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

Enhancement Of Bioavailability Enhancement Of Bioavailability Of Atorvastatin Calcium byOf Atorvastatin Calcium by Micro Micro

and Nanoparicle preparation- and Nanoparicle preparation- Design and EvaluationDesign and Evaluation

Dr.K.SenthilkumaranDr.K.Senthilkumaran

INTI International University INTI International University CollegeCollege

S.Dinesh kumar, M.NappinnaiS.Dinesh kumar, M.Nappinnai Department of Pharmaceutics, Department of Pharmaceutics,

C.L.Baid Metha College of Pharmacy,C.L.Baid Metha College of Pharmacy, Chennai, India.Chennai, India.

IntroductionIntroduction

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

Bioavailability studiesBioavailability studies provide other provide other useful pharmacokinetic information useful pharmacokinetic information related to distribution, elimination related to distribution, elimination and metabolism of drugs in the body.and metabolism of drugs in the body.

Bioavailability dataBioavailability data may also provide may also provide information indirectly about some information indirectly about some properties of a drug substance - such properties of a drug substance - such as membrane permeability as membrane permeability

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

SIGNIFICANCE OF BIOAVAILABILITY SIGNIFICANCE OF BIOAVAILABILITY STUDIESSTUDIES

Development of a suitable dosage Development of a suitable dosage form for a new drug entityform for a new drug entity

Determination of influence of Determination of influence of excipients, patient related factors, on excipients, patient related factors, on the efficiency of absorption.the efficiency of absorption.

Development of new formulations of Development of new formulations of the existing drugs.the existing drugs.

Development of suitable dosage form Development of suitable dosage form for a new drug.for a new drug.

METHODS FOR ASSESSING METHODS FOR ASSESSING

BIOAVAILABILITYBIOAVAILABILITY Pharmacokinetic methodsPharmacokinetic methods: based on the : based on the

plasma concentration of the drug and on plasma concentration of the drug and on the assumption that the pharmacokinetic the assumption that the pharmacokinetic profile reflects the therapeutic profile reflects the therapeutic effectiveness of a drug effectiveness of a drug

Pharmacodynamic methods:Pharmacodynamic methods: involves involves direct measurement of drug effect on a direct measurement of drug effect on a pathophysiologic process as a function of pathophysiologic process as a function of time. time.

REASONS FOR POOR BIOAVAILABILITYREASONS FOR POOR BIOAVAILABILITY

Poor aqueous solubility of the drug Poor aqueous solubility of the drug

Poor stability of the dissolved drug at Poor stability of the dissolved drug at physiological pH.physiological pH.

Poor permeation through the Poor permeation through the biomembranesbiomembranes

Extensive presystemic metabolism. Extensive presystemic metabolism.

METHODS TO ENHANCE METHODS TO ENHANCE

BIOAVAILABILITYBIOAVAILABILITY To enhance the dissolution rate To enhance the dissolution rate

of a drug:of a drug: Micro encapsulationMicro encapsulation Use of surfactantsUse of surfactants Solute-solvent complexationsSolute-solvent complexations Solid dispersionsSolid dispersions Molecular encapsulations with Molecular encapsulations with

cyclodextrinscyclodextrins

In the present studyIn the present study

Atorvastatin calcium is reported to Atorvastatin calcium is reported to have low bioavailability of 14% have low bioavailability of 14%

The low systemic availability is The low systemic availability is attributed to presystemic clearance attributed to presystemic clearance in gastro-intestinal mucosa in gastro-intestinal mucosa

The drug is rapidly absorbed after The drug is rapidly absorbed after oral administration and maximum oral administration and maximum plasma concentrations occur within 2 plasma concentrations occur within 2 hours. hours.

SCOPE OF THE PRESENT SCOPE OF THE PRESENT WORKWORK

Atorvastatin calcium is formulated as Atorvastatin calcium is formulated as colloidal particles for oral drug colloidal particles for oral drug delivery with an aim to improve its delivery with an aim to improve its bioavailability. bioavailability.

Biodegradable polymer poly- lactide Biodegradable polymer poly- lactide co- glycolide was used to prepare the co- glycolide was used to prepare the colloidal particles. colloidal particles.

ApproachesApproaches

Incorporation of the drug into Incorporation of the drug into colloidal particles may protect it from colloidal particles may protect it from the first pass effect, which may lead the first pass effect, which may lead to an increased bioavailability to an increased bioavailability

Prolonging the release may also Prolonging the release may also improve the bioavailability.improve the bioavailability.

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

METHODOLOGYMETHODOLOGY

Preparation of colloidal particles of Preparation of colloidal particles of atorvastatin by using poly lactide- atorvastatin by using poly lactide- co-glycolide PLGA-resomer RG co-glycolide PLGA-resomer RG 50:50 H by solvent evaporation 50:50 H by solvent evaporation techniques. techniques.

