Religious Intolerance —

The Ku Klux Klan and the Holocaust• World War II/ Fascism and Communism• Nuclear and Biological Weapons 1950-1990• Cuban Missile Crisis and Viet-Nam War•Terrorism and 9/11• Global Pollution and Climate Change• Global Disease, Poverty, and Hunger• Energy, Housing, and Economic Turmoil

Henao, An Overview of Aerosol Immunization, Meeting of the WHO Steering Committee on New Delivery Systems, 2004

Henao-Restrepo and Papania, NIH Vaccines Conference, Rockville, Dec 2003

Dry Powder Measles Vaccine Team

Aktiv-Dry LLC, Prime Contractor; Bob Sievers ,Principal Investigator; funded by a grant from the Foundation for the National Institutes of Health in the Grand Challenges in Global Health initiative.Subcontractors:

Serum Institute of India BD TechnologiesUniversity of ColoradoNational Jewish Medical and Research CenterJohns Hopkins UniversityUniversity of KansasRPC Formatec

Consultants:John Carpenter, School of Pharmacy, CU Health Sciences CenterPaul Rota and Mark Papania, US Centers for Disease Prevention and Control (CDC)University of Maryland Vaccine Research CenterWinston Consulting Witham Consulting

Project Objectives• Synthesize a stable respirable microparticulate dry powder

measles vaccine by CAN-BD • Develop at least 2 simple, very inexpensive unidose active

DPI devices that can deliver well dispersed fine particles• Install GMP Bubble Dryer at the Serum Institute of India in

Pune, India (now operational)• Assess excipient and vaccine safety pre-clinically in rodents

and Non-Human Primates, then clinically in Phase I trials in India

• Deliverable: Successful completion of Phase I clinical trials for improved needle-free inhalable dry powder measles vaccine

GRAND CHALLENGE 3: NEEDLE-FREE DELIVERY

• Maintain viral activity of attenuated Edmonston-Zagreb measles vaccine through stabilization in glassy dry respirable microparticles upon drying, then in a state of suspended animation for many months.

• The aggregate, upon dispersion from an active dry powder inhaler, must deposit in the moist respiratory tract where microparticles will rapidly dissolve in mucosa and replicate many-fold over a few days to stimulate immune responses.

• As the immune responses develop, the viremia must subside and the antibodies persist.

GRAND CHALLENGE 3: NEEDLE-FREE DELIVERY

Needed:

• Well-formulated free flowing stable microparticles with less than 1% residual moisture, with high virus titers, dissolved within ~5 seconds.

• Inexpensive uni-dose blister packs or containers to protect vaccine from contamination, decomposing, reaction with water, oxidants and UV for 2 years, 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.

• Stabilize and make dry powders with larger fine particle fraction

• Package and seal individually as unit doses to:- Extend lifetime- Pre-measure the dose- Protect powder against moisture, oxidants, and

UV light- Protect vaccine against contamination- Reduce bioburden and eliminate pathogens- Reduce vaccine wastage- Allow serial administration of vaccines- Allow formulation in optimal matrix for each

vaccine

STRATEGIES EMPLOYEDFNIH GRANT 1077

• Prepare vaccine aerosol precursors in manufacturing plant rather than in the field

• Provide dispersion and aerosolization on site with only muscle power, and without line current or batteries

• Separate and re-use aerosolizer and disperser; then incinerate each reservoir and mask or mouthpiece

• Avoid carrying water to the field (vaccine ~10 times lighter).

STRATEGIES EMPLOYEDFNIH GRANT 1077 (continued)

Immunization in Difficult Circumstances

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• Powders inherently more stable than liquids• No line current or batteries required• Lower risk of disease transmission• Less vaccine wastage• More difficult to contaminate; no water or reconstitution

needed, so easier to carry• No skin puncture or contact with blood• No re-use of needles

Summary of Some Dry Powder Advantages over Liquid Vaccines

Measles Aerosol Vaccines in Humans

3 million humans immunized effectively with wet mist aerosol measles vaccine in Mexico (Valdespino, De Castro, et al.)

