AGRAZINC 100

1. AGRAZINC 100 Pharmacological Mode of Action

2. AGRAZINC 100 • Zinc, especially, ZnO which is one of inorganic zinc products is very effective to prevent and cure the digestive disorder such as ‘diarrhea’ in young animal (piglet, calf etc.), even in matured animal • Also, Zinc is very important trace mineral involved in natural immune system

3. Bivalent cation minerals and the mucus on intestinal surface • Bivalent cation: Zinc(Zn++), Copper(Cu++), Magnesium(Mg++) etc. • On the other hand, Mucus/intestinal surface is consist of ‘glycoprotein’ and ‘glycolipid’. And, all of them show a negative charge • Bivalent cation compounds become free ions in GIT and bind to mucus through the ionic bond • To cope with this barrier in digestion of mineral, ‘Organic minerals’ were developed

4. Differences between ZnO and Organic Zinc • ZnO: can be free ion easily (Zn++) • organic Znic: strong bond • To get any effects such as anti-viral, antibiotic and reduction in excessive immune response (allergy etc) in GIT, Zinc complex should release free ions easily.

5. AGRAZINC 100 Preventing pathogenic bacteria [GIT; gastro intestinal tract]

6. E. coli and GIT dysfunction • Most important reason for GIT dysfunction like a diarrhea is E. coli; K88, K99 etc. • E. coli bind to specific receptors consist of ‘Glycoprotein’ and ‘Glycolipid’ on intestinal surface and exert toxicity on the epithelial cell in typical condition.

7. Effects of ZnO and CuSO4 • In effects of ZnO and CuSO4 on GIT… • ZnO and CuSO4 increased the performance in production and growth rate, when it’s inclusion rate was increased. • Both of them suppressed the activity of E. coli in pharmacological level in diets

8. Mucus layer: the 1st barrier to the pathogen Intestinal epithelial cell Depiction of normal condition in GIT and settle down on mucus layer [Mathew. 2001]

9. Effects of some countermeasures for E. coli • High inclusion level of Zinc(ZnO) could reduce the activity of beneficial bacteria [Ole Hojberg et al, 2005]. • AGRAZINC 100 reduced the activity and number of E. coli, but increased the activity and number of beneficial bacteria in GIT. • Lactobacilli can also bind to specific receptors of E. coli on intestinal surface competitively. So, it prevents excessive proliferation of E. coli in GIT. [Mathew, 2001]

10. AGRAZINC 100: Effects on E. coli • AGRAZINC 100 showed reduction in number of E. coli significantly [China, 2007]. From AGRANCO’s China trial, Zinc ion seems to have an affinity for specific receptors for E. coli on the intestinal surface than beneficial bacteria E. coli, log10 Lactobacillus acid., log10 [Dalian Polytechnic Univ. China, 2007]

11. Zn++ Zn++ E. coli Beneficial bacteria Logical approach to MOA of Zinc Ion(Zn++) • Zinc ion, bivalent cation, seems to have an affinity for E.coli’s binding sites on intestine more than other beneficial microorganisms. • With this hypothesis, Zinc ion seems to be bound to ‘glycoprotein’ and ‘glycolipid’ on intestinal surface preferentially that are the binding sites of E. coli. • Then, E. coli cannot find any binding sites to be adhered and channel to penetrate and exert toxicity in target tissue. And, move along with excreta. • As we have seen in many other trial, if ZnO was supplied more than specific level (unknown), it could suppress not only E. coli, but also beneficial bacteria in GIT. First is E. coli, second is Beneficial bacteria

12. Logical approach to MOA of Zinc Ion(Zn++) Graph 1 Graph 2 Lactic acid conc., umol/g wet digesta pH value ZnO which have bypassed stomach would be ionized gradually and Zinc ions would be attached to receptors consist of glycoprotein/glycolipid on intestinal surface or ‘Cell adhesion molecules’ on epithelial cells. Meanwhile, Zinc ions would be attached to specific binding sites for E. coli competitively. However, too much higher level of zinc ions flux could block the docking sites for other beneficial bacteria, and then, it could suppress the activity of these beneficial bacteria such as Lactobacilli (Graph 1) After then, the amount of intact ZnO, which could be ionized, would be reduced to the minimum level to suppress E. coli gradually. Then, the activity of beneficial bacteria would be increased (Lactic acid, it’s by-product, would be increased) [Ole Hojberg et al, AEM.71.5.2267-2277. 2005]

13. The limit of ZnO and effects of AGRAZINC 100 • The effect of pharmacological level of ZnO is well known. However, it also effects on beneficial bacteria. • Still, there need to study what incorporation level (a guide line) do not depress the activity of beneficial bacteria in certain conditions, while it depress typical pathogen such as E. coli. • AGRAZINC 100 release Zn ion where it should be released in order to protect gut health effectively from the pathogen. Then, AGRAZINC 100 can also promote any beneficial bacteria.

14. Lactobacilli and factors produced by lactobacilli also can block E. coli attachment sites K88 E. coli lactobacillus Zn++ Zn++ E. coli attachment to intestinal surface glycoproteins Free Zn++ released from AGRAZINC 100 attach to E. coli binding sites competitively. Then, AGRAZINC 100 block E. coli attachment on intestinal surface How AGRAZINC100 works in GIT?