Determination of drug content, drug Determination of drug content, drug loading and encapsulation loading and encapsulation efficiency of prepared colloidal efficiency of prepared colloidal particles. particles.

Determination of drug – polymer Determination of drug – polymer interaction by FT-IRinteraction by FT-IR

In vitroIn vitro release studies of release studies of dissolution release kinetic analysis.dissolution release kinetic analysis.

In vivo In vivo release study of atorvastatin release study of atorvastatin calcium colloidal particles calcium colloidal particles

Determination of pharmacokinetic Determination of pharmacokinetic parameters. parameters.

In vivoIn vivo pharmacodynamic study pharmacodynamic study

DRUG PROFILEDRUG PROFILE

Structure:Structure:

Molecular formula: (C 33 H 34 F N 2O 5) 2 Ca. 3 H 2 OMolecular 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

PREPARATION OF COLLOIDAL PARTICLES OF PREPARATION OF COLLOIDAL PARTICLES OF ATORVASTATIN CALCIUMATORVASTATIN CALCIUM

Atorvastatin calcium was prepared into Atorvastatin calcium was prepared into colloidal particles with polylactide – co- colloidal particles with polylactide – co- glycolide resomer (RG 50:50H) by solvent glycolide resomer (RG 50:50H) by solvent evaporation method. evaporation method.

100 mg of PLGA was weighed and 100 mg of PLGA was weighed and dissolved in a mixture of dichloro methane dissolved in a mixture of dichloro methane and ethanol in ratio (4:1) and ethanol in ratio (4:1)

100 mg of atorvastatin calcium was added 100 mg of atorvastatin calcium was added to the polymer solution and mixed well. to the polymer solution and mixed well.

The above mixture was added drop wise to The above mixture was added drop wise to the (0.4%) preheated 40oC) polyvinyl the (0.4%) preheated 40oC) polyvinyl alcohol in distilled water. alcohol in distilled water.

Tween 80 was used for emulsion formation Tween 80 was used for emulsion formation and stabilization. and stabilization.

This mixture was continuously This mixture was continuously homogenized to yield a oil in water homogenized to yield a oil in water emulsion.emulsion.

The emulsion was stirred continuously for The emulsion was stirred continuously for 3 hours to allow the organic solvent to 3 hours to allow the organic solvent to evaporate and the hardening of evaporate and the hardening of atorvastatin calcium colloidal particles to atorvastatin calcium colloidal particles to be completed.be completed.

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

S.NoS.No FormulationFormulationDrug : Drug :

PolymerPolymerMethods of Methods of preparationpreparation

Harvesting MethodHarvesting Method

11 F IF I 1:11:1SOSO CC MM LL

HOHO CC MM LL

22 F IIF II 1:21:2SOSO CC MM LL

HOHO CC MM LL

33 F IIIF III 1:31:3SOSO CC MM LL

HOHO CC MM LL

44 F IVF IV 1:41:4SOSO CC MM LL

HOHO CC MM LL

55 F VF V 1:51:5SOSO CC MM LL

HOHO CC MM LL

66 F IVF IV 1:61:6SOSO CC MM LL

HOHO CC MM LL

The formulation trials of The formulation trials of Drug :polymerDrug :polymer

Percentage yield of colloidal particles

S.No.S.No.FormulationFormulation Drug : PolymerDrug : Polymer

Percentage yield of colloidal particlesPercentage yield of colloidal particles

SOSO HOHO

CC LL CC LL

11 FF11 1:11:1 5050 4545 6565 7676

22 FF22 1:21:2 5555 4444 6464 7575

33 FF33 1:31:3 4848 4040 6565 7070

44 FF44 1:41:4 4949 4242 7575 7373

55 FF55 1:51:5 5050 5959 7070 7171

66 FF66 1:61:6 5050 4545 6969 7070

Determination of Drug content in Determination of Drug content in colloidal particlescolloidal particles

Determination the amount of Determination the amount of atorvastatin calcium present in the atorvastatin calcium present in the 100 mg colloidal particles was 100 mg colloidal particles was calculated by using standard calculated by using standard calibration graph of atrovastatin calibration graph of atrovastatin calcium in methanol. calcium in methanol.

