FORM-2
THE PATENTS ACT, 1970
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
AQUASOME FORMULATIONS FOR CANCER
THERAPY
GURUDEV M. HIREMATH, BASAVARAJ K. NANJWADE AND F. V. MANVI
"SHIV SHRADHDHA", BAGICHA VISTAR, MALPUR-383 345, GUJARAT, INDIA
The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed

FIELD OF THE INVENTION
The invention relates to aquasome formulations for effective treatment of cancer. It specifically relates to site specific drug delivery formulations in the form of aquasome for administration of anticancer agent. More specifically it relates to site specific drug delivery formulations for administration of antineoplastic such as etoposide nanoparticles in the form of aquasome and process for preparation of such aquasomes.
BACKGROUND OF THE INVENTION
Nanoparticles offer unique opportunities to improve the performance of both imaging and therapeutic agents because of their properties related to their dimension and physical-chemical reactivity. Their size of about 20-100 nm allows them at the same time to pass through cell membranes and to incorporate large amounts of drugs, which, being hidden within the particle, are devoid of direct side effects, protected from degradation and thus show improved performance. They are a self-assembling system, relatively easy to produce. Finally, their high surface/volume ratio makes them more efficient carriers. Nanoparticles currently used in therapeutic and diagnostic applications include liposomes, polymeric nanoparticles, polymeric micelles, protein nanoparticles, ceramic nanoparticles for imaging, viral nanoparticles, metallic nanoparticles for tumor ablation and imaging, dendrimers, carbon nanotubes and nanofibers. They allow drugs, as well as DNA and RNA molecules to be carried. The general advantages of nanoparticles are mentioned below:
1) Small size & relatively narrow size distribution which provide biological opportunities for site-specific drug delivery by nanoparticles.
2) Controlled release of active drug over a long period can be achieved.
3) Protection of incorporated drug against chemical degradation.
4) Possible sterilization by autoclaving or gama irradiation.
5) Nanoparticles can be lyophilized as well as spray dried.
6) No toxic metabolites are produced.
7) Relatively cheaper & stable.
8) Ease of industrial scale production by hot dispersion technique.
9) Side effect can be minimized.
10) Surface modification can easily be accomplished & hence can be used for site-specific drug delivery system.
11) They offer better therapeutic effectiveness and overall pharmacological response/unit dose.

12) Possess better stability and high drug entrapment efficiency as compared to microsphere.
13) The system can be used for various routes of administration including oral, parental, intra-ocular etc.
14) Particle size and surface characteristics of nanoparticles can be easily manipulated to achieve both passive and active drug targeting after parental administration
The term drug delivery refers to a system that can transport and deliver a drug precisely and safely to its site of action. The field of drug delivery is developing rapidly; research is being conducted to study nearly every part of the body as a potential route for administrating both classical and novel medicines. The final aim of pharmacy and medicine is the delivery of any drug at the right time in a safe and reproducible manner to a specific target at the required level. This is the reason for the research has been focusing more and more on targeted drug delivery system. m traditional drug delivery system, only small amount of drug reaches at tumor site and since many anticancer drugs have some toxicity they can kill some helpful bacteria or normal cells in normal organs. The targeted drug delivery system can overcome these shortcomings and deliver the drug right to the target, without hurting to normal organs and tissue. Although oral route is very common for drug administration adequate peptide and protein drug delivery has not been attained by this route. Nasal delivery shows a poor absorption of polar compound and it is not more suitable for administration of the cytotoxic drug. Parentral route is associated with pain and cardio vascular side effects. Localized delivery of drugs suffers from limited drug diffusion within cancerous tissue and sometimes undesirable interaction between drug and delivery vehicle. Chemotherapy is an integral part of cancer treatment. Despite of advance in oncology drug discovery, conventional chemotherapeutic agents still exhibit poor specificity to target tumor tissue and are often restricted by dose-limited toxicity. The combination of nano-technology and targeted drug delivery may provide a more efficient and less harmful solution to overcome the drawbacks of traditional drug delivery and by delivering chemotherapeutics right to the target site without hurting to normal organs and tissues.
The site-specific targeted drug delivery negotiates an exclusive delivery to specific preidentified compartments with maximum intrinsic activity of drugs and concomitantly reduced access of drug to irrelevant non-target cells. The controlled rate and mode of drug delivery to pharmacological receptor and specific binding with target cells, as well as bioenvironmental protection of the drug to the site of action are specific features of targeting. Invariably, every event stated contributes to higher drug concentration at the site of action and hence lowers concentration at non-target tissue where toxicity might-crop-up. The high drug concentration at

