NURJAYA SUMIRAN

◦ Composed of 51 amino acid residues.◦ Molecular weight of 5808 Da.◦ Most important regulatory hormones in control glucose homeostasis.◦ Made up of two chains: A chain – 21 amino acids

B chain – 30 amino acids◦ Water-soluble, unstable peptide.

two disulphide bridge

•Bovine insulin-differs only three amino acid•Porcine insulin-one amino acid from human.•Synthesized in the pancreas within the β-cells of the Islets of Langerhans from the endocrine part of the pancreas.•2 % of endocrine portion of the total mass of pancreas.•β-cells constitute 60-80% of Islets of Langerhans cells.•Clinical use-from cow, horse, pig or fish pancreases.

general term referring to all states characterized by hyperglycemia. Type 1 – autoimmune-mediated destruction of insulin producing β-cells in the

pancreas resulting in absolute insulin deficiency. Type 2 – multifactoral syndrome with combined influence of genetic susceptibility

and influence of environmental factors, the best known being obesity, age, and physical inactivity, resulting in insulin resistance in cells requiring insulin for glucose absorption.

Most common non-communicable disease. 4th or 5th leading cause a death in most developed countries.

Modes of administration◦ Subcutaneous-needles, insulin pump, or by repeated-use

insulin pens with needles

◦ Transdermal

◦ Intranasal◦ Buccal◦ Oral

retinopathy nephropathy

gangrene neuropathic ulcer

Why choose oral delivery?◦ Higher patient compliance.

Avoid the pain and discomfort associated with injections.

◦ Less expensive to produce-no need sterile conditions. [2]◦ Reduced risk of cross-infection and

needle stick injuries.

Physiological and morphological barriers against protein or peptide delivery:

i. Proteolytic enzymes in the gut lumen (pepsin, trypsin, chymotrypsin)

ii. Proteolytic enzymes at the brush border membrane (endopeptidase)

iii. Mucus layeriv. The bacterial gut florav. Epithelial cell lining itself.

Strategies◦ Enteric coating. ◦ Protease inhibitors. [3]◦ Permeation enhancers. ◦ Bioadhesive nanoparticles. [7]

Definition Nanoparticles for pharmaceutical purposes are defined as

solid colloidal particles ranging in size from 1 to 1000 nm (1m).

consist of macromolecular materials and can be used therapeutically as drug carriers, in which the active principle (drug or biologically active material) is dissolved, entrapped, or encapsulated, or to which the active principle is adsorbed or attached. [Encyclopedia of Pharmaceutical Technology]

Hypothesis:

Schematic illustration of the presumed mechanism of the paracellular transport of insulin to the bloodstream using the prepared nanoparticles

Insulin loaded Nanoparticles

Nanoparticles preparation

Nanoparticles preparation

Physicochemical characterization

Size

Zeta potential

Drug content

Association efficiency

FTIR

DSC

SEM

Biological efficacy

Blood glucose lowering

Insulin plasma level Absorption mechanism

Spectrophotometer

Intestinal uptake

Physicochemical characterizationpH

Insulin monomer contains many ionizable groups due – ◦ 6 amino acids residues capable of attaining +ve charge

polyelectrolyte◦ 10 a.a attaining a –ve charge. [11]

Isoelectric point (PI)insulin = 5.3◦ > pH 5.3 – insulin –ve charge◦ < pH 5.3 – insulin +ve charge [11, 18, 19]

DC, AE, DSC, FTIR, SEM [11, 13, 18, 20, 21, 24]o 500 nm, -15 mV, pH 4.8, AE 85 %. [11]o pH 3.0, AE 90%. [18].

Biological efficacy- oral administration in streptozotocin diabetic rats

Insulin plasma level◦ Pan et al., 2002 - 21IU/kg insulin np effective up to 15h

- 250-400 nm / +ve charge np - AE 80 %

Serum glucose level◦ Ma et al., 2005 – 50 &/or 100 U/kg ins np (60%) up to 11h

- [4.28 U/ml] np at pH 5.3 & 6.1 / +ve charge np

◦ Cui et al., 2006 – 20 IU/kg intragastric (57.4%) 8h – 12h - 200 nm / AE 90%

◦ Lin et al., 2007 – 30 UI/kg ins np (50%) 4h-6h - ~150 nm / +ve charge

Absorption mechanism

Schematic drawing of mucus (MU) covered absorptive enterocytes (EC) and M cells (MC) in the small intestine. Lymphocytes (LC) and macrophages (MP) from underlying lymphoid tissue can pass the basal lamina (BL) and reach the epithelial cell layer which is sealed by tight junctions (TJ). Possible translocation routes for NP are (I) paracellular uptake, (II) endocytotic uptake by enterocytes and (III) M cells.

