WO2014123406A1

WO2014123406A1 - A composition for treating leukemia

Info

Publication number: WO2014123406A1
Application number: PCT/MY2014/000013
Authority: WO
Inventors: Rasedee@Mat ABDULLAH; Heshu Sulaiman RAHMAN; Ahmad Bustamam Abdul; How CHEE WUN; Yeap SWEE KEONG
Original assignee: Universiti Putra Malaysia (UPM)
Current assignee: Universiti Putra Malaysia (UPM)
Priority date: 2013-02-06
Filing date: 2014-02-05
Publication date: 2014-08-14
Anticipated expiration: 2015-08-06
Also published as: EP2953637A1

Abstract

The present invention relates to a composition for treating leukemia. The composition comprises of effective amount of zerumbone and a pharmaceutically acceptable carrier characterized in that the carrier is a nanostructure lipid carrier. The composition is safe for administration in human body and shows enhanced bioavailability. The present invention also relates to a method for preparing the composition.

Description

A COMPOSITION FOR TREATING LEUKEMIA

Technical Field

[1] The present invention relates to a composition for treating leukemia. Particularly, the composition is extracted from plant and comprises of a carrier.

Background Art

[2] Zerumbone ( ZER) is a crystalline, monocyclic, sesquiterpene, phytochemical substance that was first isolated as a major compound in 1956 from the essential volatile oil of rhizomes of edible wild ginger known as Zingiber zerumbet (L.) smith, which is widely spread in South East Asia (Kitayama et al., 2003). Whereas its structure was determined in 1960, and later was characterized by NMR and X-ray (Aggarwal et al., 2008). ZER is a natural dietary compound having lipophilic characteristic.

[3] Recently, several pharmacological potentials of ZER have been identified through several in vivo and in vitro test models. As a result of these studies, it was found that ZER possesses antitumor, anti-inflammatory, antioxidant, antimicrobial,

antinociceptive, hepatoprotective, and immunomodulatory activities at different doses/ concentrations. However, its underlying molecular mechanisms are poorly understood. Regarding antitumor activities, it has been found that ZER possesses antiproliferative properties to several cancer cell lines such as the cervical (HeLa), breast (MCF-7), colon (COLO205, LS 174T, LS 180 and COLO320DM), liver (HepG2), ovary (Caov-3 and AS52), pancreatic (PANC-28, MIA PaCa-2, and AsPC-1), and white blood cells (P-388D1, HL-60, NB4 and CEMss). As well as in vivo, it suppresses the proliferation of neoplastic colon, breast, cervix, skin, lung, liver and blood cells, while minimally affecting normal cells ( Nakamura et al., 2004; Sadhu et al., 2007; Abdul et al., 2008; Abdel Wahab, et al., 2009; Elhassan et al., 2010; Eltayeb et al., 2011). However, other main actions of ZER include significant suppression of tumor promoter

12-0-tetradecanoylphorbol-13-acetate (TPA) induced Epstein-Bar virus (EBV) activation in Raji cells (Murakami et al., 1999). Moreover, ZER was able to suppress free radicals (superoxide) anion generation from both NADPH oxidase and xanthine oxidase (XO) in cancer cell lines (Murakami et al., 2002; Keong et al., 2010). Furthermore, ZER showed strong inhibitory effects on platelet aggregation in human whole blood induced by arachidonic acid (AA), collagen and ADP (Jantan et al., 2008). Additionally, ZER has been reported as a modulator for osteoclastogenesis induced by RANKL and breast cancer (Sung et al., 2009), while, itcould not prevent cisplatin-induced clastogenesis in male Sprague Dawley rat (Al-Zubairi et al., 2011).

[4] There are many publications reported on zerumbone and Zingiber species. In a granted US patent, US 7588788 disclosed a method of preparing a nutraceutical formulation comprising the step of solvent extraction from the root of Zingiber zerumbet Sm, and the use of this formulation to regulate the immune system, and more specifically to prevent or to treat an allergic disorder.

[5] In another granted US patent, US 6274177 disclosed a method of preparing an extract from Zingiber officinale, which is potent in anti-inflammation and anti-platelet aggregation, includes the following steps: a) preparing a crude liquid from rhizomes of ginger by extraction with an organic solvent or by distillation with steam; b) introducing the crude liquid to a reverse phase chromatography column, and eluting the column with water, a first eluent and a second eluent having a polarity weaker than that of the first eluent but stronger than that of chloroform, so that a first eluate resulting from elution of the first eluent and a second eluate resulting from elution of the second eluent are obtained; c) removing the first eluent from the first eluate by evaporation, so that a first concentrated eluate is obtained and is able to used as the potent extract; and d) removing the second eluent from the second eluate by evaporation, so that a second concentrated eluate is obtained and is able to used as the potent extract.