the target site is a result of relative cellular uptake of the drug vehicle, liberation of drug and efflux of free drug from the target site
Drug delivery systems (DDS) for cancer therapeutics have now been used by millions of patients and have resulted in the creation of new therapies as well as significantly improving existing ones. The development of smart targeted nanoparticles that can deliver drugs at a sustained rate directly to cancer cells may provide better efficacy and lower toxicity for treating primary and advanced metastatic cancer. Most of the low molecular weight anticancer drugs penetrate tissue and distributes throughout the body but with no selectivity towards tumour. In the targeted delivery approach such drugs are coupled with high molecular weight polymers which make drugs more stable in the bloodstream because high molecular weight polymer can get entry into the cell through endocytosis. Because of leaky microvasculature nature of tumour tissue such drug accumulates in tumour tissue. In another approach targeted drug delivery is achieved by use of liposomes to encapsulate drug which circulates throughout the blood stream and delivers the drug at target by diffusion or degradation of liposomes. The important advantages of such a system are reduced side effects and increased drug carrying capacity.
Several problems have been identified which require alterations in targeting strategies particularly, in vivo. These include:
1. Rapid clearance of targeted systems especially antibody targeted carriers.
2. Drug- antibody inactivation during conjugation.
3. Immune reactions against intravenously administered carrier systems.
4. Target tissue heterogeneity.
5. Problems of insufficient localizations of targeted systems into tumor cells.
6. Down regulation and sloughing of surface epitopes.
7. Diffusion and redistribution of released drug leading to non-specific accumulation. Aquasomes are nanoparticulate carrier system but instead of being simple nanoparticles these are three layered self assembled structures, comprised of a solid phase nanocrystalline core coated with oligomeric film to which biochemically active molecules are adsorbed with or without modification. Alternatively aquasomes are called as "bodies of water", their water like properties protect and preserve fragile biological molecules, and this property of maintaining conformational integrity as well as high degree of surface exposure are exploited in targeting of bio-active molecules like peptide and protein hormones, antigens and genes to specific sites. These carbohydrate stabilize nanoparticles of ceramic are known as "aquasomes" which was first developed by Nir Kossovsky. The pharmacologically active molecule incorporated by co-

polymerization, diffusion or adsorption to carbohydrate surface of pre formed nanoparticles. These three layered structure are self assembled by non-covalent bonds.
The drug delivery vehicle aquasome is colloidal range biodegradable nanoparticles, so that they will be more concentrated in liver and muscles. Since the drug is adsorbed on to the surface of the system without further surface modification they may not find any difficulty in receptor recognition on the active site so that the pharmacological or biological activity can be achieved immediately. In normal system, the calcium phosphate is a biodegradable ceramic. Biodegradation of ceramic in vivo is achieved essentially by monocytes and multicellular cells called osteoclasts because they intervene first at the biomaterial implantation site during inflammatory reaction. Two types of phagocytosis were reported when cells come in contact with biomaterial; either calcium phosphate crystals were taken up alone and then dissolved in the cytoplasm after disappearance of the phagosome membrane or dissolution after formation of heterophagosomes. Phagocytosis of calcium phosphate coincided with autophagy and the accumulation of residual bodies in the cell.
1. Aquasomes possess large size and active surface hence can be efficiently loaded with
substantial amounts of agents through ionic, non co-valent bonds, van der waals forces and
entropic forces. As solid particles dispersed in aqueous environment, exhibit physical properties
of colloids.
2. Aquasomes mechanism of action is controlled by their surface chemistry. Aquasomes deliver contents through combination of specific targeting, molecular shielding, and slow and sustained release process.
3. Aquasomes water like properties provides a platform for preserving the conformational integrity and bio chemical stability of bio-actives.
4. Aquasomes due to their size and structure stability, avoid clearance by reticuloendothelial system or degradation by other environmental challenges.
APPLICATIONS OF AQUASOMES:
1. Aquasomes as red blood cell substitutes, haemoglobin immobilized on oligomer surface because release of oxygen by haemoglobin is conformationally sensitive. By this toxicity is reduced, hemoglobin concentration of 80% achieved and reported to deliver blood in non linear manner like natural blood cells.