The same procedure was triplicated The same procedure was triplicated for each formulation for each formulation

Drug content of Colloidal Particles

S.No.S.No. Formulation all homogenized sample Formulation all homogenized sample (HO)(HO)

Amount of Drug in 100 mg of colloidal particlesAmount of Drug in 100 mg of colloidal particles

Centrifugation Centrifugation (mg)(mg)

Lyophilisation Lyophilisation (mg)(mg)

11 FF11 6060 5555

22 FF22 6262 5959

33 FF33 6161 6060

44 FF44 7575 7878

55 FF55 6565 7474

66 FF66 7272 7070

Figure 1 Drug content of Colloidal particles

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Lyophilised

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

Particle size analysisParticle size analysis

The particle size of formulated The particle size of formulated colloidal micro particles were colloidal micro particles were analyzed by Particle size analyzer.analyzed by Particle size analyzer.

FTIR spectra of the drug, PLGA and FTIR spectra of the drug, PLGA and the drug loaded PLGA colloidal the drug loaded PLGA colloidal particles were obtained. particles were obtained.

In vitroIn vitro release studies release studies

The sample equivalent to 100 mg of The sample equivalent to 100 mg of atorvastatin calcium was placed in 10 ml atorvastatin calcium was placed in 10 ml of phosphate buffer pH 7.2 it was of phosphate buffer pH 7.2 it was maintained at 37°C and magnetically maintained at 37°C and magnetically stirred at 100rpm.stirred at 100rpm.

2 ml of sample aliquot was with drawn at 2 ml of sample aliquot was with drawn at different time intervals and filtered different time intervals and filtered through a 0.45 mm filter through a 0.45 mm filter

The dissolution media was then The dissolution media was then replaced with 2 ml of fresh buffer. replaced with 2 ml of fresh buffer.

The atorvastatin calcium The atorvastatin calcium concentration was determined by concentration was determined by HPLC using ODS, C18 column with HPLC using ODS, C18 column with flow rate of 1ml/min at 250 nm flow rate of 1ml/min at 250 nm

In vitro Dissolution study of colloidal particles

S. No.S. No.Sampling time Sampling time

in coursein course

Concentration mcg/ml Concentration mcg/ml (from HPLC (from HPLC

chromatogram)chromatogram)

Concentration in Concentration in sampling fluid (2 sampling fluid (2

ml)ml)mcgmcg

Cumulative Cumulative amount of drug amount of drug

releasereleasemgmg

Cumulative drug Cumulative drug release percentagerelease percentage

%%

11 15 mins15 mins 4.504.50 450450 2.252.25 2.882.88

22 30 mins30 mins 6.536.53 32653265 16.7716.77 21.5121.51

33 45 mins45 mins 8.828.82 44104410 25.7625.76 33.0333.03

44 1 hr1 hr 10.0010.00 50005000 33.1233.12 41.1941.19

55 2 hr2 hr 11.1911.19 55955595 41.1041.10 52.6952.69

66 3 hrs3 hrs 11.9311.93 59655965 48.5448.54 62.2462.24

77 6 hrs6 hrs 12.6912.69 63456345 56.4156.41 72.3272.32

88 8 hrs8 hrs 13.6313.63 68156815 65.1165.11 83.4783.47

99 24 hrs24 hrs 20.2620.26 81048104 78.3678.36 100100

In vitroIn vitro drug release study of colloidal drug release study of colloidal particlesparticles

Figure 4 In vitro release study of F4

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In vivoIn vivo Pharmacokinetic Study of Pharmacokinetic Study of

atorvastatin colloidal particlesatorvastatin colloidal particles Procedure was carried with the Procedure was carried with the

approval from animal ethical approval from animal ethical committee committee

CPCSEA/IAEC approved detail 10/14-CPCSEA/IAEC approved detail 10/14-CLBMCP/2005-2006.CLBMCP/2005-2006.

Male rabbits weighing about 1 kg to Male rabbits weighing about 1 kg to 1.5 kg were used in this study1.5 kg were used in this study

The animals were housed under The animals were housed under standard environmental conditions standard environmental conditions (23°C (23°C 2°C, 55 2°C, 55 5% relative 5% relative humidity; 12 hours light/ dark cycle). humidity; 12 hours light/ dark cycle).

Prior to oral administration, the Prior to oral administration, the rabbits were starved for 24 hours rabbits were starved for 24 hours and are allowed free access to tap and are allowed free access to tap water only. water only.

The animals (12) were divided into The animals (12) were divided into two groups of 6 animals in each two groups of 6 animals in each group.group.

Group- I received standard drug of Group- I received standard drug of atorvastatin calcium 2.4mg/kg doseatorvastatin calcium 2.4mg/kg dose

Group – II received F4 HO, L particles Group – II received F4 HO, L particles equivalent to 2.4 mg/kg equivalent to 2.4 mg/kg

1ml blood sample were with drawn 1ml blood sample were with drawn from the marginal ear vein of the from the marginal ear vein of the rabbit at regular time interval.rabbit at regular time interval.

The serum samples were separated The serum samples were separated by centrifugation and estimated by by centrifugation and estimated by using HPLC method. using HPLC method.

