Formulation and Evaluation of Liposomal Formulation and Evaluation of Liposomal Drug Delivery System for DocetaxelDrug Delivery System for Docetaxel

Presented by : Reg No: 08P08207

M. Shanmukha Srinivas, IInd M.Pharm,

Under the guidance of :

Mr. GNK Ganesh M.Pharm.,

Lecturer,

Department of Pharmaceutics,

JSSCP, Ooty.

Introduction:Introduction:The goal of any drug delivery system is to provide a therapeutic amount of drug to the proper site in the body, to achieve promptly and then maintain the desired drug concentration.

Liposomes were first produced in England in 1961 by Alec D. Bangham. Liposomes are “microscopic, fluid-filled pouch whose walls are made of layers of phospholipids identical to the phospholipids that make up cell membranes”.

Liposomes: An ideal “Drug carrier” for anticancer drugs:

Anticancer drugs are known to produce serious side effects like myocardiopathy and pulmonary toxicity to other healthy tissues. Therefore targeting such type drugs to the cancerous cell is essential.

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The alternative is to use simple functional molecules which transport the drug to specific site and release it to perform task.

Liposomes are non-toxic, biodegradable microcapsule made up of one or multiple lipid bilayer membranes. Chemicals of interest can be entrapped inside the aqueous compartment of liposomes or can be incorporated into the lipid bilayer.

Liposomes have been proved as suitable vehicles for selective drug delivery and controlled drug release.

WHY?WHY?

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Site- avoidance delivery:Liposomes are taken up poorly by tissues such as heart, kidney and GI tract, which are major sites for toxic side-effects of a variety of anti neoplastic drugs.

Site-specific targeting:Reduce exposure to normal tissues.

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Formation of Liposome

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Schematic illustration of liposomes of different size and number of lamellae. SUV: Small unilamellar vesicles; LUV: Large unilamellar vesicles; MLV: Multilamellar vesicles; MVV: Multivesicular vesicles.

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General structure of Phospholipid

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Drug encapsulation in liposomes

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Accumulation of liposomes within solid tumours — (right) liposome extravasation from the disorganised tumour vasculature and (left) liposomes in normal tissue

Objective:Objective:

The main objective of the work is to prepare and evaluate the Docetaxel liposomes . The further objective of the work are as below

To study the effect of various stabilizers on drug entrapment efficacy.

To reduce the side effects.

To target the site of action.

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Scope of workScope of work The Docetaxel being hydrophobic in nature is solubilized in a 50:50 mixture of Cremophor EL(a polyethoxylated castor oil) and ethanol. Cremophor EL has been associated with a number of side effects, including hypersensitivity, nephrotoxicity and neurotoxicity. It also alter the biochemical properties of lipoproteins with partial mediation of the cytotoxic activity of docetaxel in primary cultures of tumor cells from patients.

To overcome these problems, an alternative approach is needed. In the present study docetaxel liposomes were formulated using various biolipids. The formulation can be delivered via iv using buffer pH 7.4 as vehicle which in turn overcome the side-effects of Cremophor EL. As the efficacy of the drug increases with increase in concentration, the effect of stabilizers on the entrapment of drug is studied. Being lipoid in nature the therapy may have better cell entrapment. Thus we assume the formulations may alter the patient compliance.

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WHY?

Neutral liposomes:Lack of surface charge can reduce physical stability of liposomes by increasing their aggregation. Do not interact significantly with cells.

Charged liposomes: Influence the extent of liposome interaction with cells and also accelerate their plasma clearance after systemic administration

Literature Review:Literature Review:Marc J. Ostro., et al described the methods to reduce the dosage of drug with the help of liposomes and their potential advantages and uses in different types of diseased states. In this they confirmed that liposomes are better dosage form than conventional dosage forms. They have also stated the drug can be targeted by active and passive targeting and the uses of both passive and targeting of liposomes.

Antoaneta V., et al studied about cholesterol and other sterols are important components of biological membranes and are known to strongly influence the physical characteristics of lipid bilayers. Although this has been studied extensively in fully hydrated membranes, little is known about the effects of cholesterol on the stability of membranes in the dry state.