Revaccination protection from mucosal pulmonary aerosol superior to SC injection in S. Africa (Dilraj, et al.)

Nasal administration of large droplets gave poor seroconversion results in Chile (Simon, Levine, et al.)

WHO Wet Mist Aerosol Group has been administering wet mists of SII Edmonston-Zagreb measles vaccine with 3 devices in Phase I clinical trials in India.

System description• Leads to high seroconversion• Live virus titer per loaded dose at end of shelf life =

1000 CCID50

• Filled mass of powder per dose = 10 mg• Cost of device and powder is less than or equal to

$0.26 adjusted for inflation• The delivery system will be needle-free• Safe for human administration• Meet WHO + UNICEF requirements

Product Specifications

The Principle of the New CAN-BD Process

• In CAN-BD, the vaccine formulation solution/suspension in water is mixed intimately in a low volume tee with CO2 at 80-100 bar to form an emulsion.

• The emulsion, stabilized with myo-inositol or other sugars and the other traditional excipients in the SIIL vaccine (excepting sorbitol), is rapidly expanded to atmospheric pressure through a flow restrictor to generate aerosols of microbubbles and microdroplets.

• The aerosol plume is dried at temperatures below 50-70 °C as it mixes with a sheath of nitrogen in the drying chamber.

• Dry fine powders are collected on filters and packaged to exclude moisture (aluminum foil blister packs or capsules with Al-film/ polymer laminates for packaging).

Carbon Dioxide Assisted Nebulization with a Bubble Dryer®

Drug Solution or Vaccine Suspension

Near-critical orSupercritical CO2

(80 to 100 atm)

Flow Restrictor TubeID = 75 m

10 cm

CAN-BD Nebulizer

Emulsion of productstream in liquid CO2

CO2 flashes uponrapid 100X

decompression toatmospheric

pressure

Restrictor Tip

Tuberculosis Antibiotics

Ciprofloxacin HCl

Moxifloxacin HCl

Rifampin Capreomycin Amikacin

Drying T (C) 60 60 30 60 60

Solvent Water Water Ethyl Acetate

Water Water

Mean aero dia (µm) 1.2 1.1 0.78 1.3 1.2

95% cutoff (µm) 2.2 1.9 1.3 2.6 2.2

% < 5 um 89 56 86 77 84

Retained Activity 93% ± 20% 81% ± 43% 109% ± 34%

95% ± 7% 104% ± 7%

AKTIV-DRY

Assembly: Raul and Mihesh

CAN-BD Generated Particles of Measles Vaccine Formulation

Myo-inositol based placebo (M50)

Physical Properties of Powders Dried by CAN-BD

• Other variants of myo-inositol formulations also have similar properties– Fine Particle Fractions

• About 45-50% < 5.8 µm and about 20% < 3.3 µm

– Glass Transition Temperature (Tg)• Onset Tg = 45 - 60 °C; and Midpoint Tg = 50 - 65 °C

Moisture Content

< 5.8 mﾵ < 3.3 mﾵ Onset ( C)ﾡ Midpoint ( C)ﾡ (%)

Placebo 44 2 (n=4)ﾱ 19 2 (n=4)ﾱ 61 2 (n=4)ﾱ 65 4 (n=4)ﾱ 1.0 0.3 (n=4)ﾱActive 50 1 (n=2)ﾱ 21 2 (n=2)ﾱ 53 61 --

Placebo 43 19 60 61 0.9

Active 51 5 (n=2)ﾱ 25 4 (n=2)ﾱ 46 5 (n=4)ﾱ 53 4 (n=4)ﾱ 0.8 0.6 (n=4)ﾱ

Glass Transition Temperature (Tg)Formulation ID

Fine Particle Fractions(% of loaded mass)

M50

M35man15

Replication of Live Measles Virus Vaccine in Cotton Rat Lungs (K.O. Kisich)

