bioneedle(biodegradable mini implant) delivery of vaccine Best seminar from First semester student of niper ahmedabad : seminar given by Vishal Goyani, Department of Pharm.Analysis,NIPER-Ahmedabad

1. Bioneedles as alternative delivery system for hepatitis B vaccine VISHAL N. GOYANI PHARMACEUTICAL ANALYSIS

2. Presentation outline Introduction Research paper Materials and methods Results & Discussion Conclusion

3. INRODUCTION

4. 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

5. 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

7. Bioneedles™ Mini implant Biodegradable polymer Vaccine incorporated Thermostable 16 mm long & 1.2 mm wide Volume of 4.0 to 4.5 μl

8. Bioneedles delivery IM or SC Bioneedle Applicator < 1 millisecond Pain-free application Polymer dissolves Vaccine released

9. Why Bioneedle™ ? Ultra portability No degradation No need for cold chain Advantage to developing countries (African countries) reaching even the most remote children

10. 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

11. 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

12. Waste elimination Contaminated needles, syringes, and vials require proper waste disposal Bioneedle™ no waste

13. 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

14. RESEARCH PAPER

16. 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

17. Materials and methods

18. 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 BrenntagBiosector, Denmark Bioneedles: thermoplastic starch by injection moulding Bioneedle Technologies Group's proprietary procedures

19. 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

20. 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-trehalosedi-hydrate then freeze dried

21. 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

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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.

28. Results & Discussion

29. effect of freeze drying and theroute of immunization on the IgG titer Fig. 1. IgG titers in mice (n=8)

30. Adjuvant

31. Adjuvant:IgG2a/IgG1

32. Dose response

33. The effect of delivery route

34. Antigenicity of stressed antigen

35. Structure of stressed antigen

36. Immunogenicity of stressed antigen

37. Conclusion

38. 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

39. 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

40. 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 other common 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: prevalence and associations, Am. J. Trop. Med. Hyg. 68 (2003) 341–344. [3] A. Taddio, A.L. Ilersich, M. Ipp, A. Kikuta, V. Shah, Physical interventions and injection techniques for reducing injection pain during routine childhood immunizations: systematic review of randomized controlled trials and quasirandomized controlled 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 and prevalence 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 strategies and future trends, Expert Rev. Vaccin. 5 (2006) 739–745. [6] E. Mallet, B.H. Belohradsky, R. Lagos, et al., A liquid hexavalent combined vaccine against diphtheria, tetanus, pertussis, poliomyelitis, Haemophilusinfluenzae type B and 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, new possibilities, Bioessays 29 (2007) 591–604.

41. Thank You

44. 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

45. 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.

47. Patent application title: PARENTERAL FORMULATION Inventors: GijsbertusGerardusPetrus Van De WijdevenAgents: FOLEY AND LARDNER LLP;SUITE 500Assignees: BIONEEDLE TECHNOLOGIES GROUP B.V.Origin: WASHINGTON, DC USIPC8 Class: AA61F200FI USPC Class: 424426 Patent application number: 20100080839