2. Aquasomes used as vaccines for delivery of viral antigen i.e. Epstein-Barr and Immune deficiency virus to evoke correct antibody, objective of vaccine therapy must be triggered by conformationally specific target molecules.
3. Aquasomes have been used for successful targeted intracellular gene therapy, a five layered composition comprised of ceramic core, polyoxyoligomeric film, therapeutic gene segment, additional carbohydrate film and a targeting layer of conformationally conserved viral membrane protein.
4. Aquasomes for pharmaceuticals delivery i.e. insulin, developed because drug activity is conformationally specific. Bioactivity preserved and activity increased to 60% as compared to i.v. administration and toxicity not reported.
5. Aquasomes also used for delivery of enzymes like DNAase and pigments/dyes because enzymes activity fluctuates with molecular conformation and cosmetic properties of pigments are sensitive to molecular conformation.
Cancer is the end product of a multi step process that occurs over many years. "Cancer refers to any one of a large number of diseases characterized by the development of abnormal cells that divide uncontrollably and have the ability to infiltrate and destroy normal body tissue". Among the novel drug delivery systems that have been devised over the years are many particulates-carries systems; for example microspheres, nanoparticles, lipoproteins, micellular systems and liposomes. The Drug delivery remains a challenge in management of cancer. Cancer drug delivery is no longer simply wrapping up cancer drugs in a new formulation for different routes of delivery. The focus is on targeted cancer therapy. The newer approaches to cancer treatment not only supplement the conventional chemotherapy and radiotherapy but also prevent the damage to normal tissues and prevent drug resistance.
Recently there has been immense interest in using polymeric particles for the sustained or controlled release of protein and peptide drugs because of their ease of fabrication, relatively simple administration and versatility. The successful implementation of nanoparticles for drug delivery depends on their ability to penetrate through several anatomical barriers, sustained release of their content and their stability in nanometer size. However, the scarcity of safe polymer with regulatory approval and their high cost have limited the wide spread application of nanoparticles to clinical medicine. To overcome these limitation of polymeric nanoparticles & due to the reason that many other carriers like prodrug 8c liposomes utilized are prone to destructive interaction between drug & carrier. In such case aquasomes prove to be worthy

carrier, carbohydrate coating prevents destructive denaturating interaction between drug & carrier.
Etoposide is an antineoplastic agent, which is used in the treatment of small cell lung cancer, drug resistant testicular cancer, lymphoma and several types of leukemia. Etoposide shows variables bioavailability upon oral administration. Intravenous administration of etoposide is limited by its lipophilicity. Further, vehicles required to increase its aqueous solubility for intravenous administration are often associated with adverse effects such as hypotension, anaphylaxis, and bronchospasm etc. Etoposide also causes dose limiting hematological toxicity. Previous formulation of Etoposide such as oral gelatin capsules and i.v. infusions had problems. Oral dosage forms of etoposide were ampoule, lipophilic capsule, and hydrophilic soft gelatin capsule. These oral formulations cause poor bioavailability (mean 57%), and absorption was nonlinear with increasing doses in the clinically used ranges. Poor solubility in water and limited stability in solutions led to complex i.v. formulations of etoposide. The current etoposide i.v. formulation (etoposide injection) is supplied in 5ml vials (Cipla, India; Biochem, India) containing etoposide (100 mg), Benzyl alcohol (0.15 g) and ethyl alcohol (30.5% v/v). Benzyl alcohol may cause serious side effects, especially in neonates. The formulation is a concentrate that needs to be diluted in large volumes before infusion, and care is necessary as the drug may precipitate. The infusion requires careful control as high rates may cause hypotension and cardiac arrest. Therefore, there is a need for formulation of etoposide, which would eliminate benzyl alcohol as vehicle for the i.v. formulation and increase drug stability.
The other problem associated with the administration of etoposide is the drug's inherent toxicity. The major dose-limiting factor is myelosupression. Neutropenia is seen around day eight to ten with thrombocytopenia usually occurring two to three days later. Leukopenia occurs more frequently than thrombocytopenia and is often moderate to severe. There is some evidence that larger dose of etoposide results in higher risk of developing subsequent acute myeloid leukemia (AML) or secondary acute non-lymphoblastic leukemia. Children with acute lymphocytic leukemia treated with high cumulative doses of epipodophyllotoxins using either weekly or twice-weekly schedules of administration have a relatively high risk of developing secondary AML. The cumulative dose of etoposide and the schedule of administration may play an important role in the induction of therapy-related leukemia. Therefore, a more targeted drug delivery system, which may reduce the toxicity, decrease the need for large doses and frequent dosing schedules, is needed.
Presently lot of interest has been generated by various drug delivery systems like microsphere, liposomes, niosomes, nanocapsules and nanoparticle. Nanoparticle is the latest trend in cancer

