Bioneedles as alternative delivery system for hepatitis B vaccine

VISHAL N. GOYANIPHARMACEUTICAL ANALYSIS

Presentation outline

•Introduction•Research paper•Materials and methods•Results & Discussion•Conclusion

INRODUCTION

Vaccinations

•Vaccination is the administration of antigenic material to produce immunity  to a disease

•About 1 billion vaccinations every year •Cost-effective life-saver for children•Smallpox eradicated•The conventional way

for vaccine delivery is SC or IM route by using syringe & needle

Vaccine delivery technologies•Auto-disable syringes•Prefilled syringes or droppers•Needle-free injections (jet injector)•Biodegradable implants

(Bioneedles™)•Microneedles•Transdermal delivery•Buccal /sublingual delivery•Inhalation delivery•Reconstitution devices

Bioneedles™

•Mini implant •Biodegradable polymer •Vaccine incorporated •Thermostable •16 mm long & 1.2 mm wide •Volume of 4.0 to 4.5 μl

Bioneedles delivery

•IM or SC•Bioneedle Applicator • < 1 millisecond • Pain-free application • Polymer dissolves • Vaccine released

Why Bioneedle™ ?

•Ultra portability•No degradation•No need for cold chain•Advantage to developing countries (African countries) reaching

even the most remote children

Advantage at vaccination center•No reconstitution•No need for clean workplace•Dry delivery •No risk of contamination•No vaccine wastage •1000 vaccinations/hour •20x faster campaign•Less medical staff

Oh dear, No fear, bioneedle is hear

•No needle fear because pain-free •No infection through needle-stick injury•No infection through contamination•Prevents 25 million infections of HepB,

HepC, HIV per year from reuse

Waste elimination

•Contaminated needles, syringes, and vials require proper waste disposal

•Bioneedle™ no waste

Hepatitis B Vaccine

•For the prevention of hepatitis B virus infection

•Vaccine contains one of the viral envelope proteins, hepatitis B surface antigen

•DOSE: A course of three vaccine injections

•MOA: Antibody to HBsAg is established in the bloodstream which then provide immunity to hepatitis B infection

RESEARCH PAPER

INTRODUCTION

•Bioneedles as alternative delivery system for hepatitis B vaccine

•New system should at least as effective as the conventional delivery system

•LPS-derived LpxL1 compared to Al(OH)3

as an adjuvant and the heat stability and dose reduction has been evaluated for liquid and Bioneedle formulations

Materials and methods

Materials•Yeast derived, commercial GMP grade,

hepatitis B surface antigen (HBsAg)•Control vaccine: Engerix-B 20 μg/mL from

GSK• lpxL1 LPS from ImsaVac, Netherlands•Al(OH)3, Alhydrogel 2% from Brenntag

Biosector, Denmark•Bioneedles: thermoplastic starch by

injection moulding Bioneedle Technologies Group's

proprietary procedures

Formulations

•Different formulation: 1) HBsAg 2) HBsAg + Al(OH)3

3) HBsAg +lpxL1

•All formulations contained 2 μg HBsAg•Adjuvants either 50 μg Al(OH)3 or 0.5 μg lpxL1•Liquid formulations (L) 1) Subcutaneous injections :500μl 2) Intramuscular injections :100μl

Cont..

•The freeze dried formulations(F) :500 μl freeze dried using a Leybold GT 4/6

freeze dryer•Bioneedles (B) were filled, by using a

specially designed filling apparatus, with liquid formulations containing 5% w/w of D-trehalose di-hydrate then freeze dried

Hepatitis B antigen ELISA

•For HBsAg quantification•Bioneedles were dissolved by incubation in 1 ml water by using α-Amylase & pullulanase•Commercial kit of Abbott, Murex HBsAg version 3 was used

UV Circular dichroism

•Used to investigate the secondary structure of antigen

•Chirascan™ (Applied Photophysics, UK)•Liquid and freeze dried formulations were

stored for 3 weeks at 4 °C, 37 °C, 50 °C and 60°C prior to the measurements

•concentration of antigen 40 μg/ml at 25 °C•Scan region of 200–260 nm•Scan velocity was 0.1 nm/s•Chirascan™CDNN software was used to

provide an estimate of the secondary structure

In vivo studies

•BalB/C mice of 10–14 g•Prior to immunization, all animals were

anesthetized with a mixture of 1-isoflurane, N2 and O2

•For the dose response study, the hepatitis B doses used were 2 μg, 1 μg, 0.5 μg and 0.1 μg

•Liquid formulations were either injected I.M in the rear leg, S.C. in the groin

Cont..

•Freeze dried formulations were reconstituted In 500 μl water prior to subcutaneous injection

•Bioneedles were implanted in mice by using a sterilized trocar with mandrin S.C not I.M

•Two weeks after primary immunization, blood was collected

•Four weeks after the booster vaccination, all mice were sacrificed by bleeding

IgG, IgG1 and IgG2a ELISA•Polystyrene microtiter plates were coated HBsAg

diluted in phosphate buffered saline (PBS)•The plates were incubated overnight at room

temperature. The plates were washed with tap water containing Tween 80.