[6] Formulation of poorly soluble drugs is a general intractable problem in pharmaceutical field, especially those compounds poorly soluble in both aqueous and organic media. Approximately about 40% or more of the drugs in the pipeline and up to 70% of molecules coming from synthesis have solubility problems and consequently poor oral bioavailability, as well as delivery problems (Lipinski, 2000; Cornelia et al., 2008; Lei et al., 2008; Seenivasan et al., 2011 ). Poor solubility of the drug results in low concentration gradient between the gut and blood vessel leads to a limited transport during oral administration. Similarly, parenteral administration as microsuspensions frequently cannot lead to sufficient drug levels due to the limited solute volume at the injection site (Gao et al., 2008). Therefore, the urgent need for poorly soluble drugs is to efficiently and safely increase their saturation solubility in the body fluid (Souto et al., 2004; Abbasalipourkabir et al., 2011b). Thus, some formulation approaches based on solid lipids were invented to tackle the problems, such as incorporating drugs into nanocarriers and generating drug nanoparticles (Jens and Rainer, 2008; Patidar et al., 2010). The successful implementation of nanoparticles for drug delivery relies on their ability to penetrate through several anatomical barriers, sustained release of their contents and their stability in the nanometer size (Souto et al., 2004; Abbasalipourkabir et al., 2010; Lacatusu et al., 2011). For this purpose, different types of nanocarriers such as solid lipid nanoparticles (SLN), nanostructuredlipid carriers (NLC), as well as lipid drug conjugates were developed (Koping-Hoggard et al., 2005; Shaji and Jain, 2010; Abbasalipourkabir et al., 2011b; Mistry et al., 2011).

[7] Lipid nanoparticles are a carrier system with a number of desirable features such as low toxicity, easy biodegradation of particulate matrix, non toxic degradation product, high ability to incorporate lipophilic and hydrophilic drugs, controlled release of incorporated drug, easy scale up and lastly low cost (Pardeike et al., 2011). In this respect, NLC, the second new generation solid lipid nanoparticle (SLN) is a good and alternative type of delivery system that holds great promise for reaching the goal of controlled and site specific drug delivery were introduced at the end of the 1990s to overcome some of the potential difficulties and drawbacks of SLN (Miiller et al., 2000a; Radtke et al., 2005; Xiang et al., 2008; Kathy et al., 2009; Bhatt and Pethe, 2010; Girish et al., 2011).

[8] There is still a need in the art to provide a composition for treating leukemia

having enhanced bioavailability and biodistribution as well as safe for administration in human body.

Disclosure of Invention

Technical Problem

[9]

Technical Solution

[10]

Advantageous Effects

[11] According to a first aspect of the present invention, there is provided a composition for treating leukemia comprises of an effective amount of zerumbone and a pharmaceutically acceptable carrier characterized in that the carrier is a nanostructure lipid carrier. Provision of the nanostructure lipid carrier is advantageous as it enhances water solubility and bioavailability of the zerumbone towards target cell.

[12] Preferably, the zerumbone is extracted from Zingiber zerumbet.

[13] Preferably, the the nanostructure lipid carrier comprising solid lipid and liquid lipid.

[14] Preferably, the composition is suitable for parenteral administration or oral administration.

[15] According to a second aspect of the present invention, there is provided a composition for treating leukemia of Claim 1 comprises of:

[16] a) providing Zingiber zerumbet rhizome;

[17] b) extracting essential oil from the rhizome;

[18] c) crystalling the oil to form zerumbone crystal;

[19] d) adding the zerumbone crystal to a molten lipid forming a mixture;

[20] e) dispersing the mixture in aqueous surfactant to obtain pre-emulsion;

[21] f) homogenizing the pre-emulsion to obtain a nanoemulsion; and

[22] g) cooling the nanoemulsion to obtain the composition.

Description of Drawings

[23] Figure 1: illustrates DSC scan thermograms of a) bulk lipid, b) ZER and c) ZER-

NLC.

[24] Figure 2: illustrates X-ray diffraction patterns of a) ZER, b) HPO and c) ZER-

NLC.

[25] Figure 3: illustrates in vitro release profile in PBS, pH 7.4/SDS (2%) of a) ZER from NLC and b) ZER.

[26] Figure 4: illustrates cytotoxicity assessed by MTT assay after 72 hrs of a) ZER-

NLC treated Jurkat cell, b) ZER treated Jurkat cell and c) ZER free NLC treated Jurkat cell.

Detailed Description of The Preferred Embodiment

[27] The present invention relates to a composition for treating leukemia. The composition comprises of an effective amount of zerumbone and a pharmaceutically acceptable carrier.

[28] The zerumbone is extracted from Zingiber zerumbet. Preferably, the zerumbone is extracted from essential oil of fresh Zingiber zerumbet rhizome.

[29] The pharmaceutically acceptable carrier used in the composition is a nanostructure lipid carrier. The nanostructure lipid carrier comprises of solid lipid and liquid lipid. Utilization of the nanostructure lipid carrier enhances the solubility of the zerumbone in aqueous environment thus enhances bioavailability and biodistribution of the composition towards cell of human body. Moreover, utilization of the liquid lipid enhances loading capacity of the zerumbone into the nanostructure lipid carrier.