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Jorge J. C. S., et al reported the methods of liposomal formulation for encapsulating the enzyme (L-asparaginase). In this study they formulated liposomes with natural phospholipids (egg yolk lecithin).

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Rassoul Dinarvand ., et al prepared PEGylated liposomal formulation of docetaxel has been developed with the purpose of improving the docetaxel solubility without any need to use tween80 that is responsible for hypersensitivities following administration. The PEGylated liposomal formulation of docetaxel were prepared by dried thin film hydration technique.

Harris shoaib M ., et al developed a once-daily sustained release matrix tablet of ibuprofen using hydroxypropyl methylcellulose (HPMC) as release controlling factor and evaluated drug release parameters as per various release kinetic models. Different dissolution models were applied to drug release data in order to evaluate release mechanisms and kinetics.

Ramesh Panchagnula., described the source, chemistry, synthesis and solubility of paclitaxel. And he described the current. Approaches focused on developing formulations that are devoid of Cremophor® EL, the possibility of large-scale preparation; and stability for longer periods of time.

Plan of work:Plan of work:

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STAGE 1:Preformulation studiesStandard calibration curve of docetaxel in UVStandard calibration curve of docetaxel in HPLCCompatibility studies

STAGE 2:Preparation of docetaxel liposomesPreparation of drug loaded liposomes by dried thin lipid film hydration method.Preparation of charged and neutral liposomes.

STAGE 3:Physicochemical characterization of liposomesParticle size analysisZeta potentialSEM studies

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STAGE 4:In vitro characterizationPercentage of drug contentStudy on in vitro drug release from neutral and charged liposomes Release kinetics

STAGE 5:Short term stability studies

DRUG PROFILE:DRUG PROFILE:DRUG : Docetaxel Trihydrate Docetaxel , a diterpenoid synthesised from paclitaxel (which is derived from needles and bark of the Pacific Yew tree (Taxus brevifolia) )

Molecular formula : C43H53NO14

Molecular weight : 807.879 g/mol

Bioavailability : orally 8±6%

Metabolism : Hepatic

Half life: 48-72 hours

Excretion : Biliary

Therapeutic considerations:Routes : IV

Mechanism of Action: potent inhibitor of cell replication and microtubule inhibitor

Clinical use: Docetaxel is approved by the FDA for the treatment of ovarian, breast and lung cancers. It is also used in the treatment of Kaposi’s sarcoma. 17

LIPID PROFILE:LIPID PROFILE:Name : Soybean lecithin

Synonym : Phosphatidylcholine

Description:colour : Yellowish brownconsistency : agglomeratesIodine value : 85-95%solubility : soluble in both aqueous and organic phase.

Uses : Lecithin is used as a food supplement and for medical uses

Chemistry : Lecithin is composed of phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine cholesterol.

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2. Preformulation studies:2. Preformulation studies:Standard calibration curve of Docetaxel: The UV absorbance’s of docetaxel standard solutions in the range of 10-50 µg/ml of drug in 50:50 of acetonitrile and buffer pH 3.0 showed linearity at λmax 227nm .The linearity was plotted for absorbance(A) against concentration (C) with R² value 0.996 and with the slope equation y=0.0187x-0.0039.

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Standard calibration curve of Docetaxel in HPLC:

A stock solution of (1mg/ml) of standard drug was prepared, later required dilutions were made with a mixture of acetonitrile: phosphate buffer pH 3.0 (56:44). The standard chromatogram of docetaxel showed in Figure and the calibration curve showed below. HPLC DATA:Mobile phase: Methanol: PBS (pH 3.0)Flow rate: 1ml/minSample injected: 20µlConcentration range: 10-50 µg/mlColumn: C18

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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min

-0.50

-0.25

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

mAU(x10)

0

10

20

30

40

50

60

70

80

90

psi

B.Press.(Status)A.Press.(Status)227nm4nm (1.00)

DT

/5.9

54/3

25791

Standard chromatogram of Docetaxel

Compatibility studies:

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The compatibility between the drug and the selected lipid and other chemicals was evaluated using FTIR peak matching method. There was no appearance or disappearance of peaks in the drug-lipid mixture, which confirmed the absence of any chemical interaction between the drug , lipid and other chemicals.