•Serum was added in series of three fold dilutions (in PBS)

•After 2 h of incubation at 37 °C the plates were washed and goat anti mouse immunoglobulin conjugated to horse radish peroxidase was added

Cont..

•The conjugate was incubated for 1.5 h at 37 °C.

•After washing the plates, 100 μl TMB substrate solution was added to each well.

•The reaction was stopped after 10 min with 2 M H2SO4 and the absorbance was measured at 450 nm.

•The titre was determined as the dilution factor at which the absorbance was 50% of the maximum absorbance

Statistics

•Antibody titers are expressed as the mean log10 titer of eight or ten independent observations plus the standard errors of the means.

•Antigenic recoveries are expressed as the mean of three independent observations, plus the mean standard errors of the means.

•Statistical evaluations are done with a Student t test (one way ANOVA) by using a two tailed distribution and a two sample unequal variance.

Results & Discussion

effect of freeze drying and theroute of immunization on the IgG titer

Fig. 1. IgG titers in mice (n=8)

Adjuvant

Adjuvant:IgG2a/IgG1

Dose response

The effect of delivery route

Antigenicity of stressed antigen

Structure of stressed antigen

Immunogenicity of stressed antigen

Conclusion

•The study shows that hepatitis B was successfully formulated with Bioneedles

•Bioneedles with lpxL1, when delivered subcutaneously, induce higher IgG titers after one vaccination than conventional vaccine

•After a booster vaccination comparable IgG titers are induced as with the conventional liquid vaccine but with higher IgG2a/IgG1 ratios

•Even better results are expected when bioneedle can be tested IM

•Bioneedles with lpxL1 also showed much better heat stability than the conventional liquid vaccine

•Although these advantages are mainly attributed to lpxL1, the Bioneedle has the additional advantages like:

▫Ultra-portability ▫Thermo-stability▫Pain-free▫Contamination-free▫No waste

Reference

[1] R.M. Jacobson, A. Swan, A. Adegbenro, S.L. Ludington, P.C. Wollan, G.A. Poland,Making vaccinesmore acceptable—methods to prevent andminimize pain and othercommon adverse events associated with vaccines, Vaccine 19 (2001) 2418–2427.[2] Y. Nir, A. Paz, E. Sabo, I. Potasman, Fear of injections in young adults: prevalenceand associations, Am. J. Trop. Med. Hyg. 68 (2003) 341–344.[3] A. Taddio, A.L. Ilersich, M. Ipp, A. Kikuta, V. Shah, Physical interventions andinjection techniques for reducing injection pain during routine childhoodimmunizations: systematic review of randomized controlled trials and quasirandomizedcontrolled trials, Clin. Ther. 31 (Suppl 2) (2009) S48–S76.[4] S. Wright, M. Yelland, K. Heathcote, S.K. Ng, G. Wright, Fear of needles—nature andprevalence in general practice, Aust. Fam. Physician 38 (2009) 172–176.[5] J.U. Igietseme, F.O. Eko, Q. He, C.M. Black, Combination vaccines: design strategiesand future trends, Expert Rev. Vaccin. 5 (2006) 739–745.[6] E. Mallet, B.H. Belohradsky, R. Lagos, et al., A liquid hexavalent combined vaccineagainst diphtheria, tetanus, pertussis, poliomyelitis, Haemophilus influenzae type Band hepatitis B: reviewof immunogenicity and safety, Vaccine 22 (2004) 1343–1357.[7] G. Kersten, H. Hirschberg, Antigen delivery systems, Expert Rev. Vaccin. 3 (2004)453–462.[8] M.A. Aziz, S. Midha, S.M. Waheed, R. Bhatnagar, Oral vaccines: new needs, newpossibilities, Bioessays 29 (2007) 591–604.

Than

k You

Immunogenicity & Antigenicity• Antigenicity is the ability of a chemical

structure (referred to as an Antigen) to bind specifically with certain products of adapttyive immunity: T cell receptors or Antibodies(a.k.a. B cell receptors).

• immunogenicity refers to the ability of an antigen to induce an adaptive immune response.

• Thus an antigen might bind specifically to a T or B cell receptor, but not induce an adaptive immune response

•mandrin A stiff wire or stylet inserted into a soft catheter to give it shapeand firmness while passing  through a hollow tubular structure

•An antibody titer is a measurement of how much antibody an organism has produced that recognizes a particularepitope, expressed as the greatest dilution ratio (or its reciprocal) that still gives a positive result. ELISA is a common means of determining antibody titers.

• Patent application title: PARENTERAL FORMULATION

• Inventors:  Gijsbertus Gerardus Petrus Van De Wijdeven Agents:  FOLEY AND LARDNER LLP;SUITE 500

Assignees:  BIONEEDLE TECHNOLOGIES GROUP B.V. Origin: WASHINGTON, DC US IPC8 Class: AA61F200FI USPC Class: 424426 Patent application number: 20100080839