[30] Advantageously, incorporation of the nanostructure lipid carrier shows high entrapment efficiency of the zerumbone without losing efficacy and potency. Moreover, the nanostructure lipid carrier provides controlled release of the zerumbone in the cell.

[31] The composition further comprises of an antibody. The composition could be

added with specific antibody to react with target tissue and organ. Therefore, this enhances specificity of the composition in treating leukemia.

[32] Preferably, the composition is suitable for parenteral administration and oral administration.

[33] Upon administration in the body, the composition inhibits proliferation of leukemic cell and induces death of leukemic cell by apoptosis. The composition shows cytotoxicity activity by inhibit proliferation of the leukemic cell without exerting cytotoxicity activity on normal cell. This shows that the composition has specific activity towards leukemic cell and therefore is safe for administration in human body.

[34] The present invention also relates to a method for preparing the composition for treating leukemia. The method comprises the step of first, providing Zingiber zerumbet rhizome. Second, extracting essential oil from the rhizome. Third, crystalling the oil to form zerumbone crystal. Fourth, adding the zerumbone crystal to a molten lipid forming a mixture. Fifth, dispersing the mixture in aqueous surfactant to obtain pre- emulsion. Sixth, homogenizing the pre-emulsion to obtain a nanoemulsion and seventh, cooling the nanoemulsion to obtain the composition.

[35] The molten lipid in the fourth step comprises of solid lipid and liquid lipid.

Preferably, the solid lipid is selected from the group of hydrogenated palm oil and phosphatidylcholine while olive oil is used as liquid lipid. Utilization of the hydrogenated palm oil results in enhanced stability of the composition and also increases entrapment efficiency of the zerumbone in the nanostructure lipid carrier. Those lipids are selected to be used as the carrier as it is not toxic and does not show side effect, therefore is safe for health. Moreover, those lipids are relatively inexpensive and easily available in the market. Therefore, the composition could be produced with lower cost.

[36] Preferably, the surfactant used in the fifth step is selected from the group of

sorbitol, polysorbate-80, tween-80 and thimerosal. The surfactant functions as stabilizer that maintains size of the formed nanostructure lipid carrier in nanometer.

[37] The method further comprises of adding an antibody. Addition of specific antibody to the composition would enhance specificity of the composition in treating leukemia where the antibody would react with target tissue and organ.

[38] Advantageously, the nanostructure lipid is used for preparation of medicament for treatment of leukemia.

[39] Alternatively, the zerumbone could be replaced by other compound but not limited to Galanas A, Galanas B, Shagol and Zingerol.

[40] Advantageously, the composition could be mass produced in a short period of time.

[41] The present invention will be explained in more detail through the examples

below. The examples is presented only to illustrate the preferred embodiments of the present invention and not intended in any way to limit the scope of the present invention.

[42]

[43] Example

[44]

[45] ZER Extraction

[46] Pure ZER crystals were prepared from extracted essential oil of fresh Z. Zerumbet rhizomes by steam hydrodistiUation procedure according to a method described earlier (Abdel Wahab et al., 2010) in a yield of 1.3 g/kg. In brief, fresh rhizomes were initially cleaned, washed, sliced and later placed in a steam distillator containing tap water and heated immediately. The outlet of the device connected to special glass ware

(Dienstag) in order to collect the distillate contains the volatile oil. The collected volatile oil was crystallized spontaneously using absolute n-hexane (100%) (Sigma Aldrich, USA) and the solution was left standing in fume hood (Novaire, USA) to evaporate. Recrystalization was performed also using absolute hexane (100%) 3 times to obtain pure ZER crystals. The purity of ZER (99.96%) was determined using high performance liquid chromatography (HPLC) system (Waters, USA) according to an established method mentioned elsewhere (El-tayib et al., 2010). Pure crystals of ZER were collected in clean glass vials and kept at 4#C for further analysis.

[47] Lipid Dispersion Preparation

[48] The lipid phase that was composed of hydrogenated palm oil, phosphatidylcholine

(Lipoid S 100) and olive oil at the ratio of 7:3:3 were melted by heating to approximately 10°C above the melting point of the lipid matrices to avoid lipid memory effect, using Model 830 circulating water bath (Protech, Malaysia). Then, 400 mg of ZER added into the molten lipid, which has a yellowish-milky colored solution.

[49] Aqueous Dispersion Preparation

[50] This step was including the dissolve of sorbitol, polysorbate-80 and thimerosal of about 4.75g, l.Og and 0.005%, respectively in bidistilled water after completion the volume to 100 ml. Subsequently, this aqueous surfactant was heated to the same temperature as that of the lipid matrices. Teflon-coated magnet was used for stirring in this procedure.

[51] ZER-NLC Emulsion Formation

[52] Previously prepared 0.4% (w/v) ZER in lipid melt was dispersed in an aqueous surfactant solution to obtain hot pre-emulsions, using high-speed stirring in Ultra- Turrax® (IKA/Staufen, Germany) at 13000 rpm for 10 min. The obtained hot pre- emulsions were then homogenized in a high-pressure homogenizer EmulsiFlex® (Avestin, Inc. /Ottawa, Canada) at 1000 bar for 20 cycles at 60#C. So far, the hot oil- in-water nanoemulsion obtained was immediately filled and sealed into siliconized glass vials and allowed to permit subsequent recrystalization of the lipid phase after cooling at room temperature (25#C) to finally form the active-loaded ZER-NLC.