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Chemicals Mixture of Docetaxel trihydrate, Soy lecithin and

Cholesterol

Docetaxel TrihydrateCarbonylgroups(C=O) – 1737 & 1710 Amine groups (NH) –

3373.61Hydroxyl groups (OH) –

3493.2

1737.92 & 1710.92 3373.61 3493.2

CholesterolKetone groups(C=O) –

1670.41Hydroxyl groups (OH) –

3396.76Aromatic (C-C) –

1465.95

1674.98 3396.76 1465.98

Soy lecithinCarbonyl groups(C=O) –

1739.85Hydroxyl groups (OH) –

3410.26Carboxylic acids –

3192.80

1737.92 3470.06 3190.37

There was no interaction between the Drug , cholesterol and soy lecithin

RANGE : 4000 – 400 cm-1

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Chemicals Mixture of Docetaxel trihydrate, Soy lecithin,

Cholesterol and Stearylamine

Docetaxel TrihydrateCarbonylgroups(C=O) – 1737 & 1710 Amine groups (NH) – 3373.61Hydroxyl groups (OH) – 3493.2

1737.92 & 1710.92 3373.61 3493.2

CholesterolKetone groups(C=O) –

1670.41Hydroxyl groups (OH) –

3396.76Aromatic (C-C) –

1465.95

1654.98 3396.76 1465.98

Soy lecithinCarbonyl groups(C=O) –

1739.85Hydroxyl groups (OH) –

3410.26Carboxylic acids –

3232.80

1737.92 3470.06 3190.37

StearylamineAmines (NH) –

3333.10 3335.57

RANGE : 4000 – 400 cm-1

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Chemicals Mixture of Docetaxel trihydrate, Soy lecithin,

Cholesterol and Dicetylphosphate

Docetaxel TrihydrateCarbonylgroups(C=O) – 1737 & 1710 Amine groups (NH) – 3373.61Hydroxyl groups (OH) – 3493.2

1737.92 & 1710.92 3373.61 3493.2

CholesterolKetone groups(C=O) –

1670.41Hydroxyl groups (OH) –

3396.76Aromatic (C-C) –

1465.95

1659.58 3396.76 1465.98

Soy lecithinCarbonyl groups(C=O) –

1739.85Hydroxyl groups (OH) –

3410.26Carboxylic acids –

3232.80

1737.92 3437.06 3224.67

DicetylphosphateHydroxyl groups (OH) – 3437Amine groups (N-H) – 1631

3440.03 1635.29

RANGE : 4000 – 400 cm-1

Formulation and Optimization of Formulation and Optimization of liposomesliposomes

Procedure for the preparation of Docetaxel liposomes: Docetaxel liposomes were prepared by using dried film hydration tecnique. Accurately weighed drug and other chemicals were dissolved in 10ml of chloroform and stirred in mechanical stirrer to form a homogenous mixture. The mixture was dried in rotary evaporator with vacuum of about 25mm Hg at 25°c. The process was continued until all the chloroform gets evaporated to get a dried thin film on the inner surface of the vacuum flask. Add 10ml of PBS pH 7.4 and rotated at 25°c without vacuum, to get a homogenous liposomal suspension of multi lamellar vesicles (MLVs).

