1. LECTURE 4

2. INTRODUCTION Irreversible type of indirect cholinomimetics are phosphate esters which are very stable to hydrolysis. Cholinomimetics Direct Indirect Reversible Irreversible

3. 2- Irreversible AChEIs Mechanism of action: -They inhibit AChE by the same mechanism as the carbamate inhibitors except that they leave the enzyme esterified as phosphate esters. -The rate of hydrolytic regeneration of the phosphorylated enzyme is much slower than that of the carbamylated enzyme (in hours).

4. Indirect Cholinomimetics Inactive

5. 2- Irreversible AChEIs Why referred to as irreversible inhibitors? 1- because the duration of action is very long >>> death occurs before regeneration takes place. 2- because the phosphorylated ACHE can undergo a process known as aging.

6. 2- Irreversible AChEIs

7. Applications 1- Medical (Isofluorophate) 2- Insecticides (Parathion) 3- Warfare agents (Sarin)

8. 2- Irreversible AChEIs Aging is the result of cleavage of one or more of the phosphoester bonds while the AChE is phosphorylated. Phosphorus atom become much less electrophilic >>> will not undergo hydrolytic regeneration to give the active form of AChE. Only those phosphorus-derived AChEIs that possess at least one phosphoester group undergo this aging process.

9. Isofluorophate -It is an irreversible AChEI & a powerful miotic agent which can effectively reduce eye pressure. Uses: Chronic glaucoma(topical).

10. Parathion - It contains Sulfur atom bonded to the Phosphorous atom. - It is a very weak AChEI - Parathion is rapidly bioactivated by microsomal oxidation in insects (but not in human)to afford the corresponding oxo dv. which is a powerful AChEI:

11. Isofluorophate Disadvantage: Its vapor is highly toxic (nerve gas)and it is recommended that only solutions in arachis oil can be used therapeutically.

12. SARIN - It is a colorless, odorless heavy lipophilic liquid - It is an illegal chemical warfare - After phosphorylation, only one aging reaction takes place, and then the enzyme becomes completely refractory to regeneration.

13. SARIN - It is lethal even at very low conc. -People who absorb a non-lethal dose, but do not receive immediate medical treatment, may suffer of permanent neurological damage

14. SARIN Symptoms of sarin exposure could be summarized in what is known as SLUDGEM syndrome: Which is a summary of the pathological effects indicative of massive discharge of peripheral nervous system.

15. SARIN SLUDGEM (cont.) 1-Salivation: stimulation of salivary gland 2-Lacrimation: stimulation of lacrimal gland 3-Urination: relaxation of int. sphincter of urethra 4-Defecation: relaxation of int. anal sphincter 5-GIT upset: diarrhea 6-Emesis: vomiting 7-Miosis: stimulation of pup. constrictor muscles or 8-Muscle spasm: Stimulation of skeletal muscle

16. Antidote for irreversible AChEIs The problem required the design of reagents capable of: 1- efficiently catalyzing phosphate ester hydrolysis (strong nucleophile)and regenerating active AChE. 2- being safe enough for use as therapeutic agents.

17. PRALIDOXIME - Pralidoxime (PAM) is an oxime derivative 2-pyridinealdoxime chloride - It is a strong nucleophile and safe at the same time.

18. CHEMICAL SYNTHESIS PAM is synthesized by reacting picolinaldehyde (2-formyl pyridine) with hydroxylamine, giving pyridine-2-aldoxime, which is further reacted with an alkyl halide, giving the desired pralidoxime:

19. MODE OF ACTION 1-In organophosphate poisoning, an organophosphate binds to just one end of the acetylcholinesterase enzyme [ the anionic site ], blocking its activity. Pralidoxime is able to attach to the other half [ the unblocked, esteric site ] of the acetylcholinesterase enzyme.

20. MODE OF ACTION 2-It then binds to the organophosphate, the organophosphate changes conformation, and loses its binding to the acetylcholinesterase enzyme.

21. MODE OF ACTION 3-The conjoined poison / antidote then unbinds from the site, and thus regenerates the enzyme, which is now able to function again.

22. LIMITATIONS 1- It must be given within a short period of time, after enzyme phosphorylation.. Why? 1- because of the aging process. 2- It is only effective in organophosphate toxicity 2- (i.e. it does not have an effect if the AChE is carbamylated )

23. LIMITATIONS 3- It can not cross the blood-brain barrier to regenerate phosphorylated AChE in the brain, 3- this is why atropine, is concomitantly administered with pralidoxime during the treatment of organophosphate poisoning