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.
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
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.