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. 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. 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. Naproxen

5. Methodology

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. 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. 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. 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. 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. 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. 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. 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. Statistical Analysis
• Analysis of variance (ANOVA)
• Bonferroni-Dunn post hoc pair-wise comparison

15. RESULTS

16. Chemical Analysis

17. Chemical Hydrolysis

18. Kinetic Data of Hydrolysis

19. Comparison of Plasma Conc. Profile

20. Comparative Pharmcokinetic Parameter

21. Carrageenan Paw Inflammation Inhibition

22. Carrageenan Air Pouch Model

23. Gastric Injury Model

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. 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. Thank You !!!!!!!!!!!