therapy. It helps to formulate the product with maximum therapeutic value and minimum or negligible range of side effect. Nanoparticles ranging in size between 10 and 1000 nm. They are manufactured from synthetic/ natural polymers and ideally suited to optimize drug delivery and reduced toxicity. The successful implementation of nanoparticles for drug delivery depends on their ability to penetrate through several anatomical barriers, sustained release of their content and their stability in nanometer size. However, the scarcity of safe polymer with regulatory approval and their high cost have limited the wide spread application of nanoparticles to clinical medicine. To overcome these limitation of polymeric nanoparticles & due to the reason that many other carriers like prodrug & liposomes utilized are prone to destructive interaction between drug & carrier. In such case aquasomes prove to be worthy carrier, carbohydrate coating prevents destructive denaturating interaction between drug & carrier. Conventional formulations of etoposide are limited by its lipophilicity. Further, vehicles required to increase its aqueous solubility for intravenous administration are often associated with adverse side effect. Dose required for anti-tumor effect is toxic to normal tissue so it also causes dose limiting hematological toxicity.
There are reports available on nanoparticles of etoposide for targeted drug delivery, the interested ones include LH Reddy and RSR Murthy [AAPS PharmSciTech 2005; 6 (2) Article 24, pages E158-E166] which reports the Etoposide-Loaded Nanoparticles Made from Glyceride Lipids. LH Reddy et al. [AAPS PharmSciTech 2006; 8(2) Article 29, pages E254-E62] reported the preparation and release behavior of testosterone (TST) encapsulated surfactant free nanoparticles of poly (DL-lactide-co-glycolide). Surfactant-free PLGA nanoparticles were successfully prepared by both the dialysis and the solvent diffusion method. The PLGA nanoparticles prepared using solvent diffusion method had a smaller particle size and higher loading efficiency than the dialysis method but the nanoparticle yield was lower. Panchaxari Dandagi et. al. [Indian Journal of Novel Drug delivery 3(1), Jan-Mar, 2011, 43-51] reported the study of polymeric biodegradable nanoparticles (NPs) of Etoposide (ETP) which were prepared by modified spontaneous emulsification solvent diffusion method by using polylactic-co-glycolic acid (PLGA) as biodegradable matrix.
The patents of interest include United States Patent 7,220,428 and 7,521,066 disloses the etoposide loading in lipid nanoparticles with the 1 to 30% of etoposide addition in the pharmaceutical composition.
Though there are reports in the art for the nanoparticles of etoposide with lipid for targeted drug delivery but there is no report on the etoposide aquasome with oligomer coating in inorganic core for the effective targeted delivery for treatment of caner. Therefore, to overcome the inherent

drawbacks associated with conventional formulations of etoposide, the present inventors aim is to encapsulate etoposide in biodegradable polymers such as oligomer in inorganic core to formulate them as aquasomes, which will have following advantages: a) Increased aqueous solubility, b) Reduce risk of unwanted side effects, c) Enhanced patient convenience and compliance, d) Increased bioavailability and hence smaller dosage form size, e) Controlled drug release potential and f) More effective cancer treatment.
OBJECTS OF THE INVENTION
The primary object of the present invention is the development of aquasome formulations for
treatment of cancer.
Another object of the invention is to develop the drug delivery formulations in the form of
aquasome containing etoposide nanoparticles.
It is yet another object of the invention is to develop a process for preparation of aquasomes
loaded with etoposide nanoparticles for treatment of cancer.
It is yet another object of the present invention for the preparation of aquasomes loaded with
etoposide nanoparticles for treatment of cancer by immobilizing etoposide onto hydroxyapetite
core.
STATEMENT OF THE INVENTION
Aquasome formulation for cancer therapy comprising: a) therapeutically effective amount of anticancer agent; b) oligomer and c) inorganic core. The anticancer agent is antineoplastic which is etoposide ranges from 0.01 to 0.1% (w/v).and the oligomer is lactose ranges from 10 to 90% (w/v) with a inorganic core is hydrated calcium phosphate (brushite) ranges from 0.0005 to 0.002%) (w/v). Aquasome formulations having the particle size less than 250 nm. Process for preparation of etoposide loaded aquasome by lyophilization method comprising: a) coating of lactose by resuspending the inorganic core into distilled water and mixing with lactose with continuous mechanical agitation to form the polyhydroxylated core; b) loading of etoposide dissolved in acetone at a dispersion of lmg/ml of the polyhydroxylated core; and c) filtering and lyophilization of fonned nanoparticles.