[53] Particle size (PS) and Polydispersity Index (PDI) Measurement

[54] Photon correlation spectroscopy (PCS), which also known as dynamic light

scattering is the most powerful technique for routine measurements of PS and particle width distribution has been used for this purpose. Thus, 3 days following production, the aqueous ZER-NLC was dispersed in a fixed amount of filtered double-distilled water to avoid multi-scattering phenomena and then placed into a golden cuvette. After that, the analysis was done; the mean PS (diameter, nm +SD) and PDI (size distribution) of ZER-NLC were calculated with the Malvern software (Zetasizer Nano ZS, Malvern, UK). The measurements were carried out in triplicates (n=3) and standard deviations (SD) calculated at a fixed scattering angle of 90° at a room temperature (25°C) (Mukherjee et al., 2010; How et al., 2011).

[55] The average PS and PDI of nanocarriers are important features of NLC from which the stability of the drug-loaded NLC can be predicted. Therefore, optimal production parameters of ZER-NLC dispersion are important to be investigated to obtain small particle size and narrow size distributions. To this regard, the average PS and PDI which indicates size distribution within the produced NLC population was determined more precisely in a quantitative manner via PCS at 52.68+0.105nm and

0.290+0.004 Ιμιη respectively. However, an increase to the amount of ZER into the NLC system did not have any influence on the PS and PDI of the formula (Wa Kasongo et al., 2011). In reference to this, the morphology of the particles was studied within 7days of NLC production using TEM analysis. It was indicated that ZER-NLCs had nanometer- size, irregular, round to spherical and relatively uniform shapes, with a narrow size distribution of particles but invisible free drug crystals, which is known as zero-dimensional materials (Mathur et al., 2010). On the contrary, it was found that the sizes of the ZER-NLCs measured by PCS were smaller than that measured using TEM. This discrepancy is possibly due to the differences in measurement principals, measuring conditions and methods used between these two techniques. PCS technique measures the Brownian displacement, which is related to the size of the particles, whilst in TEM, particles are exposed to high vacuum electron beam column (Burgess, 2006).

[56] Zeta potential (ZP) Measurement

[57] ZP is an indirect measurement of the thickness of the diffusion layer; hence, it can be used to predict long term stability (Venkatesh et al., 2011). Thus, the particle elec- trophoretic movement was measured by Laser Doppler Electrophoresis technique and using Zetasizer Nano ZS (Malvern, UK) for analysis. Precisely, the measurements were conducted in triplicates after appropriate dilution of freshly prepared particles in bidistilled water to get optimum kilo counts per second (Kcps) of 50-200 for measurements (Thatipamula et al., 2011).

[58] Particles in the nanosuspensions exhibited negative charges on the surface that are expressed as ZP, and is considered as the key factor to predict and evaluate the stability of colloidal dispersion. It was currently concluded that ZP of nanosuspensions greater than +30mV are required for full electrostatic stabilization of the nanoparticles (Thatipamula et al., 2011). The result showed particles of ZER-NLC with ZP of # 15.03+1.242 mV, hence allowing the particles to be stable. Generally, ZP measurements are based on the electrophoretic mobility of the drug in aqueous medium. Interestingly, suspensions with higher ZP appeared to allow less particle flocculation and aggregation possibly due to electrostatic repulsion (Mulla et al., 2010; Seenivasan et al., 2011).

[59] Transmission Electron Microscopy (TEM)

[60] TEM with smaller size limit detection and good validation for other methods

provides a way to directly observe the shape and surface morphology of nanoparticles has been applied. Briefly, a drop of diluted ZER-NLC dispersions was placed onto the surface of a carbon-coated copper grid and following the removal of excess liquid using a hydrophilic filter membrane. Upon drying at 25#C in less than 1 min, the grid with mesh size of 300 was then negatively stained with 2% phosphotungstic acid (PTA) (w/v) for lmin and was allowed to dry at room temperature. The ZER-NLC sample was placed onto sample holders, probed with TEM (Hitachi H-7100, Japan) and then the image was captured (AL-Haj et al., 2008b; Xin et al., 2010).

[61] Lyophilization of ZER-NLC

[62] Lyophilization is the best way to increase physical as well as chemical stability of nanoparticles over a long storage time and for improvement of nanoparticles incorporation into pellets, tablets and capsules (Abbasalipourkabir et al., 2011a). The Lyophilized ZER-NLC was prepared for both DSC and WXRD screening tests by freeze-drying method using freeze dryer (Christ, Germany). Briefly, 8ml of NLC dispersion containing ZER was poured into a plastic petridish and frozen at -80#C (Thermoforma, USA). After that, the plate was wrapped with paraffilm, several holes were made at the top cover part, and subsequently freeze-dried for 24hrs at -55#C (Waard et al., 2009; Eltayeb et al., 2011).

