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 two disulphide B chain 30 amino acids bridge Water-soluble, unstable peptide.

Bovine insulin-differs only three amino acid Porcine insulin-one amino acid from human. Synthesized in the pancreas within the -cells of the Islets of Langerhans from the endocrine part of the pancreas. 2 % of endocrine portion of the total mass of pancreas. -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 -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

neuropathic ulcer gangrene

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 layer iv. The bacterial gut flora v. 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
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 characterization

pH 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]

> 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].

Isoelectric point (PI)insulin = 5.3

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 % 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

Serum glucose level

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 Peyers patches mostly abundant in the ileum.

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.

Size dependence of NP absorption

Xu et al., 2006 Particle < 10m can be taken by M cells & transported to Peyers patches. Most microparticles > 5 m remain in the Peyers patches but < 5 m are transported through the different lymphatics. Jung et al., 2000 Intestinal uptake increased of 100 nm np compared > particles of 1 & 10 m. Identical uptake in Peyers 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

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

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).

Damge et al., 2007

In situ isolated intestinal loop

A - unlabelled B not significant label in Peyers patches C fluorescent in the lumen intestine, next to villi apical pole D small fluorescent in Peyers patches

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 m in A, B, C and 50 m in D.

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.

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Thank you