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Novel Tocopherol Naproxen Ester Pro Drug


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Novel Tocopherol Naproxen Ester Pro Drug

  1. 1. Design, Synthesis and Anti-inflammatory Activity of Novel -Tocopherol Naproxen Ester Prodrug Madan Baral B. Pharm. School of Health and Allied Sciences Pokhara University, PO Box 427, Lekhnath-12, Kaski, NEPAL
  2. 2. OVERVIEW  Introduction  Methodology • General Procedure of Synthesis • Chemical Analysis • Pharmacokinetic Evaluation – In Vivo Bioavaibility Study in Rabbit • Pharmacodynamic Evaluation – Carrageenan Paw Inflammation – Carrageenan Air Pouch Model – Gastric Injury Model • Statistical Analysis  Results and Discussion  Conclusion
  3. 3. Introduction • Naproxen : Non-steroidal anti-inflammatory drugs (NSAIDs) • Therapeutic Uses: Treatment of pain, inflammation and fever, mostly arthritic pain • Side effects : Stomach irritation to ulceration and bleeding • Causes: – blockage of prostaglandin biosynthesis in the GI tract – direct action of free carboxylic groups in NSAIDs and production of reactive oxygen species (ROS) is increased in NSAIDs therapy • Objective : Overcome the oxidative degradation of ROS • Naproxen esters containing tocopherol was designed and synthesized. • In vitro and in vivo tests suggested prodrugs exhibited anti-inflammatory activity with a strong and significant reduction in GI lesion.
  4. 4. Naproxen
  5. 5. Methodology
  6. 6. General Procedure of Synthesis of Naproxen Prodrug Stirring 20 min. Naproxen DCC Acetonitrile Stirring 48 Hr. Tocoph erols Acetonitrie Triethyla mine Filtrate + Ethylacetate Susp. Mixture Distillation Drying Extraction Ester Prodrug
  7. 7. Chemical Analysis • 1H NMR • Electron Impact (EI–MS) mass spectra • HPLC separation • Elemental analysis (C, H, N) • Log D calculation using ACD/Labs software • Thinlayer chromatography • Column chromatography
  8. 8. Chemical and Enzymatic Hydrolysis • Chemical hydrolysis: at 37±0.1 C in buffer phosphate solution at pH 8.0 containing 50% of 2-hydroxypropyl)-β-cyclodextrin (HP-β-CD) • Enzymatic hydrolysis: initiated by diluting 1:10 (v/v) appropriate amounts of a stock solution of each prodrug in methanol (1 mg/ml) with a preheated buffer phosphate solution at pH 8.0 containing 5% dimethyl-β- cyclodextrin (DM-β-CD) and porcine liver esterase at 37±0.1. • • Hydrolysis samples are taken and analyzed by HPLC for naproxen, α- or -tocopherol and the remaining ester prodrug. influence of methanol and DM-β-CD on enzyme activity. • A new HPLC method was developed to simultaneously analyze the synthesized prodrug and the corresponding tocopherol in hydrolysis studies.
  9. 9. Reverse Phase column C18 equipped with direct-connect guard column used for further analysis Methanol 100% 2ml/min 273 nm Methanol+ 0.05 H3PO4 1 ml/ml 315 nm Mobile Phase Flow rate Detection Limit • First Method •Second Method
  10. 10. Pharmacokinectic Evaluation • Male New Zealand albino rabbits (Charles River, Calco,Italy) 2-3 kgs kept under standard laboratory conditions for about 2 weeks • 3 group of 6 male rabbits • Cannulated via central ear artery using polyethylene tubing 15 min prior to oral administration of test drugs for the evaluation of oral bioavaibility. • Blood sample taken were immediately analyzed for the concentration vs. time profile with several different pharmacokinetic parameters • While the plasma concentration naproxen was determined by HPLC method using indomethacin as internal standard.
  11. 11. Pharmacodynamic Evaluation Carrageenan Paw Inflammation • Male Sprague-Dawley rats, six in group taken • Orally administered with freshly prepared test compounds, VE-α-NPX, VE- -NPX, Naproxen or vehicle (5 % Tween 80) were orally administered to each group at 10 mg/kg (Naproxen molar equivalents) 1 hr. prior to the carrageenan injection (50 μl 1% carrageenan in saline) into the right hind paw pad • Paw volume determined and calculated as % inhibition to access the anti-inflammatory activity
  12. 12. Carrageenan Air Pouch Model • Male Sprague-Dawley rats, six in group taken • Air pouch was created by a subcutaneous injection of sterile air into the intrascapular area • Oral administration of test compounds 1 hour prior to the carrageenan injection into the pouch • 4hr later inflammatory exudates collected to determine the prostaglandin E2 (PGE2) levels by
  13. 13. Gastric Injury Model • Freshly prepared test compounds, VE-α-NPX, VE- - NPX, Naproxen or vehicle (5 % Tween 80) were orally administered to rats, six for each group • Killed 3 hour postdosing and stomachs were removed and examined for visible mucosa damage by an observer unaware of the treatment • Measure of all hemorrhagic lesions calculated for gastric damage score, which was the sum of the lengths of all lesions in a stomach
  14. 14. Statistical Analysis • Analysis of variance (ANOVA) • Bonferroni-Dunn post hoc pair-wise comparison
  15. 15. RESULTS
  16. 16. Chemical Analysis
  17. 17. Chemical Hydrolysis
  18. 18. Kinetic Data of Hydrolysis
  19. 19. Comparison of Plasma Conc. Profile
  20. 20. Comparative Pharmcokinetic Parameter
  21. 21. Carrageenan Paw Inflammation Inhibition
  22. 22. Carrageenan Air Pouch Model
  23. 23. Gastric Injury Model
  24. 24. Conclusion • The ester prodrugs of Naproxen VE-α-NPX and VE- -NPX are promising anti-inflammatory agents in the treatment of chronic inflammatory diseases and inflammation- associated disorders due to protective effect against gastric injury.
  25. 25. Reference • Spadaro A, Bucolo C, Ronsisvalle G and Pappalardo M (2009); Design, Synthesis and Antiinflammatory Activity of Novel - Tocopherol Naproxen Ester Prodrug, Journal of Pharmaceutical Sciences and Research1(4), 88-95.
  26. 26. Thank You !!!!!!!!!!!