DETAILED DESCRIPTION OF THE INVENTION ETOPOSIDE:

Molecular structure and identity of Etoposide (ETO)
Formula: C29H32O13;
Mol. Weight: 588.557g/mol. Etoposide is 4'-demethyl-epipodophyllotoxin-9-[4,6-0-(7?)-
ethylidene-beta-D glucopyranoside];
Category: Antineoplastic
Dose: The recommended dosage of etoposide is 50-100mg/m2 per day i.v. for five consecutive days or 100 mg/m day on days 1, 3 and 5. The admixed drug should be given by intravenous infusion over a period of not less than 30min. If oral dose form is preferred, twice the relevant i.v dose should be given.
Description: White, odorless and amorphous powder.
Solubility: Very soluble in Dichloromethane, Methanol and Chloroform, slightly soluble in Ethanol and sparingly in Water and Ether.
Storage: Preserve in tight, light-resistant containers.
Standards: Etoposide contains not less than 95.0% and not more than the equivalent of 105.0% of C29H32O13 calculated on the anhydrous basis.
Preformulation studies: Preformulation study is the process of optimizing the delivery of drug through determination of physicochemical properties of the new compound that could affect drug performance and development of an efficacious, stable and safe dosage form. It gives the information needed to define the nature of the drug substance and provide a framework for the drug combination with pharmaceutical excipients in the dosage form. Hence, preformulation studies were performed on the obtained sample of drug for identification and compatibility studies.

Examples:
Preparation of Aquasomes: The general procedure consists of an inorganic core formation, which will be coated with Lactose forming the polyhydroxylated core that finally will be loaded by Etoposide, our model drug.
Lactose coating; A sample of l.0mg of the inorganic cores was resuspended into 1.0 ml bi-distilled water and was added to a 100 ml solution of Lactose, whose concentrations were 0.04, 0.06, 0.08, 0.1M and 0.2M (13.69, 20.53, 27.38, 34.22 and 68.44 g) each. After a period of 90 min of mechanical agitation the dispersion was filtered through a membrane filter (pore size 0.22 urn) of nitrocellulose, and then lyophilizised producing the polyhydroxylated nanoparticles. Drug loading; A solution 0.06M of Etoposide (3.5 mg) in acetone was added at a dispersion of lmg/ml of the polyhydroxylated cores after a mechanical agitation, which was maintained for 90 min. The dispersion was filtered and freeze dried. The formulations are given in Table 1.

Characterization of Etoposide loaded Aquasomes:
Particle Size and Surface morphology: Particle size of different batches of aquasome was determined by dynamic scattering particle size analyzer (Nanotrac Particle Analyzer 150, Microtrac Inc., PA, USA). The range of the analyzer is 0.8 nm to 6.54 (am. Particles suspended in a dispersing fluid are subject to random collisions with the thermally excited molecules of the dispersing fluid resulting in Brownian motion. The velocity and direction of the resulting motion are random but the velocity distribution of a large number of mono-sized particles averaged over a long period will approach a known functional form, in this case the size distribution of the

particles. In the Nanotrac, light from a laser diode is coupled to the sample through an optical beam splitter in the Nanotrac probe assembly. The interface between the sample and the probe is a sapphire window at the probe tip. The sapphire window has two functions! Firstly, it reflects the original laser back through the beam splitter to a photo detector. This signal that has the same frequency as the original laser acts as a reference signal for detection, offering heterodyne detection. Secondly, the laser passes through the sapphire window and is scattered by the particles, which are in suspension but moving under Brownian motion. The laser is frequency shifted according to the Doppler Effect relative to the velocity of the particle. Light is scattered in all directions including 180 degrees backwards. This scattered, frequency shifted light is transmitted through the sapphire window to the optical splitter in the probe to the photo detector. These signals of various frequencies combine with the reflected signal of un-shifted frequency (Controlled Reference) to generate a wide spectrum of heterodyne difference frequencies. The power spectrum of the interference signal is calculated with dedicated high-speed FFT (Fast Fourier Transform) digital signal processor hardware. The power spectrum is then inverted to give the particle size distribution.
Particle size analysis: In order to analyze particle size drug loaded lyophilized nanoparticles were dispersed in deionized water, vortexed for 10 min and sonicated for 5 min before sampling. Particle size was determined by laser scattering light using Malvern Laser Analyzer Instruments (IISc, Bangalore, India).
The particle sizes of all five formulations were listed in Table 3. Particle size of nanoparticles of etoposide was found to be 220.4 nm, 192.4 nm, 176.7 nm, 158.6 nm and 154.2 nm for formulations Fl, F2, F3, F4, and F5 respectively. Particles of all formulations were in nanosize having smooth spherical surface.
Zeta Potential: The zeta potential of a particle is the overall charge that the particle acquires in a particular medium. Knowledge of the zeta potential of a Aquasome preparation can help to