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DOCETAXEL

DISSOLVED IN CHLOROFORM

UNDER MAGNETIC STIRRING

LIPID AND OTHER

CHEMICALS

Homogenous Mixture

Chloroform evaporated in

Rotary vacuum evaporator

Dried thin Film formed

Dissolved in pH 7.4

Liposomal suspension

formedFreeze drying

Liposomes

Ratio of ingredients

Types of Liposomes

Neutral

Positive

Negative

Lecithin:cholesterol: stearyl amine:Dicetyl

phosphate

5:5:0:0 4.5:4.5:1:0 4.5:4.5:0:1

6:4:0:0 5:4:1:0 5:4:0:1

7:3:0:0 6:3:1:0 6:3:0:1

8:2:0:0 7:2:1:0 7:2:0:1

9:1:0:0 8:1:1:0 8:1:0:1

4:6:0:0 4:5:1:0 4:5:0:1

3:7:0:0 3:6:1:0 3:6:0:1

2:8:0:0 2:7:1:0 2:7:0:1

1:9:0:0 1:8:1:0 1:8:0:1

Table no:1The composition and ratios of lecithin, cholesterol and stabilizers for different types of liposomes .

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Type of liposom

es

Drug Soy lecithin

Cholesterol

Stearyl amine

Dicetyl phospha

te

Neutral 2 8 2 - -

Positive 2 7 2 1 -

Negative 2 7 2 - 1

Table no :2 The composition and ratios of Drug, lecithin, cholesterol and stabilizers for optimized batches.

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120 mg in 10 ml of pH 7.4

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Negatively charged liposomal suspension form

Positively charged liposomal suspension form

Neutral liposomal suspension form

Powder form Powder form Powder form

Particle size analysisParticle size analysis Particle size and size distribution of liposomes in the extruded suspension were determined by laser light scattering (Zetasizer ZS, Malvern, UK).

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Sample Particle size (µm)

Zeta potential

(mV)

Poly Dispersi

ve index(Pd

i)

Neutral 20 16.12 0.351

Positive 14 24.66 0.347

Negative 11 -25.21 0.635

Table no:3 Physicochemical characteristics of the neutral and charged liposomes

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SEM images

Neutral liposome Positive liposome

Negative liposome

Percentage of drug Percentage of drug contentcontent Liposomes containing drug equivalent to 20mg was centrifuged and the supernatant was diluted with aliquot amount of ACN and Phosphate buffer pH 3.0 and the concentration was determined by UV-Visible spectrophotometer. The amount of drug loaded was determined by the formula:

Drug loading = Total amount of drug in solution – amount drug present in supernatant % of drug content = (amount of drug loaded / label claim) x 100

Table no:4 Percentage of drug content of Docetaxel in charged and neutral liposomes are as below

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S. No Type of liposome Percentage of drug content

1 Neutral 88.14±0.22

2 Positive 82.8±0.95

3 Negative 84.23±0.65

In vitroIn vitro drug release profile drug release profileThe in vitro release of drug from the liposomal formulation was determined using the membrane diffusion technique. Medium: phosphate buffer pH 7.4 (200ml)Temperature: 37±0.5ºC

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HPLC DATA:Mobile phase: Methanol: PBS (pH 3.0)Flow rate: 1ml/minSample injected: 20µlConcentration range: 10-50 µg/mlColumn: C18

Time (hrs) Cumulative % drug release

Neutral Negative Positive1 0.9 2.3 1.82 1.6 4.1 3.93 3.3 6.2 5.34 4.7 8.3 7.46 7.9 12.4 11.58 11.1 16.7 15.8

10 13.9 20.6 19.612 16.8 23.9 22.114 20.4 27.7 25.416 24.2 31.5 31.120 30.6 38.3 36.724 36.4 45.6 43.136 47.3 57.9 54.948 58.2 69.1 64.5

Table no:5 Cumulative % drug release profile of neutral, negative and positive liposomes

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Cumulative % drug release Vs Time(hrs) of neutral, negative and positive liposomes

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Release kinetics

Type of liposome

s

Zero-order (R²)

First-order (R²)

Higuchi (R²)

Korsmeyer –

Peppas (n)

Neutral 0.9814 0.9991 0.9881 1.130

Positive 0.9671 0.9973 0.9879 0.9402

Negative 0.9728 0.9977 0.9773 0.914

The release kinetics of neutral and charged liposomes were studied. All formulations follow first order release kinetics i.e the release from system where release rate is concentration dependent and follow case II transport when it applied to the Korsmeyer – Peppas Model for mechanism of drug release.

