Formulation and Evaluation of Liposomal Drug 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: 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”. <ul><li>Liposomes: An ideal “Drug carrier” for anticancer drugs: </li></ul><ul><li>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. </li></ul>
<ul><li>The alternative is to use simple functional molecules which transport the drug to specific site and release it to perform task. </li></ul><ul><li>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. </li></ul><ul><li>Liposomes have been proved as suitable vehicles for selective drug delivery and controlled drug release. </li></ul>
WHY? 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.
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.
Accumulation of liposomes within solid tumours — (right) liposome extravasation from the disorganised tumour vasculature and (left) liposomes in normal tissue
Objective: <ul><li>The main objective of the work is to prepare and evaluate the Docetaxel liposomes . The further objective of the work are as below </li></ul><ul><li>To study the effect of various stabilizers on drug entrapment efficacy. </li></ul><ul><li>To reduce the side effects . </li></ul><ul><li>To target the site of action . </li></ul>
Scope 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.
WHY? <ul><li>Neutral liposomes: </li></ul><ul><li>Lack of surface charge can reduce physical stability of liposomes by increasing their aggregation. </li></ul><ul><li>Do not interact significantly with cells. </li></ul><ul><li>Charged liposomes: </li></ul><ul><li>Influence the extent of liposome interaction with cells and also accelerate their plasma clearance after systemic administration </li></ul>
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. 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) .
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: <ul><li>STAGE 1: </li></ul><ul><li>Preformulation studies </li></ul><ul><li>Standard calibration curve of docetaxel in UV </li></ul><ul><li>Standard calibration curve of docetaxel in HPLC </li></ul><ul><li>Compatibility studies </li></ul><ul><li>STAGE 2: </li></ul><ul><li>Preparation of docetaxel liposomes </li></ul><ul><li>Preparation of drug loaded liposomes by dried thin lipid film hydration </li></ul><ul><li>method. </li></ul><ul><li>Preparation of charged and neutral liposomes. </li></ul><ul><li>STAGE 3: </li></ul><ul><li>Physicochemical characterization of liposomes </li></ul><ul><li>Particle size analysis </li></ul><ul><li>Zeta potential </li></ul><ul><li>SEM studies </li></ul>
<ul><li>STAGE 4: </li></ul><ul><li>In vitro characterization </li></ul><ul><li>Percentage of drug content </li></ul><ul><li>Study on in vitro drug release from neutral and charged liposomes </li></ul><ul><li>Release kinetics </li></ul><ul><li>STAGE 5: </li></ul><ul><li>Short term stability studies </li></ul>
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 : C 43 H 53 NO 14 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.
LIPID PROFILE: Name : Soybean lecithin Synonym : Phosphatidylcholine Description: colour : Yellowish brown consistency : agglomerates Iodine 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.
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 λ max 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.
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/min Sample injected: 20µl Concentration range: 10-50 µg/ml Column: C 18
Compatibility studies: . 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.
RANGE : 4000 – 400 cm -1 Chemicals Mixture of Docetaxel trihydrate, Soy lecithin and Cholesterol Docetaxel Trihydrate Carbonylgroups(C=O) – 1737 & 1710 Amine groups (NH) – 3373.61 Hydroxyl groups (OH) – 3493.2 1737.92 & 1710.92 3373.61 3493.2 Cholesterol Ketone groups(C=O) – 1670.41 Hydroxyl groups (OH) – 3396.76 Aromatic (C-C) – 1465.95 1674.98 3396.76 1465.98 Soy lecithin Carbonyl groups(C=O) – 1739.85 Hydroxyl groups (OH) – 3410.26 Carboxylic 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 Chemicals Mixture of Docetaxel trihydrate, Soy lecithin, Cholesterol and Dicetylphosphate Docetaxel Trihydrate Carbonylgroups(C=O) – 1737 & 1710 Amine groups (NH) – 3373.61 Hydroxyl groups (OH) – 3493.2 1737.92 & 1710.92 3373.61 3493.2 Cholesterol Ketone groups(C=O) – 1670.41 Hydroxyl groups (OH) – 3396.76 Aromatic (C-C) – 1465.95 1659.58 3396.76 1465.98 Soy lecithin Carbonyl groups(C=O) – 1739.85 Hydroxyl groups (OH) – 3410.26 Carboxylic acids – 3232.80 1737.92 3437.06 3224.67 Dicetylphosphate Hydroxyl groups (OH) – 3437 Amine groups (N-H) – 1631 3440.03 1635.29
Formulation and Optimization of liposomes 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).
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 formed Freeze drying Liposomes
Table no:1The composition and ratios of lecithin, cholesterol and stabilizers for different types of liposomes . Ratio of ingredients Types of Liposomes Neutral Positive Negative Lecithin:cholesterol: stearyl amine:Dicetylphosphate 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 :2 The composition and ratios of Drug, lecithin, cholesterol and stabilizers for optimized batches. 120 mg in 10 ml of pH 7.4 Type of liposomes Drug Soy lecithin Cholesterol Stearyl amine Dicetyl phosphate Neutral 2 8 2 - - Positive 2 7 2 1 - Negative 2 7 2 - 1
Negatively charged liposomal suspension form Positively charged liposomal suspension form Neutral liposomal suspension form Powder form Powder form Powder form
Particle size analysis Particle size and size distribution of liposomes in the extruded suspension were determined by laser light scattering (Zetasizer ZS, Malvern, UK). Table no:3 Physicochemical characteristics of the neutral and charged liposomes Sample Particle size (µm) Zeta potential (mV) Poly Dispersive index(Pdi) Neutral 20 16.12 0.351 Positive 14 24.66 0.347 Negative 11 -25.21 0.635
SEM images Neutral liposome Positive liposome Negative liposome
Percentage of drug content 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 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 vitro drug release profile The 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 HPLC DATA: Mobile phase: Methanol: PBS (pH 3.0) Flow rate: 1ml/min Sample injected: 20 µ l Concentration range: 10-50 µ g/ml Column: C 18
Cumulative % drug release Vs Time(hrs) of neutral, negative and positive liposomes
Release kinetics 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. Type of liposomes 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
First order release model of Docetaxel liposomal formulations. Zero order release model of Docetaxel liposomal formulations.
Korsmeyer – Peppas Model for mechanism of drug release Higuchi release model of Docetaxel liposomal formulations
Stability studies: The stability of the lyophilized docetaxel liposome was evaluated after storage at 4 0 C 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 . Table no:6 Effect of temperature on percentage of drug content at 4ºC 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: 7 Effect of temperature on percentage of drug content at room temperature 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
Conclusion: <ul><li>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: </li></ul><ul><li>In vitro cytotoxicity studies </li></ul><ul><li>Long term stability studies </li></ul><ul><li>In vivo pharmacological work on animals </li></ul><ul><li>In vivo pharmacokinetic studies on animals </li></ul><ul><li>In vivo studies in human volunteers </li></ul>.
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