predict the fate of the Aquasomes in vivo and to assess the stability of the systems. Measurement of the zeta potential of samples in the Zetasizer Nano ZS (Malvern instruments, U.K) is done using a combination of laser Doppler velocimetry and phase analysis light scattering (PALS) by a patented technique called M-3 PALS to measure the particle electrophoretic mobility. In this technique, a voltage is applied across a pair of electrodes at either end of a cell containing the particle dispersion. Charged particles are attracted to the oppositely charged electrode and their velocity is measured and expressed in unit field strength as their electrophoretic mobility.The Aquasome samples were diluted 1 in 10 with PBS and measurements were carried out at 25°C. Particles with zeta potentials more positive than + 30 mV or more negative than - 30 mV are normally considered stable.
The stability of the drug delivery system systems was assessed by measuring the zeta potential of the particles by Zetasizer Nano ZS. If all the particles in suspension have a large positive or negative zeta potential then they will tend to repel each other and there will be no tendency for the particles to come together. Zeta potential values of five aquasomes formulations are presented in the Table 3. Incorporation of charged components Stearyl amine (positive) and diacetyl phosphate (negative) contributed to positive and negative zeta potential of aquasomes. Zeta potential was found to be -38.7 ± 2.46, +44.8 ± 3.12, -68.4 ± 2.62, +54.8 ± 2.26, +55.6 ± 2.26for formulations F-l, F-2, F-3, F-4 and F-5 respectively. From the above results, it was confirmed that all formulations (F-l to F-5) having more positive (> +30 mV) and negative (> -30 mV) zeta potential was considered to be stable.
Fourier transform infrared analysis (FT-IR): FT-IR spectral measurement for pure Etoposide drug, Lactose, physical mixtures of Etoposide and Lactose, Etoposide loaded Aquasome formulation (F-l to F-5) were taken at ambient temperature using Thermo Nicolet, Japan FT-IR spectrophotometer (STIC, Cochin, India). Samples were mixed with KBr and vacuum packed to obtain pellets of the material. All the spectra acquired are scanned between 400 and 4000 cm"1 at

a resolution of 4 cm"1. The FT-IR spectra obtained for pure etoposide, lactose solution, formulation Fl, F2, F3, F4 and F5 are given in Figure 1. The characteristic peaks of the pure drug were compared with the peaks obtained for formulation and tabulated in Table 2.

Differential Scanning Calorimetric analysis (DSC): Differential scanning calorimetry (DSC) analysis was conducted to ascertain the compatibility of drug with the polymer using Mettler Toledo DSC 822 (STIC, Cochin, India). Pure Etoposide, pure Lactose, physical mixture and drug loaded nanoparticles formulations were weighed (4.00-6.00 ± 0.1 mg) and placed in sealed water, frozen in liquid nitrogen and lyophilized. The coolant was liquid nitrogen. The samples were scanned at rate 10°C/min, from 10°C to 230°C. An empty aluminum pan was used as reference. The generated thermograms of DSC samples endothermic peaks of pure Etoposide were 269°C, pure Lactose at 220.0°C and in case of nanoparticles formulations Fl to ¥5 were observed around 220.0°C as shown in Figure 2. X-ray diffraction analysis (XRD): X-ray diffraction patterns of Etoposide, pure Lactose,
physical mixture and Etoposide loaded aquasome formulations were determined using a X-ray
diffractometer (Bruker AXS D8) equipped with a rotating target X-ray tube and a wide-angle
goniometer (STIC, Cochin, India). The X-ray source was Ka radiation from a copper target with
graphite monochromater. The X-ray tube was operated at a potential of 40 kV and a current of 30
mA. The range (29) of scans was from 0 to 70°C and the scan speed was 0.04o per min at
increments of 0.02°. The characteristics diffraction peaks observed for the pure drug,
charbohydrate, inorganic core and nanoparticles formulations (F-l to F-5) are given in Figure 3.