[63] Differential Scanning Colorimetry (DSC)

[64] DSC can be used to determine the speciation of crystallinity and polymorphism of bulk material, drug and drug-nanoparticles through the measurement of glass and melting point temperatures with their associated enthalpies (Mukherjee et al., 2010). For this purpose, thermal characteristics of HPO, ZER and lyophilized ZER-NLC were studied by Mettler DSC 822e (Mettler Toledo, Greifensee, Switzerland). Approximately about lOmg of bulk lipid, ZER and lyophilized ZER-NLC were place in pin-holed bottomed, sealed aluminum pans with lids and heated, while an empty aluminum pan was used for reference purposes. Then, the DSC curves were recorded at temperature ranging between 20#- 80#C with a constant linear heating rate of 5°C/min with ultra high pure dry nitrogen. The analysis was repeated three times and expressed as the mean of three determinations. Lastly, the enthalpies were calculated using the Mettler Star software (AL-Haj et al., 2008a; How et al., 2011, Wa Kasongo et al., 2011).

[65] Generally, 3 different polymorphic structures are exhibited by the lipid molecules; the unstable #-, the metastable #'-, and the most stable #-modification. The latter or at least, predominantly #-modification are exhibited by most bulk lipids (Souto and Miiller, 2007). The thermal curves of HPO and ZER exhibited endo thermic peaks at 59.42°C and 66.4#C as seen in Figure la and Figure lb respectively, indicating stable arrangement of bulk lipid (#-modification) in the formulations (Muhlen et al., 1998). On the other hand, the melting endothermic peak of the ZER-NLC appeared at very lower temperature (57.09#C) as seen in Figure lc, which was in fact lower than the bulk lipid attributed to the #'-modification (AL-Haj et al., 2008a). This decrease in melting temperature of nanoparticles with the bulk lipid has been attributed to their small size and presence of surfactants. However, depressed melting point exhibition of bulk lipid upon transformation into the nanoparticulate form has been demonstrated earlier (Hunter, 1986). Hence, HPO used in the formulation of ZER-NLC resulted in a 9.31°C depression in melting point of nanosuspensions, which was due to its less- ordered arrangements, the need for lesser amount of energy to overcome the lattice force within the materials and possibly the small size of nanoparticles (Jenning et al., 2000). Generally, the melting points of colloidal system were distinctly decreased by about 10-15#C, which can be assigned to the colloidal dimensions of the particles in particular to their large surface to volume ratio and not to the recrystalization of the lipid matrices in a metastable polymorph possessing a lower melting point (Wan et al., 2008). [66] Wide- Angle X-ray Diffraction (WXRD)

[67] The geometric scattering of radiation from crystal planes within nanoparticle

dispersion for assessing the degree of crystallinity can be determined by WXRD. Thus, an X-ray diffractometer (Philips, Germany), equipped with a copper anode (Cu K#) (#= 1.5406A) radiation was used for lyophilized ZER-NLC crystallinity detection. Powder samples of HPO, ZER and lyophilized ZER-NLC about 10mm in length were placed onto the top of X-ray plates, exposed to 45kV voltage, 40mA current at room temperature with a scanning speed of 5°/min and scanning range of 2# respectively. Consequently, the X-ray diffractogram patterns were recorded over the range of 20-80° (Eltayeb et al., 2011; How et al., 2011).

[68] The obtained results showed that ZER produced diffraction pattern with a distinctive sharp peak, indicating that it had a crystalline structure as seen in Figure 2a. Similarly, a relatively sharp peak was observed clearly in the HPO, but with less intensity as seen in Figure 2b. However, diffraction peaks for ZER-NLC were found to be broader and less intense as seen in Figure 2c than that of HPO with diffused X- ray scattering pattern, which reveals its amorphousness. The X-ray diffraction patterns of ZER-NLC were characterized by large diffraction peaks, hence showing no presence of characteristic peaks resembling that of neither pure crystalline ZER nor HPO. This obtained result indicates that ZER no longer present as a crystalline material, and its NLC complexes exist in the amorphous state. Thus, the formulation in this current study had produced ZER-NLC of low crystallinity and molecular order, in agreement to previously reported incorporation of solid and liquid lipid combination of NLC that favours less crystallization structure.

[69] ZER-NLC Stability Detection

[70] To monitor the short term stability as a function of storage conditions, a volume of

5ml of ZER loaded NLCs dispersion was filled into amber colored glass vials after 3days of preparation. Subsequently, these vials were stored at different temperatures including refrigerator, room temperature and controlled incubator of about 4#C, 25#C, and 40#C respectively. After 30days, the average PS, PDI, ZP, and EE of the nanoparticles against storage time were measured. Samples were estimated in triplicates (Hu et al., 2006; Xiang et al., 2008; Qiang and Hongxia, 2010; Thatipamula et al., 2011).