•Low hygroscopicity; water content of formulation less than 1%

•Widely distributed naturally in human cells

•Phase I Clinical trial showed oral administration of 18 g/day for three months had no serious adverse effects (S. Lam et al. Cancer Epidemiol Biomarkers Prev 2006; 15 (8))

•Costs less than $8000 per metric ton; not animal-derived; isolated from rice in Asia

Chemical Structure of Myo-inositol

Myo-inositol --newly shown to be stabilizing

CAN-BD ProcessingVaccines Live attenuated measles virus

Live attenuated influenza virusHepatitis B surface antigen(HBsAg)

Antibodies PRIMATZED® anti-CD4Human IgGAnti-human lambda light chain

Proteins Alpha-1-antitrypsinTrypsinogenLactate dehydrogenaseLysozymeInsulinAlkaline phosphatase

Nucleotides siRNA duplexAptamersDNA single strand

Formulation Constituents

SugarsBuffers: Tricine, sodium potassium phosphate, sodium acetateSurfactants: Palmitic acid, stearic acid, Tween 20, Tween 80, Pluronic F68Amino acids: Arginine, methionine, leucine, glycine, etc.Metal chelating agents: EDTA, DTPA

Antibiotics MoxifloxacinTobramycinAmoxycillinDoxycyclineCefazolinCiprofloxacinAmikacinCapreomycinRifampin

Antivirals siRNA Relenza

Other PEGPVPHydrolyzed gelatinSodium chlorideDPPC (lung surfactant)Salbutamol (asthma drug)Albuterol sulfateCromolyn sodiumPhytosterols* (nutraceutical)Naproxen*Budesonide*Betamethasone*Amphotericin B*Cyclosporin*Myo-inositol and other sugars

*Hydrophobic

Aktiv-Dry PuffHaler™ DPI System

TWO SEPARABLE COMPONENTS --ONE REUSABLE AND ONE DISPOSABLE

• Aerosolization assembly (~ 500 uses per day)- Pliable polymeric bulb - Equipped with pressure release valve- Compartment to hold blister

• Patient Interface (use only once)- Collapsible plastic bag reservoir- Permeable filter facemask or mouthpiece with vents

PuffHaler® Aerosol Delivery System

• Utilizes air expansion through a pressure release valve

Top View

Side View

Some Advances to Celebrate

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

• Pressure release valves and syringes facilitate dispersion and create stable aerosol clouds comparable to newly FDA-approved active DPI.

• Greatly reduced cost and complexity of DPI‘s.

• Reduced water content of powders to < 1%.

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

“GRAND CHALLENGE”Bob Sievers

Campan MarbleLourdes, France

Gift installed in Norlin Historic Quadrangle

at University of Colorado

In gratitude for the support by the

Foundation for the National Institutes of

Health

Fehsenfeld-Sievers Publications

1. “Development of a Semi-Continuous Method for the Measurement of Nitric Acid Vapor and Particulate Nitrate and Sulfate,” S.M Buhr, M.P. Buhr, F.C. Fehsenfeld, J.S. Holloway, U. Karst, R.B. Norton, D.D. Parish, and R.E. Sievers, Atmospheric Environment, 29:19, 2609-2624 (1995).

2. "Assessment of Pollutant Emission Inventories by Principal Component Analysis of Ambient Air Measurements," M.P. Buhr, M. Trainer, D. D. Parrish, R.E. Sievers, and F.C. Fehsenfeld, Geophys. Res. Lett., 19, 10, 1009-1012 (1992).

3. "Contribution of Organic Nitrates to the Total Reactive Nitrogen Budget at a Rural Eastern U.S. Site," M.P. Buhr, D. D. Parrish, R. B. Norton, F.C. Fehsenfeld and R.E. Sievers, J. Geophys. Res., 95, 9809-9816, (1990).