15. AGRAZINC 100 Preventing Virus

16. Infection routes of Virus • The viral infection through the gut and respiratory system(throat, lungs etc.) is commenced from the binding (penetration) between Virus and the host cell. • Virus can get into the host cell through CAMs (Cell Adhesion Molecules). There are IgSF CAMs(ICAM-1, VCAM-1, etc), Selectins, Integrins and Cadherins

17. Capsid of Virus CAMs of Host cell Infection routes of Virus • Nucleic acid of Virus is covered with a certain protein called ‘Capsid’ seems like a “canyons’” or “pockets” pattern. • This capsid will be attached to a certain receptor (ICAM-1, Integrins, etc.) just like a ‘key and lock’ formation

18. Inside of Host cell Capsid of Virus CAMs of Host cell channel Infection routes of Virus, CAMs • CAMs (Cell Adhesion Molecules) • CAMs are proteins located on the cell surface involved with the binding with other cells or with the extracellular matrix (ECM) in the process called cell adhesion • Rotavirus utilize different integrins (glycoproteins) as cell receptors. • The outer end of ICAM-1 have a narrow, wedge-shaped segment that can reach and bind with side chains on the canyon floor of rhinovirus. • CAMs allow the virus to penetrate the cell and replicate. -Wikipedia-

19. Zinc and cell adhesion molecules • In common cold virus(rhinovirus), Zinc ion is a competitive inhibitor of ICAM-1 in both rhinovirus particles and nasal epithelium. • Zinc ions are small, positively charged spheres with an affinity for CAMs that can easily reach the virus canyon floor • Zinc ions may block the binding between Virus and CAMs on the cell membrane. Therefore, it may reduce viral infection of intestinal epithelial cells. [Darrell Hulisz, J Am Pharm Assoc 44(5):594-603, 2004]

20. Development of ICAM (Intercellular Adhesion Molecule) Development of ICAM-1 Flourescent-dyed ICAM-1 and ‘wedge’ liked micro projection Normal cell surface with ICAM-1 (wt-IC1) has micro projections, but other cell surface without inner cell domain(ICAM-1 [IC1ᅀCTD])has no projections Cell membrane without ICAM-1 development Cell membrane with ICAM-1 development TOP VIEW A micro projection in ICAM-1 developed cell Surface of ICAM-1 developed cell SIDE VIEW Jun, Chang-Duk et al, Molecular Biology of the Cell Vol. 18, Issue 6, 2322-2335, June 2007

21. CAMs, Infection, Immune and Zinc ion (Zn++) • CAMs provide a channel to immune cells and pathogen in blood to adhere them on epithelial cell line. Therefore, these immune cells can move into target tissue. • This kind of method can be applied for both of virus and bacteria or immune cell • It means; if these kind of channel were blocked, we could reduce the rate of viral infection in host cells and excessive immune cells flux by increment of the permeability of the cell into the target area/tissue that can lead ‘inflammation’ • Zinc ions may attach glycoproteins/glycolipids on the cell membrane, such as CAMs. Then, it may prevent the binding among epithelial cells and other cells or extracellular matrix

22. AGRAZINC 100 Zn++ : preventing allergy in GIT

23. Immune response.. • In young animals, allergenic factors (ANFs in soy, etc) can induce allergenic reaction in GIT, even in matured animal. • These induce excessive immune reaction on intestinal epithelium, such as ‘hemorrhage’, ‘inflammation’

24. The basic of inflammatory response (immune) • Mast cell • A mast cell is a resident cell of areolar connective tissue (loose connective tissue) and contains many granules rich in histamine and heparin • Once allergen is bound with IgE on a mast cell, ‘mast cell’ is activated and release ‘histamine’, ‘leukotriene’ and ‘Prostaglandine(PG)’ • Histamine increase a permeability of blood vessel. Then, it makes immune cells move into target tissue. • At the same time, ‘mast cell’ release Zn++ as a natural “circuit breaker” to prevent a excessive immune response; regulate the amount of immune cell movement.

25. A permeability of tissue and immune response Zn ions attach to CAMs and regulate the amount of immune cell movement into tissue Michael J. Holtzman et al, Washington Univ. School of Medicine, 2001

26. AGRAZINC 100 : reduction in inflammation and a excessive immune response • Zinc ions released from AGRAZINC 100 are... • acting right on time and place: Once zinc ions are absorbed at intestinal epithelium, they regulate immune cell flux into intestinal epithelium to prevent a allergy-like excessive immune response. • How? • Once Zn ++ of AGRAZINC 100 attach to CAMs (Cell Adhesion Molecules), they can block the binding between CAMs and immune cells that lead inflammation

27. AGRAZINC 100 Differences in types of zinc complex

28. Types and effects of zinc complex • Until now, we have studied ‘mode of action’ are related to AGRAZINC 100 • However, all of zinc complexes are performing same effects were shown in previous slides effectively. • To bring such effects in GIT, zinc ion must be released easily right on time and place. It means too tight bond in a zinc complex is not effective such as chelated minerals.

29. Types and effects of zinc complex

30. AGRAZINC 100 summery • Glycoproteins, Glycolipids and CAMs are very important in AGRAZINC 100 • AGRAZINC 100 attaches to these receptors competitively to prevent pathogen exert their own toxicity on epithelium • AGRAZINC 100 reduces the activity of E. coli as a pathogen, but increase the activity of beneficial bacteria in GIT. • AGRAZINC 100 may reduce a excessive immune response like a ‘allergy’ induced by ANFs (anti nutritional factors). • AGRAZINC 100 bypasses ‘stomach’ by 100% and commence releasing of zinc ions from the beginning of intestine (duodenum).