[71] It was found that the particle size of ZER-NLC increased by 40nm, PDI was

0.536+0.025, ZP was -17.43+0.416mV, while EE was lowered by 4.5% after 1 month storage at 40#C. However, very slight changes of mean particle size, PDI, ZP and EE had been found for those samples stored between 5-8#C and left on the bench at room temperature prior to analysis. This obtained data imply that ZER-NLC stored refrigerated and at room temperature were physically stable with no flocculation or coalescence of the droplets at these conditions.

[72] HPLC Analysis [73] ZER (lmg) was accurately weighed using sensitive balance Model ALPS-AL204

(Mettler Toledo, UK) and diluted to 1ml volume with methanol in a 10ml conical flask. After that, a series of standard working solutions of ZER were prepared giving the final concentrations ranging as follows: 2.5, 5, 10, 20 and ^g/ml.

[74] HPLC determination was performed according to an established method mentioned previously (El-Tayeb et al., 2010) using a validated Reverse phase HPLC (RP-HPLC) system, which consisted of a mobile phase delivery pump, auto sampler, UV detector and a workstation (Waters Alliance, USA). The stainless steel 4μιη particle size (4.6 mm I.D. x 100 mm length) analytical symmetry column (Merck, Germany) packed with a dimethyl octylsilyl (C18)-bonded amorphous silica stationary phase was used at room temperature. The mobile phase composed of a binary mixture of HPLC graded absolute methanol (MeOH) and potassium dihydrogen phosphate (KH2P04) buffer (pH 6.0) (0.001%) at a ratio of 70:30 (v/v) was freshly prepared for each run and degassed before use. The column temperature was set to 50#C (ambient temperature). The injection volume was ΙΟμΙ with a flow rate of lml/min. The samples were estimated with UV detection at a wavelength of 250nm.

[75] The HPLC was calibrated with standard solutions of 2.5-40μg/ml of ZER. Briefly,

ZER samples were chromatographed at room temperature and the retention times of ZER were eluted at 2.145 min. following which, a calibration curve was plotted. A good linear relationship was observed between the concentration of ZER and the peak area of ZER with an acceptable correlation co-efficient (r² = 0.9984) for quantitative analysis. The required studies were carried out to estimate the precision and accuracy of the HPLC method (Madishetti et al., 2010; Thatipamula et al., 2011).

[76] Determination of Entrapment Efficiency (EE) and Drug Loading Capacity

(DL)

[77] The amount of drug incorporation in NLCs influences the release characteristics of the drug; hence it is very important to measure the amount of encapsulated drug per unit weight of nanoparticles (Mistry et al., 2011). The EE of ZER-NLC was estimated after separation of the free ZER and solid lipids from the aqueous medium by ultrafiltration. For this purpose, Centrisart filter tubes (Sartorius AG, Goettingen, Germany) were used, which consisted of a filter membrane with a molecular weight cut-off of 300 kDa at the base of the sample recovery chamber. Three milliliter aliquot of undiluted ZER-NLC sample was placed in the outer chamber and the sample recovery chamber was fitted on top of the sample. The unit was closed and centrifuged at 20,000 x g for 15 minutes using a Model 32 R universal centrifuge (Hettich,

Germany). The principle behind this process was that ZER-NLC were separated from the aqueous phase and remained in the outer chamber, and the aqueous phase filtered into the sample recovery chamber through the membrane. Hence, the amount of ZER in the aqueous phase was evaluated by a validated RP-HPLC system. The amount of encapsulated active was calculated according to Wa Kasongo et al (2011), by taking the difference between the total amounts used to prepare the dispersion and the amount of ZER that remained in the aqueous phase following the ultrafiltration process.

[78] EE% = ((Total amount of ZER) - (Free amount of ZER) / Total amount of ZER) x

100

[79] Whilst, the following equation was used for determining the DL% capacity of

ZER-NLC (Xin et al., 2010).

[80] DL% = (Total amount of ZER encapsulated into NLC / Total amount of lipid used in ZER-NLC formulation) x 100

[81] The result showed that approximately 3.88mg free ZER in aqueous ZER-NLC dispersion was obtained and hence, anticipated that 396.12mg of 400mg ZER were successfully encapsulated into NLC. Thus, the amount of ZER being entraped into nanoparticles was satisfactorily high (99.03%w/w).

[82] High EE of the nanosuspension is due to the high lipophilicity of ZER and its low water solubility. The EE is an important parameter that can affect drug releasing characteristics and therefore must form the integral part of the formulation development process (Joshi and Patravale, 2007). It was found that the loading capacity of ZER- NLC was 7.922%, probably due to the high solubility of ZER in HPO and olive oil. Thus, the presence of HPO and olive oil have enhanced the loading capacity of ZER in this nanosuspension of ZER-NLC.

