Design, Synthesis and Anti-inflammatory Activity of Novel -Tocopherol Naproxen

Ester Prodrug

Madan BaralB. Pharm.

School of Health and Allied SciencesPokhara University, PO Box 427, Lekhnath-12, Kaski,

NEPAL

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

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.

Naproxen

Methodology

General Procedure of Synthesis of Naproxen Prodrug

Stirring20 min.

Naproxen

DCC

Acetonitrile

Stirring48 Hr.

Tocopherols

Acetonitrie

Triethylamine

Filtrate + Ethylacetate

Susp. Mixture

Distillation Drying ExtractionEster

Prodrug

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

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.

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

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.

Pharmacodynamic EvaluationCarrageenan 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

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 immunoassay

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

Statistical Analysis

• Analysis of variance (ANOVA)

• Bonferroni-Dunn post hoc pair-wise comparison

RESULTS

Chemical Analysis

Chemical Hydrolysis

Kinetic Data of Hydrolysis

Comparison of Plasma Conc. Profile

Comparative Pharmcokinetic Parameter

Carrageenan Paw Inflammation Inhibition

Carrageenan Air Pouch Model

Gastric Injury Model

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.

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.

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