4. "Monoterpene Hydrocarbons in the Nighttime Troposphere," J.M. Roberts, C.J. Hahn, F.C. Fehsenfeld, J.M. Warnock, D.L. Albritton, and R.E. Sievers, Environ. Sci. Technol., 19, 364 (1985).

5. "Measurements of Anthropogenic Hydrocarbon Concentration Ratios in the Rural Troposphere: Discrimination between Background and Urban Sources," J.M. Roberts, R.S. Hutte, F.C. Fehsenfeld, D.C. Albritton and R.E. Sievers, Atmos. Env., 19, 1945 (1985).

6. "Measurements of Hydrocarbon Concentration Ratios and NOx Concentrations in the Rural Troposphere: Estimation of Air Mass Photochemical Age and Discrimination between Background and Urban Sources," J.M. Roberts, F.C. Fehsenfeld, D.L. Albritton, and R.E. Sievers, "Environmental Impact of Natural Emissions," V.P. Aneja, Ed., APCA Specialty Conf., Research Triangle Park, NC, 233-250 (1984).

7. "Measurements of Aromatic Hydrocarbon Ratios and NOx Concentrations in the Rural Troposphere: Observation of Air Mass Photochemical Aging and NOx Removal," J.M. Roberts, F.C. Fehsenfeld, S.C. Liu, M.J. Bollinger, C. Hahn, D.L. Albritton and R.E. Sievers, Atmospheric Environment, 18, 2421-2432 (1984).

8. "Measurement of Monoterpene Hydrocarbons at Niwot Ridge, Colorado," J.M. Roberts, F.C. Fehsenfeld, D.L. Albritton, and R.E. Sievers, J. Geophysical Research, 88, C15, 10667-10678 (1983).

9. "Conversion of NO2, HNO3, and N-Propyl Nitrate to NO by a Gold-Catalyzed Reduction with CO," M.J. Bollinger, R.E. Sievers, D.W. Fahey, and F.D. Fehsenfeld, Anal. Chem., 55, 1980-1986 (1983).

10. "Sampling and Analysis of Monoterpene Hydrocarbons in the Atmosphere with Tenax GC Porous Polymer," J.M. Roberts, F.C. Fehsenfeld, D.L. Albritton, and R.E. Sievers, Chapter 23 in "Identification and Analysis of Organic Pollutants in Air," Ann Arbor Science Publishers, Ann Arbor, MI (1983).

11. "Techniques for the Measurements of Atmospheric Hydrocarbons at a Remote Rural Site," J.M. Roberts, P.D. Goldan, D.L. Albritton, F.C. Fehsenfeld, and R.E. Sievers, "Proceedings of Symposium on Pollutant Monitoring of Ambient Air and Stationary Sources, Raleigh, NC, May 4-7, 1982."

12. Chapter in Book Entitled "Electron Capture-Theory and Practice in Chromatography," Elsevier Publishing Co., 1981 "Selective Electron Capture Sensitization," M.P. Phillips, P.D. Goldan, F.C. Fehsenfeld, and R.E. Sievers.

13. "Vinyl Chloride Detection at Sub-ppb Levels Using a Chemically Sensitized Electron Capture Detector," P.D. Goldan, F.C. Fehsenfeld, W.C. Kuster, M.P. Phillips and R.E. Sievers, Anal. Chem., 52, 1751 (1980).

14. "Selective Electron-Capture Sensitization," R.E. Sievers, M.P. Phillips, R.M. Barkley, M.A. Wizner, M.J. Bollinger, R.S. Hutte, and F.C. Fehsenfeld, J. Chromatogr., 186, 3 (1979).

15. "Selective Electron Capture Sensitization: Enhancement of Electron Capture Detector Sensitivity to Non-Electron Attaching Compounds by Nitrous Oxide Added to the Carrier Gas," M.P. Phillips, R.E. Sievers, P.D. Goldan, W.C. Kuster, and F.C. Fehsenfeld, Anal. Chem., 51, 1819 (1979).