Enhancement Of Bioavailability Of Atorvastatin Calcium by Micro

Enhancement Of Bioavailability Of Atorvastatin Calcium by Micro and Nanoparicle preparation. Design and Evaluation Dr. K. Senthilkumaran INTI International University College

n n n S. Dinesh kumar, M. Nappinnai Department of Pharmaceutics, C. L. Baid Metha College of Pharmacy, Chennai, India.

Introduction n Bioavailability : is the true rate and extent of therapeutically active drug, which reaches the systemic circulation and is available at the site of action from an administered dosage form.

n n Bioavailability studies provide other useful pharmacokinetic information related to distribution, elimination and metabolism of drugs in the body. Bioavailability data may also provide information indirectly about some properties of a drug substance - such as membrane permeability

n Absolute bioavailability of a drug is 1 (or 100%) indicates complete absorption of drug.

SIGNIFICANCE OF BIOAVAILABILITY STUDIES n n Development of a suitable dosage form for a new drug entity Determination of influence of excipients, patient related factors, on the efficiency of absorption.

n n Development of new formulations of the existing drugs. Development of suitable dosage form for a new drug.

METHODS FOR ASSESSING BIOAVAILABILITY n n Pharmacokinetic methods: based on the plasma concentration of the drug and on the assumption that the pharmacokinetic profile reflects therapeutic effectiveness of a drug Pharmacodynamic methods: involves direct measurement of drug effect on a pathophysiologic process as a function of time.

REASONS FOR POOR BIOAVAILABILITY n n Poor aqueous solubility of the drug Poor stability of the dissolved drug at physiological p. H. Poor permeation through the biomembranes Extensive presystemic metabolism.

METHODS TO ENHANCE BIOAVAILABILITY n n n To enhance the dissolution rate of a drug: Micro encapsulation Use of surfactants Solute-solvent complexations Solid dispersions Molecular encapsulations with cyclodextrins

In the present study n n n Atorvastatin calcium is reported to have low bioavailability of 14% The low systemic availability is attributed to presystemic clearance in gastro-intestinal mucosa The drug is rapidly absorbed after oral administration and maximum plasma concentrations occur within 2 hours.

SCOPE OF THE PRESENT WORK n n Atorvastatin calcium is formulated as colloidal particles for oral drug delivery with an aim to improve its bioavailability. Biodegradable polymer poly- lactide co- glycolide was used to prepare the colloidal particles.

Approaches n n Incorporation of the drug into colloidal particles may protect it from the first pass effect, which may lead to an increased bioavailability Prolonging the release may also improve the bioavailability.

n Size reduction of the final formulation in colloidal particles improve absorption leading to improvement in bioavailability.

METHODOLOGY n n Preparation of colloidal particles of atorvastatin by using poly lactideco-glycolide PLGA-resomer RG 50: 50 H by solvent evaporation techniques. Determination of drug content, drug loading and encapsulation efficiency of prepared colloidal particles.

n n n Determination of drug – polymer interaction by FT-IR In vitro release studies of dissolution release kinetic analysis. In vivo release study of atorvastatin calcium colloidal particles

n n Determination of pharmacokinetic parameters. In vivo pharmacodynamic study

DRUG PROFILE n Structure: Molecular formula: (C 33 H 34 F N 2 O 5) 2 Ca. 3 H 2 O Molecular weight: 1209. 42 Description: White to off-white crystalline powder. Solubility : Atorvaststin calcium is very slightly soluble in water, freely soluble in methanol. Dose: 10 -40 mg daily

PREPARATION OF COLLOIDAL PARTICLES OF ATORVASTATIN CALCIUM n n n Atorvastatin calcium was prepared into colloidal particles with polylactide – coglycolide resomer (RG 50: 50 H) by solvent evaporation method. 100 mg of PLGA was weighed and dissolved in a mixture of dichloro methane and ethanol in ratio (4: 1) 100 mg of atorvastatin calcium was added to the polymer solution and mixed well.

n n n The above mixture was added drop wise to the (0. 4%) preheated 40 o. C) polyvinyl alcohol in distilled water. Tween 80 was used for emulsion formation and stabilization. This mixture was continuously homogenized to yield a oil in water emulsion.

n n The emulsion was stirred continuously for 3 hours to allow the organic solvent to evaporate and the hardening of atorvastatin calcium colloidal particles to be completed. The colloidal particles of atrovastatin calcium, were recovered by refrigerated centrifugation/membrane filtration/ lyophilisation

The formulation trials of Drug : polymer S. No Formulation Drug : Polymer Harvesting Methods of preparation SO 1: 2 3 F III 1: 3 4 F IV 1: 4 5 F V 1: 5 6 F IV 1: 6 M L C M L HO C M L SO F II C SO 2 HO SO 1: 1 L SO F I M SO 1 C C M L HO C M L

Percentage yield of colloidal particles S. No. Formulation Drug : Polymer SO HO C L 1 F 1 1: 1 50 45 65 76 2 F 2 1: 2 55 44 64 75 3 F 3 1: 3 48 40 65 70 4 F 4 1: 4 49 42 75 73 5 F 5 1: 5 50 59 70 71 6 F 6 1: 6 50 45 69 70

Determination of Drug content in colloidal particles n n Determination the amount of atorvastatin calcium present in the 100 mg colloidal particles was calculated by using standard calibration graph of atrovastatin calcium in methanol. The same procedure was triplicated for each formulation

Drug content of Colloidal Particles Amount of Drug in 100 mg of colloidal particles S. No. Formulation all homogenized sample (HO) 1 Centrifugation (mg) Lyophilisation (mg) F 1 60 55 2 F 2 62 59 3 F 3 61 60 4 F 4 75 78 5 F 5 65 74 6 F 6 72 70

n From the results of analysis of drug content, drug loading, drug encapsulation formulation F 4 was selected for further studies.