Comparison of Comparison of In vivoIn vivo pharmacokinetic release studypharmacokinetic release study

S.No.S.No. Sampling timeSampling time Atorvastatin Calcium Atorvastatin Calcium

(raw material)(raw material) Formulation F4 atorvastatin Formulation F4 atorvastatin

colloidal particle mcg/mlcolloidal particle mcg/ml 11 30mts30mts 40.940.9 61.661.6

22 1hr1hr 71.2871.28 98.898.8

33 2hr2hr 121.6121.6 129.7129.7

44 4hr4hr 110.3110.3 118.56118.56

55 8hr8hr 70.570.5 85.5485.54

66 24hr24hr 21.321.3 35.2335.23

Figure 5 In vivo pharmacokinetic release study

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Time (hrs)

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In vivoIn vivo pharmacokinetic pharmacokinetic parametersparameters

Parameters Units Standards Test – F4

C max mcg/ml 121.6 129.7

t max Hrs 2.0 2.0

AUC0- Mcg-hr/ml 1917 2449

AUMCO- (mcg-hr* hr/ml 28570 42406

MRT Hr 14.9 17.3

Pharmacodynamic StudyPharmacodynamic Study

Procedure was carried with approval Procedure was carried with approval from animal ethical committeefrom animal ethical committee

CPCSEA/IAEC approved detail 10/15-CPCSEA/IAEC approved detail 10/15-CLBMCP/2005-2006.CLBMCP/2005-2006.

The rats were divided into two The rats were divided into two groups A and B groups A and B

Group A and B received high fat diet Group A and B received high fat diet (HFD) (HFD)

group A was treated with standard group A was treated with standard drugdrug

group B received the formulation F4 group B received the formulation F4 colloidal particles of atorvastatin colloidal particles of atorvastatin calcium orally, administered.calcium orally, administered.

At the end of experimental period all At the end of experimental period all the animals were fasted over night the animals were fasted over night and blood was with drawn.and blood was with drawn.

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

In vivo Pharmocodynamic study

S.NoS.No ParametersParameters Standard Atorvastatin calcium (Raw Standard Atorvastatin calcium (Raw material) mg/dlmaterial) mg/dl

Test FTest F44 – Formulation Colloidal – Formulation Colloidal

particles mg/dlparticles mg/dl

11 TCTC 67.1 67.1 ++ 1.7 1.7 60.560.5++ 2 2

22 TGTG 5555++ 4.6 4.6 46.7 46.7 ++ 1.5 1.5

33 HDLHDL 20.920.9++ 3 3 14.514.5++ 1.0 1.0

44 LDLLDL 37.437.4++ 1.0 1.0 32.532.5++ 2.2 2.2

55 VLDLVLDL 10.2 10.2 ++ 1 1 9.1 9.1 ++ 0.6 0.6

Figure 6 In vivo Dynamic Study

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TC TG HDL LDL VLDL

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SUMMARYSUMMARY

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

Atorvastatin calcium colloidal Atorvastatin calcium colloidal particles was prepared by using particles was prepared by using emulsification solvent evaporation emulsification solvent evaporation technique to achieve maximum drug technique to achieve maximum drug content. content.

The Prepared Colloidal particle were The Prepared Colloidal particle were evaluated for drug content, drug evaluated for drug content, drug loading and encapsulation efficiency.loading and encapsulation efficiency.

F4Ho, L particles showed smooth F4Ho, L particles showed smooth spherical shape. spherical shape.

Therefore F4Ho, L were subjected to Therefore F4Ho, L were subjected to particle size analysis particle size analysis

The The in vitroin vitro drug release from F4Ho, drug release from F4Ho, L shows the maximum release L shows the maximum release studies after 24 hrs. studies after 24 hrs.

In vivoIn vivo pharmacokinetic study results pharmacokinetic study results revealed that the formulation F4 shows revealed that the formulation F4 shows better Cmax, tmax, AUC, AUMC and MRT, better Cmax, tmax, AUC, AUMC and MRT, than the standard drug.than the standard drug.

The The in vivoin vivo pharmacodynamic study shows pharmacodynamic study shows significance decrease in lipidaemic significance decrease in lipidaemic parameters in rats with formulation F4 parameters in rats with formulation F4 than these administered with standard than these administered with standard atorvastatin calcium. atorvastatin calcium.

To conclude, colloidal particles of To conclude, colloidal particles of atorvastatin calcium improved atorvastatin calcium improved bioavailability of the drug.bioavailability of the drug.

The relative Bio-availability is 1.27 The relative Bio-availability is 1.27 and it is proved by pharmacokinetic and it is proved by pharmacokinetic and pharmacodynamic studies.and pharmacodynamic studies.

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