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First order release model of Docetaxel liposomal formulations.

Zero order release model of Docetaxel liposomal formulations.

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Korsmeyer – Peppas Model for mechanism of drug release

Higuchi release model of Docetaxel liposomal formulations

Stability studies:Stability studies:The stability of the lyophilized docetaxel liposome was evaluated after storage at 40C and room temperature for 3 months storage. The percentage of drug content of the samples were determined as a function of the storage time. The liposomes stored at 4°c were found to be stable for duration of three months.

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Type of liposome

Effect of stability on percentage of drug content at 4ºC

0 day 15 days 1 month 2 month 3 month

Neutral 88.14±0.72

88.14±0.89

88.01±1.21

87.35±2.3 87.06±2.71

Positive 82.8±0.35 82.8±0.48 81.7±0.74 80.28±0.94 79.4±1.84

Negative 84.23±0.25

84.23±0.29

84.14±0.48

83.97±0.75 83.14±1.34

Table no:6 Effect of temperature on percentage of drug content at 4ºC

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Type of liposome

Effect of stability on percentage of drug content at room temperature

0 day 15 days 1 month 2 month 3 month

Neutral 88.14±1.3 87.66±1.6 86.34±2.35 79.2±3.53 71.94±4.01

Positive 82.8±0.96 81.51±1.12 80.15±2.05 74.43±2.78 68.8±3.79

Negative 84.23±0.75 83.92±0.97 82.69±1.84 76.28±2.89 70.06±3.24

Table no: 7 Effect of temperature on percentage of drug content at room temperature

Conclusion:Conclusion:

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From the executed experimental results, it could be concluded that the stabilizers like stearylamine and dicetylphosphate along with cholesterol were suitable carrier for the preparation of liposomal docetaxel. Though the preliminary data based on in vitro dissolution profile, release kinetics and stability studies proved the suitability of such formulations, still a thorough experiment will be required based on the actual animal and human volunteers. Thereafter we can find the actual mode of action of this kind of dosage form. Therefore, a future work will carried on following areas:

In vitro cytotoxicity studies Long term stability studies In vivo pharmacological work on animals In vivo pharmacokinetic studies on animals In vivo studies in human volunteers

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References: Ramachandran M, Jagesh R Bellare. Manufacturing of Liposomes; A

Review. Current Sci. 1995,68,715-724.

en.wikipedia.org/wiki/docetaxel

Tao Yang, Min-koo Choi, Hongxia Lin, Suk –Jan Chung, Chang-Koo Shim& Dae-Duk Kim. Liposome formulation of Paclitaxel with enhanced solubility and stability. Drug Delivery. 2007,14,301-308.

Vyas S P., Khar R K,. Targeted & Controlled Drug Delivery. CBC

Publishers & Distributors, 2004, New Delhi, 476.

en.wikipedia.org/wiki/lecithin.

Luigi Cattle, Brusa P, Arpicco S, Stella B, Dosio F, Cattel L. Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing docetaxel. J Control Rel. 2003; 91: 417–429.doi:10.1016/S0168-3659(03)002712

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James Swabrick., & James C. Boylan. Enclopedia of pharmaceutical Technology. Marcle Dekker. Vol 9, pages 12, 6, 1.

Jorge, J.C.S., Perez-Soler, R., Morais, J.G., & Cruz, M.E.M (1994). Liposomal plamitoyl-L-asparaginase: Characterization and biological activity. Can.Chemother. Pharmcology. 34, 230-234.

Jun Wu., QuingLiu., 2006. A folate receptor-targeted liposomal formulation for paclitaxel. Int .J. Pharm.316,148-153.

Lasic D. D., Papahadjopoulos D., In: Medical applications of liposomes, Elsevier, New York, 1998;9-12.

Lingang Zhang., Jichun Hu., & Zuhong Hu. (1997). Preparation of liposomes with a controlled assembly procedure. J. Colloid & Inerface Sci. 190, 76-80

THANK‘YOU’

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