[83] In vitro Drug Release Study

[84] This investigation is useful for the quality control as well as for the prediction of in vivo kinetics. Drug release from nanocarriers is however influenced by the structure and composition of the nanocarriers (Harivardhan et al., 2006; Joshi et al., 2008;

Mistry et al., 2011). Hence, ZER release from NLC was conducted for a period of 48 hr using in vitro kinetic method. This method is based upon the dilution and separation employs direct dispersion of lipid nanoparticles in the release medium. Thus, the release of ZER from NLC was performed using a modified Franz diffusion cell (FDC) system (Permgear, USA) having a surface area of 0.785 cm² and 5 ml capacity. A cellulose acetate membrane (Dialysis membrane having pore size of 200 nm, with molecular weight cutoff between 5000-10000 Dalton) (Hi- media, India) was adapted between the donor and receptor compartment by a rubber ring (Saboktakin et al., 2010 and 2011). Briefly, membrane was soaked in PBS 12 hrs before mounting in Franz diffusion cell. Volumes of 0.5 ml ZER-NLC (vehicle) or 0.5 ml of ZER in DMSO solution (control) were loaded to the donor compartment. The concentration of ZER in both cases was 4 mg/ml. Whilst, the receptor compartment was filled with 5 ml dialysis medium (PBS, 0.1M, pH 7.4 and sodium dodecyl sulphate (SDS) 2%) to maintain sink conditions during experiment (Gambhire et al., 2011). Continuously, the content of the cell was agitated with the help of magnetic stirrer at 500 rpm at constant temperature (37.0 + 0.5 °C). An aliquot of 500 μΐ sample was taken out from receptor compartment medium through side arm tube with stainless steel 316 syringe needle (Sigma Aldrich, USA) at time intervals of 0.5, 1, 2, 4, 6, 8, 10, 12, 24 and 48 hrs (Zhaowu et al., 2011). Immediately, the volume of each sample withdrawn was restored with same volume of fresh PBS each time to maintain constant volume throughout the study and the corresponding ZER concentration was corrected (Sanad et al., 2010; Fang et al., 2011). Samples were analyzed to evaluate the amount of ZER released from NLC using HPLC system. The experiment was carried out in triplicate (Mulla et al., 2010; Thatipamula et al., 2011). The obtained release data were evaluated by zero order, first order and Higuchi equations to assay the release kinetics (Derakhshandeh et al., 2010).

[85] The receptor compartment model contained SDS (2%) in the dialysis medium as the solvent due to ZER hydrophobicity and insufficient solubility in aqueous medium (Gambhire et al., 2011). Similarly, DMSO was used as the solvent in the donor compartment model of the control group. Our current result demonstrated that in vitro drug release kinetics of ZER from NLC across a cellulose acetate membrane followed closely the zero-order kinetic model (r²= 0.92). The results of cumulative percentage profile of ZER released from ZER-NLC dispersion and dispersion of pure ZER over 48hr are shown in Figure 3a and 3b respectively. The result further showed that ZER is released gradually from loaded NLC during a given period, whilst control group showed more rapid release, compared to the nanoparticle. The amount of ZER released at the end of 48hr period in the NLC formulation was found to be 46.7% (0.95mg), indicating that the nanosuspension could be carriers for controlling the release of ZER, Whilst, ZER released from pure drug dispersion at the end of 48hr was 90.59%

(1.81mg). Generally, the formulation with higher drug content shows higher release rate constants (Jithan and Swathi, 2010; Zhaowu et al., 2011). In contrast, the formulation with higher EE% shows slowest drug release (Sanad et al., 2010). This slow release of the drug from NLC suggests homogenous entrapment of the drug throughout the system, while a fast release might be due to large surface area of high diffusion coefficient (small molecular size).

[86] In Vitro Cytotoxic Study

[87] Human acute leukemia Jurkat T-cell line ( J.RT3-T3.5 ), commonly known as

Jurkat cell line was purchased from American Type Culture Collection (ATCC, USA). The cells were maintained in RPMI-1640 (ATCC, USA) medium; supplemented with L-glutamine (2Mm), 10% heat inactivated fetal calf serum (FCS) (ATCC, USA), lOOunits/ml penicillin, and 100μg/ml streptomycin (Sigma Aldrich, USA). Cells were cultured and grown in 75cm² culture flasks (TPP, Switzerland) at 37#C using incubator (Binder, Germany) with a humidified atmosphere of 95% air and 5% C0₂.