Particle size analysis n n The particle size of formulated colloidal micro particles were analyzed by Particle size analyzer. FTIR spectra of the drug, PLGA and the drug loaded PLGA colloidal particles were obtained.

In vitro release studies n n n The sample equivalent to 100 mg of atorvastatin calcium was placed in 10 ml of phosphate buffer p. H 7. 2 it was maintained at 37°C and magnetically stirred at 100 rpm. 2 ml of sample aliquot was with drawn at different time intervals and filtered through a 0. 45 mm filter

n n The dissolution media was then replaced with 2 ml of fresh buffer. The atorvastatin calcium concentration was determined by HPLC using ODS, C 18 column with flow rate of 1 ml/min at 250 nm

In vitro Dissolution study of colloidal particles S. No. Sampling time in course Concentration mcg/ml (from HPLC chromatogram) Concentration in sampling fluid (2 ml) mcg Cumulative amount of drug release mg Cumulative drug release percentage % 1 15 mins 4. 50 450 2. 25 2. 88 2 30 mins 6. 53 3265 16. 77 21. 51 3 45 mins 8. 82 4410 25. 76 33. 03 4 1 hr 10. 00 5000 33. 12 41. 19 5 2 hr 11. 19 5595 41. 10 52. 69 6 3 hrs 11. 93 5965 48. 54 62. 24 7 6 hrs 12. 69 6345 56. 41 72. 32 8 8 hrs 13. 63 6815 65. 11 83. 47 9 24 hrs 20. 26 8104 78. 36 100

In vitro drug release study of colloidal particles

In vivo Pharmacokinetic Study of atorvastatin colloidal particles n n n Procedure was carried with the approval from animal ethical committee CPCSEA/IAEC approved detail 10/14 CLBMCP/2005 -2006. Male rabbits weighing about 1 kg to 1. 5 kg were used in this study

n n The animals were housed under standard environmental conditions (23°C 2°C, 55 5% relative humidity; 12 hours light/ dark cycle). Prior to oral administration, the rabbits were starved for 24 hours and are allowed free access to tap water only.

n n n The animals (12) were divided into two groups of 6 animals in each group. Group- I received standard drug of atorvastatin calcium 2. 4 mg/kg dose Group – II received F 4 HO, L particles equivalent to 2. 4 mg/kg

n n 1 ml blood sample were with drawn from the marginal ear vein of the rabbit at regular time interval. The serum samples were separated by centrifugation and estimated by using HPLC method.

Comparison of In vivo pharmacokinetic release study Sampling time Atorvastatin Calcium Formulation F 4 atorvastatin (raw material) S. No. colloidal particle mcg/ml 1 30 mts 40. 9 61. 6 2 1 hr 71. 28 98. 8 3 2 hr 121. 6 129. 7 4 4 hr 110. 3 118. 56 5 8 hr 70. 5 85. 54 6 24 hr 21. 3 35. 23

In vivo pharmacokinetic parameters Parameters Units Standards Test – F 4 C max mcg/ml 121. 6 129. 7 t max Hrs 2. 0 Mcg-hr/ml 1917 2449 (mcg-hr* hr/ml 28570 42406 Hr 14. 9 17. 3 AUC 0 - AUMCO- MRT

Pharmacodynamic Study n n Procedure was carried with approval from animal ethical committee CPCSEA/IAEC approved detail 10/15 CLBMCP/2005 -2006. The rats were divided into two groups A and B Group A and B received high fat diet (HFD)

n n n group A was treated with standard drug group B received the formulation F 4 colloidal particles of atorvastatin calcium orally, administered. At the end of experimental period all the animals were fasted over night and blood was with drawn.

n The total lipids (TL), total cholesterol (TC), triglycerides (TG), phospholipids (PL) and HDl. Cholesterol were estimated in the serum, using commercially available standard kits.

In vivo Pharmocodynamic study S. No Parameters Standard Atorvastatin calcium (Raw material) mg/dl Test F 4 – Formulation Colloidal particles mg/dl 1 TC 67. 1 + 1. 7 60. 5+ 2 2 TG 55+ 4. 6 46. 7 + 1. 5 3 HDL 20. 9+ 3 14. 5+ 1. 0 4 LDL 37. 4+ 1. 0 32. 5+ 2. 2 5 VLDL 10. 2 + 1 9. 1 + 0. 6

SUMMARY n In the present study an attempt has made to increase the bio availability of the drug by preparing colloidal particles using different drug to polymer ratio to determine the suitable formation.

n n Atorvastatin calcium colloidal particles was prepared by using emulsification solvent evaporation technique to achieve maximum drug content. The Prepared Colloidal particle were evaluated for drug content, drug loading and encapsulation efficiency.

n n n F 4 Ho, L particles showed smooth spherical shape. Therefore F 4 Ho, L were subjected to particle size analysis The in vitro drug release from F 4 Ho, L shows the maximum release studies after 24 hrs.

n n In vivo pharmacokinetic study results revealed that the formulation F 4 shows better Cmax, tmax, AUC, AUMC and MRT, than the standard drug. The in vivo pharmacodynamic study shows significance decrease in lipidaemic parameters in rats with formulation F 4 than these administered with standard atorvastatin calcium.

n n To conclude, colloidal particles of atorvastatin calcium improved bioavailability of the drug. The relative Bio-availability is 1. 27 and it is proved by pharmacokinetic and pharmacodynamic studies.

n THANK YOU