Damge at al., 2007

3 possible mechanism suggested for intestinal uptake of ins np &/ or ins released fr np:

i. Uptake via a paracellular pathway

ii. Trancystosis or receptor-mediated transcytosis & transport via EC of intestinal mucosa.

iii.Lymphatic uptake via M cells of the Peyer’s patches mostly abundant in the ileum.

Size dependence of NP absorption

Xu et al., 2006◦ Particle < 10μm can be taken by M cells & transported to Peyer’s patches.◦ Most microparticles > 5 μm remain in the Peyer’s patches but < 5 μm are

transported through the different lymphatics. Jung et al., 2000

◦ Intestinal uptake increased of 100 nm np compared > particles of 1 & 10 μm.◦ Identical uptake in Peyer’s patches & EC – 100 nm.◦ Size < 500 nm required.◦ Summarized:

i. Np < 100 nm ↑ uptake by absorptive EC than np > 300 nm.ii. Uptake of np <100 nm by follicle-associated epithelia > efficient via

absorptive EC.iii. Uptake of np > 500 nm by absroptive EC unlikely event.iv. Only np < 500 nm reach general circulation.

Hydrophobicity and surface charge Jung et al., 2000

◦ Uncharged and +ve charge np consist hydrophobic PS provide affinity to absorptive EC.

◦ -ve charge PS np show only affinity to any type of intestinal tissues.

◦ -ve charge np fr more hydrophilic polymers show ↑ bioadhesive prop & absorbed by both M cells & absorptive EC.

Electrostatic interaction at mucosal surface is –ve charge. [5,19]

Ma et al., 2005◦ Administration of FITC-chitosan np dispersion by

oral gavage

Confocal micrographs of rat ilea isolated 3 h after the oral administration of FITC-chitosan nanoparticle dispersion (2.5 mL, chitosan concentration of 1.33 mg/mL).

A - between the intestinal villiB - on the surface of the intestinal enterocytesC - within the outer most layers of the intestinal enterocytes, D -transported into the tissues underlying the absorptive cells.

Damge et al., 2007◦ In situ isolated intestinal loop

Fluorescence microscopy of tissue slices through the intestinal epithelium 30 min after the intra-ileal administration of FITC-labeled insulin (A, B) or FITC-labeled insulin nanoparticles 50 IU/kg (C, D). The bar represents 200 μm in A, B, C and 50 μm in D.

A - unlabelledB – not significant label in Peyer’s patchesC – fluorescent in the lumen intestine, next to villi apical pole D –small fluorescent in Peyer’s patches

Lin et al., 2007◦ Confocal laser scanning microscope

(a) Fluorescence images of the duodenum, jejunum and ileum (after 3D reconstruction) retrieved from a rat model 3 h after oral administration of the FITC-labelled nanoparticles (NPs); (b) fluorescence images of one of the villi of the retrieved duodenum immunofluorescently stained for ZO-1 proteins and nuclei 3 h after oral administration of the FITC-labelled NPs. Control group: the group without oral administration of the FITC-labelled NPs. XY plane: the horizontal plane; XZ plane: the vertical plane.

1. Peppas, N., Kavimandan, N.J. (2006). Nanoscale analysis of protein and peptide absorption: Insulin absorption using complexation and pH-sensitive hydrogels as delivery vehicles. Euro. J. of Pharmaceutical Sciences, 29, 183-197.

2. Fasano, A. (1998). Innovatie strategies for oral deliver of drugs and peptides. Tibtech, 16, 152-157.

3. Carino., G.P., Mathiowitz, E. (1999). Oral insulin delivery. Advanced Drug delivery Rev, 35, 249-257.

4. Jorgensen L., Moeller E.H., van de Weert M., Nielsen H.M. Frokjaer S. (2006). Preparing and ealuating delivery systems for proteins. Euro. J. of Pharmaceutical Sciences, 29, 174-182.

5. Lemarchand, C, Grf, R., Couvreur, P. (2004). Polysaccharide-decorated nanoparticles. Euro. J. of Pharm. and Biopharm., 58, 327-341.

6. Saffran, M., Pansky, B., Budd, G.C., Williams, F.E. (1997). Insulin and the gastrointestinal tract. J. Controlled Rel,46, 89-98.

7. Panyam, J., Labhasetwar, V. (2003). Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Advanced Drug delivery Rev, 55, 329-347.

8. Jung, T., Kamm, W., Bretenbach, A., Kaiserling, E., Xioa, J.X., Kissel, T. (2000). Biodegradable nanoparticles for aoral delivery of pptides: is there a role for polymers to affect mucosal uptake?. Euro. J. of Pharm. and Biopharm, 50, 147-160.

9. Ma, Z., Lim, T.M., Lin, L-Y. (2005). Pharmacological activity of peroral chitosan- insulin nanoparticles in diabetic rats. Int. J. Pharm, 293, 271-280.