[88] The antiproliferative effect of ZER, ZER free nanoparticles (blank nanoparticle) and ZER-NLC on Jurkat T-cells were quantified by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay reflecting cell viability as previously described (Jun et al., 2003; Miyoshi et al., 2003; Hae et al., 2006). The stock solution and working solution of ZER was prepared by the dimethyl sulphoxide (DMSO) (Sigma Aldrich, USA) solution and RPMI-1640 complete growing medium respectively, whilst the working solution of blank nanoparticles and ZER-NLC were prepared by RPMI-1640 complete growing medium. Jurkat T-cells were allowed to grow in 25 cm³ cell culture flask until confluent and the density was determined using a hemocytometer. Then, 100 μΐ of cell suspension was seeded into each well of 96-well microculture plates (TPP, Germany) at a concentration of 2xl0⁵ cells/ml. Simultaneously, cells were treated separately with 100 μΐ ZER, ZER free nanoparticles and ZER-NLC at various concentrations in 96-well microculture plates (TPP, Germany). After incubation for 72 hrs in C0₂ incubator at 37#C, 20 μΐ of MTT (Micro culture Tetrazolium) (Sigma Aldrich, USA) solution (5mg/ml) in phosphate buffer solution (PBS, pH=7.4) (Sigma Aldrich, USA) was added into each well, covered with aluminum foil, and incubated for an additional 4 hrs in the dark to allow the active live cells to convert water-soluble yellow MTT solution into water insoluble purple formazan compound. After that time, 170 μΐ of the media containing MTT solution were aspirated. The remaining purple formazan were then lysed by adding 100 μΐ MTT solubilization solution (Sigma Aldrich, USA) after shaking for 5 min, using plate shaker (Eppendorf, USA). Consequently, the absorbance was measured using an ELISA plate reader (Universal Microplate reader) (Biotech, Inc, USA) at 570 nm after background correction at 630 nm. The IC₅₀ value (concentration at which 50% of the cells are viable and the other 50% are killed) was determined from the dose-response curve (%cell viability versus concentration of ZER, ZER free nanoparticle and ZER- NLC). Finally, the obtained values of ZER-NLC were compared to those of the ZER (control positive) and ZER free nanoparticle (Abbasalipourkabir et al., 201 lc; Eltayeb et al., 2011).

[89] The data obtained were statistically analyzed. The results were expressed in mean

+ SD. One-way analysis of variance (ANOVA) was performed using SPSS 17.0 to calculate r² for zero kinetic order.

[90] ZER anticancer activity is not affected or impaired by NLC incorporation. The IC₅₀ value of ZER alone was 5.397+0.43 ^g/ml as seen in Figure 4a, while IC₅₀ value for ZER-NLC was 5.64+0.385 μg/ml as seen in Figure 4b. It is obvious that cell viability of ZER-NLC was near similar to ZER alone at 72 hr post treatment. The reason is that ZER alone is immediately available to target cells by passive diffusion while ZER- NLC need to internalize into cells via endocytosis or phagocytosis though there are evidence of ZER in the core which are released from the nanoparticles into extracellular space (Chen et al., 2008; Xin et al., 2010). But considering the nanoparticles formulation, the release of ZER from the nanoparticles can be a fraction of a whole loaded ZER and hence, acts on the targeted cell at specific intervals. This would then allow ZER-NLC to be more effective than ZER alone in terms of anticancer activity (Dong and Feng, 2007). Cytotoxicity of non-loaded nanoparticles was concurrently conducted under the similar conditions using MTT assay. The almost negligible cytotoxic of blank NLC (IC5o= 230.769+5.55μg/m) as seen in Figure 4c is mainly due to the presence of lecithin and components of the aqueous phase used, especially the nonionic emulsifier (Abbasalipourkabir et al., 2011b).

The invention being thus described, it will be apparent that the same may be varied in many ways. Such variations are to be regarded as within the scope of the invention, and all such modifications as would be apparent to one skilled in the art are intended to be within the scope of the following claim.

Mode for Invention

[92]

Industrial Applicability

[93]

Sequence List Text

[94]

Claims

[I] 1. A composition for treating leukemia comprising an effective amount of

zerumbone and a pharmaceutically acceptable carrier characterized in that the carrier is a nanostructure lipid carrier.

[2] 2. A composition as claimed in Claim 1, wherein the zerumbone is extracted from Zingiber zerumbet.

[3] 3. A composition as claimed in Claim 1, wherein the nanostructure lipid carrier comprising solid lipid and liquid lipid.

[4] 4. A composition as claimed in Claim 1, wherein the composition further

comprising an antibody.

[5] 5. A composition as claimed in Claim 1, wherein the composition is suitable for parenteral administration or oral administration.

[6] 6. A composition as claimed in Claim 1, wherein the composition induces

apoptosis on a leukemic cell.

[7] 7. A method for preparing a composition for treating leukemia of Claim 1

comprising:

a) providing Zingiber zerumbet rhizome;

b) extracting essential oil from the rhizome;

c) crystalling the oil to form zerumbone crystal;

d) adding the zerumbone crystal to a molten lipid forming a mixture;

e) dispersing the mixture in aqueous surfactant to obtain pre-emulsion;

f) homogenizing the pre-emulsion to obtain a nanoemulsion; and g) cooling the nanoemulsion to obtain the composition.

[8] 8. A method as claimed in Claim 7, wherein the molten lipid in step (d)

comprising solid lipid and liquid lipid.

[9] 9. A method as claimed in Claim 8, wherein the solid lipid is selected from the group of palm oil and phosphatidylcholine.

[10] 10. A method as claimed in Claim 8, wherein the liquid lipid is olive oil.

[I I] 11. A method as claimed in Claim 7, wherein the surfactant in step (e) is selected from the group of sorbitol, polysorbate-80, tween-80 and thimerosal.

[12] 12. A method as claimed in Claim 7, wherein the method further comprising the step of adding an antibody.

[13] 13. Use of a nanostructure lipid for the preparation of medicament for treatment of leukemia.