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

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

3. Introduction 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. 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.

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

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

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

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

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

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

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

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

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

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

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

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

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

17. Determination of pharmacokinetic parameters. Determination of pharmacokinetic parameters. In vivo pharmacodynamic study In vivo pharmacodynamic study

18. DRUG PROFILE Structure: Structure: Molecular formula: (C 33 H 34 F N 2O 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 -40mg daily

19. PREPARATION OF COLLOIDAL PARTICLES OF ATORVASTATIN CALCIUM Atorvastatin calcium was prepared into colloidal particles with polylactide – co- glycolide resomer (RG 50:50H) by solvent evaporation method. Atorvastatin calcium was prepared into colloidal particles with polylactide – co- glycolide resomer (RG 50:50H) 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 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. 100 mg of atorvastatin calcium was added to the polymer solution and mixed well.

20. The above mixture was added drop wise to the (0.4%) preheated 40oC) polyvinyl alcohol in distilled water. The above mixture was added drop wise to the (0.4%) preheated 40oC) polyvinyl alcohol in distilled water. Tween 80 was used for emulsion formation and stabilization. Tween 80 was used for emulsion formation and stabilization. This mixture was continuously homogenized to yield a oil in water emulsion. This mixture was continuously homogenized to yield a oil in water emulsion.

21. 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 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 colloidal particles of atrovastatin calcium, were recovered by refrigerated centrifugation/membrane filtration/ lyophilisation

22. S.NoFormulation Drug : Polymer Methods of preparation Harvesting Method 1 F I 1:1 SOCML HOCML 2 F II 1:2 SOCML HOCML 3 F III 1:3 SOCML HOCML 4 F IV 1:4 SOCML HOCML 5 F V 1:5 SOCML HOCML 6 F IV 1:6 SOCML HOCML The formulation trials of Drug :polymer

23. Percentage yield of colloidal particles S.No.Formulation Drug : Polymer Percentage yield of colloidal particles SOHO CLCL 1 F1F1F1F11:150456576 2 F2F2F2F21:255446475 3 F3F3F3F31:348406570 4 F4F4F4F41:449427573 5 F5F5F5F51:550597071 6 F6F6F6F61:650456970

24. Determination of Drug content in colloidal particles 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. 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 The same procedure was triplicated for each formulation

25. Drug content of Colloidal Particles S.No. Formulation all homogenized sample (HO) Amount of Drug in 100 mg of colloidal particles Centrifugation(mg) Lyophilisation (mg) 1 F1F1F1F16055 2 F2F2F2F26259 3 F3F3F3F36160 4 F4F4F4F47578 5 F5F5F5F56574 6 F6F6F6F67270

27. From the results of analysis of drug content, drug loading, drug encapsulation formulation F4 was selected for further studies. From the results of analysis of drug content, drug loading, drug encapsulation formulation F4 was selected for further studies.

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

29. In vitro release studies The sample equivalent to 100 mg of atorvastatin calcium was placed in 10 ml of phosphate buffer pH 7.2 it was maintained at 37°C and magnetically stirred at 100rpm. The sample equivalent to 100 mg of atorvastatin calcium was placed in 10 ml of phosphate buffer pH 7.2 it was maintained at 37°C and magnetically stirred at 100rpm. 2 ml of sample aliquot was with drawn at different time intervals and filtered through a 0.45 mm filter 2 ml of sample aliquot was with drawn at different time intervals and filtered through a 0.45 mm filter

30. The dissolution media was then replaced with 2 ml of fresh buffer. The dissolution media was then replaced with 2 ml of fresh buffer. The atorvastatin calcium concentration was determined by HPLC using ODS, C18 column with flow rate of 1ml/min at 250 nm The atorvastatin calcium concentration was determined by HPLC using ODS, C18 column with flow rate of 1ml/min at 250 nm

31. 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 4.504502.252.88 2 30 mins 6.53326516.7721.51 3 45 mins 8.82441025.7633.03 4 1 hr 10.00500033.1241.19 5 2 hr 11.19559541.1052.69 6 3 hrs 11.93596548.5462.24 7 6 hrs 12.69634556.4172.32 8 8 hrs 13.63681565.1183.47 9 24 hrs 20.26810478.36100

32. In vitro drug release study of colloidal particles

33. In vivo Pharmacokinetic Study of atorvastatin colloidal particles Procedure was carried with the approval from animal ethical committee Procedure was carried with the approval from animal ethical committee CPCSEA/IAEC approved detail 10/14- CLBMCP/2005-2006. CPCSEA/IAEC approved detail 10/14- CLBMCP/2005-2006. Male rabbits weighing about 1 kg to 1.5 kg were used in this study Male rabbits weighing about 1 kg to 1.5 kg were used in this study

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

35. The animals (12) were divided into two groups of 6 animals in each group. The animals (12) were divided into two groups of 6 animals in each group. Group- I received standard drug of atorvastatin calcium 2.4mg/kg dose Group- I received standard drug of atorvastatin calcium 2.4mg/kg dose Group – II received F4 HO, L particles equivalent to 2.4 mg/kg Group – II received F4 HO, L particles equivalent to 2.4 mg/kg

36. 1ml blood sample were with drawn from the marginal ear vein of the rabbit at regular time interval. 1ml 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. The serum samples were separated by centrifugation and estimated by using HPLC method.

37. Comparison of In vivo pharmacokinetic release study S.No. Sampling time Atorvastatin Calcium (raw material) Formulation F4 atorvastatin colloidal particle mcg/ml 130mts40.961.6 21hr71.2898.8 32hr121.6129.7 44hr110.3118.56 58hr70.585.54 624hr21.335.23

39. In vivo pharmacokinetic parameters ParametersUnitsStandardsTest – F 4 C max mcg/ml121.6129.7 t max Hrs2.0 AUC 0-  Mcg-hr/ml19172449 AUMC O-  (mcg-hr* hr/ml2857042406 MRTHr14.917.3

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

41. group A was treated with standard drug group A was treated with standard drug group B received the formulation F4 colloidal particles of atorvastatin calcium orally, administered. group B received the formulation F4 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. At the end of experimental period all the animals were fasted over night and blood was with drawn.

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

43. In vivo Pharmocodynamic study S.NoParameters Standard Atorvastatin calcium (Raw material) mg/dl Test F 4 – Formulation Colloidal particles mg/dl 1TC 67.1 + 1.7 60.5+ 2 2TG 55+ 4.6 46.7 + 1.5 3HDL 20.9+ 3 14.5+ 1.0 4LDL 37.4+ 1.0 32.5+ 2.2 5VLDL 10.2 + 1 9.1 + 0.6

45. SUMMARY 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. 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.

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

47. F4Ho, L particles showed smooth spherical shape. F4Ho, L particles showed smooth spherical shape. Therefore F4Ho, L were subjected to particle size analysis Therefore F4Ho, L were subjected to particle size analysis The in vitro drug release from F4Ho, L shows the maximum release studies after 24 hrs. The in vitro drug release from F4Ho, L shows the maximum release studies after 24 hrs.

48. In vivo pharmacokinetic study results revealed that the formulation F4 shows better Cmax, tmax, AUC, AUMC and MRT, than the standard drug. In vivo pharmacokinetic study results revealed that the formulation F4 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 F4 than these administered with standard atorvastatin calcium. The in vivo pharmacodynamic study shows significance decrease in lipidaemic parameters in rats with formulation F4 than these administered with standard atorvastatin calcium.

49. To conclude, colloidal particles of atorvastatin calcium improved bioavailability of the drug. 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. The relative Bio-availability is 1.27 and it is proved by pharmacokinetic and pharmacodynamic studies.

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