10. Pan, Y., Li, Y-J., Zhao, H-Y., Zheng, J-M., Xu, H., Wei, G., Hao, J-S., Cui, F-D. (2002). Bioadhesive polysaccharide in protein delivery system: chitosan nanoparticles improve the intestinal absorption of insulin in vivo. Int. J. Pharm, 249, 139-147.

References

11. Sarmento, B., Ribeiro, A., Veiga, F., Ferreira, D. (2006). Development and characterization of new insulin containing polysaccharide nanoparticles. Colloidals and Surfaces B: Biointerfaces 53, 200-209.

12. Silva, C.M., Ribeiro, A.J., Figueiredo, I.V., Goncalves, A.R., Veiga, F. (2006). Alginate microspheres prepared by internal gelation: development and effect on insulin stability. Int. J. Pharm, 311, 1-10.

13. Cui, K., Shi, K., Zhang, L., Tao, A., Kawashima, Y. (2006). Biodegradable nanaoparticles loaded with insulin-phopholipid complex for oral delivery : preparation in vitro characterization and in vivo evaluation. J. Controlled Rel, 114, 242-250.

14. Ibrahim, M.A., Ismail, A., Fetouh, M.I., Gopferich, A. (2005). Stability of insulin during the erosion of poly(lactic acid) and poly(lactic-co-glycolic acid)microspheres. J. Controlled Rel, 106, 241-252.

15. Leobandung, W., Ichikawa, H., Fukumori, Y., Peppes, N.A. (2002). Preparation of stable insulin-loaded nanospheres of poly(ethlylene glycol) macromers and N-isopropyl acrylamide. J. Controlled Rel, 80, 357-363.

16. Kim, B., Peppas, N.A.(2003). In vitro release behavior and stability of insulin in complexation hydrogels as oral drug delivery carriers. Int. J. Pharm, 266, 29-37.

17. Fan, Y.F., Wang, Y.N., Fan, Y.G., Ma, J.B. (2006). Preparation of insulin nanoparticles and their encapsulation with biodegradable polyelectrolytes via the layer-layer adsorption. Int. J. Pharm, 324, 158-167.

18. Cheng, K., Lim, L-Y. (2004). Insulin-loaded calcium pectinate nanoparticles: effects of pectin molecular weight and formulation pH. Drug Dev and Industrial Pharmacy, 30 (4), 359-367.

19. Sarmento, B., Ferreira, D., Veiga, F., Ribeiro, A. (2006). Characterization of insulin-loaded alginate nanoparticles produces by ionotropic pre-gelation through DSC and FTIR studies . Carbohydrate Polymers, 66, 1-7.

20. Sarmento, B., Martins, S., Ribeiro, A., Veiga, F., Neufald, R., Ferreira, D. (2006). Development and comparison of different nanoparticulate polyelectrolyte complexes as insulin carriers. Int. J. Peptide Res. and Therapeutics, 12, 131-138

21. Xu, X., Fu, Y., Hu, H., Duan, Y., Zhang, Z. (2006). Quantitative determination of insulin entrapment efficiency in triblock copolymeric nanoparticles by high performance liquid chromatography. J. Pharm and Biomed Analy, 40, 266-273.

22. Wang, L-Y., Gu, Y-H., Su, Z-G., Ma, G-H. (2006). Preparation and improvement of release behavior of chitosan microspheres containing insulin. Int. J. Pharm, 311, 187-195.

23. Sarmento, B., Ferreira, D.C., Jorgensen, L., Van De Weert, M. (2007). Probing insulin’s secondary structure after entrapment into alginate/chitosan nanoparticles. Euro. J. of Pharmaceutics and Biopharmaceutics, 65, 10-17.

24. Sarmento, B., Ribeiro, A., Veiga, F., Ferreira, D. (2006). Development and characterization of new insulin containing polysaccharide nanoparticles. Colloidals and Surfaces B: Biointerfaces 53, 200-209.

25. Li, M-G., Lu, W-L., Wang, J-C., Zhang, X., Wang, X-Q., Zheng, A-P., Zhang, Q. (2006). Distribution, transition, adhesion and release of insulin loaded nanoparticles in the gut of rats. Int. J. Pharm, 329, 182-191.

26. Zhang, N., Ping, Q., Huang, G., Xu, W., Cheng, Y., Han, X. (2006). Lectin-modified solid lipid nanoparticles as carriers for oral administration of insulin. Int. J. Pharm, 327, 153-159.

27. Lin, Y-H., Chen, C-T., Liang, H-F., Kulkarni, A R., Lee, P-W., Chen, C-H., Sung, H-W. (2007). Novel nanoparticles for oral insulin delivery via the paracellular pathway. Nanotechnology, 18, 105102.

28. Damge, C., Maincent, P., Ubrich, N. (2007). Oral delivery of insulin associated to polymeric nanoparticles in diabetic rats. J. Controlled Rel, 117, 163-170.

Thank you…