Scanning Electron Microscopic analysis (SEM analysis): The shape and surface
characteristics of Aquasomes were visualized using scanning electron microscopy, JSM-848,
Joel, Japan (Materials department, Indian Institute of Science (IISc) Bangalore, India). The
nanoparticles were first dried under vacuum. Nanoparticles then were glued to aluminum sample
holders and gold coated under argon atmosphere. The coated nanoparticles were finally
characterized for surface morphology under suitable magnification. SEM photographs of selected
formulations were shown in Figure 4. The Etoposide nanoparticles have shown smooth and
spherical shape with different sizes.
Evaluation of Etoposide Nanoparticles:
Percentage entrapment efficiency: To determine Etoposide entrapment in nanoparticles,
equivalent 10.0 mg of each nanoparticles formulation were suspended in 10.0 ml containing PBS
pH 7.4. Nanoparticles suspensions were subjected to cold centrifuge at -4 DC and 14,000 rpm
using Remi centrifuge (Remi equipment Pvt. Ltd., Mumbai) for 15 min. From the supernatant 2.5
ml solution was transferred into 25 ml volumetric flask and diluted upto the mark with methanol.
The resulting solutions were analyzed for Etoposide content using double beam UV-
spectrophotometer and % entrapment efficiency was calculated using following equation.

The drug encapsulation efficiency of all four formulations was shown in Table 3. Relatively high entrapment of Etoposide in the aquasomes could be ascribed to the sparingly soluble nature of the drug. Due to the sparingly soluble nature of Etoposide, it probably gets absorbed to the lactose layer of the hydroxyapetite core. The encapsulation efficiencies increased from Fl to F5. The maximum encapsulation was found in F-5 (88.41%) and lowest encapsulation in F-l (72.62%).

In vitro drug release studies: The in vitro release studies of Etoposide nanoparticles were carried out at 37±2°C in phosphate buffer saline (PBS) pH 7.4 buffer media for a period of 90 hrs. A horizontal water bath shaker (Remi equipment Pvt. Ltd., Mumbai) was used to conduct in vitro release studies. The 30 ml screw capped bottles, containing Etoposide nanoparticles in 20 ml of PBS pH 7.4 as release medium, were fixed iu holders in water bath shaker and temperature was maintained at 37±2oC. The platform was allowed to vibrate horizontally at an average speed of 100 rpm to induce mixing in the release medium. At periodic intervals of every 1 h, 2.0 ml of the release medium was sampled and replaced with fresh 2.0 ml of release medium to provide the necessary sink condition. The samples were further diluted with methanol upto 10.0 ml, filtered by 0.22 urn membrane (Millipore, India) and analyzed by double beam UV-visible spectrophotometer for the amount of Etoposide released from nanoparticles. The cumulative percentage drug release was calculated to establish the drug release profile of the Etoposide loaded nanoparticles.

Treatment of dissolution data with different models: To analyze the mechanism of drug release from the nanoparticles the data obtained from the drug release studies was analyzed according to the equations as below:

Different Models of Release Mechanism:
Method Equations
Zero Order Qt = QO + kOt
First order In Qt = In QO + k1t
Matrix ((Higuchi Matrix) Qt = QO - kHtl/2
Korsmeyer-Peppas log (Qt/Qoo) = log k + n log t
Where, Qt: cumulative amount of drug released at any specified time (t), QO: dose of drug
incorporated in the delivery system, Qco: amount of drug release after infinite time, kO: rate
constant of zero order, kl: rate constant of first order, kH: rate constant of Higuchi matrix model,
kk: release rate constant which considers structural & geometric characteristics of the
nanoparticles, n: the diffusion exponent; indicative of the mechanism of drug release.
The 'n' value could be used to characterize different release mechanisms as mentioned below.
Different Release Mechanisms of'n' Value:
'n' Mechanism
0.5 Fickian Diffusion (Higuchi Matrix)
0.5 < n < 1 Anomalous Transport (First order)
1 Case-II Transport (Zero order release)
n > 1 Super case-II Transport
The release profile of an entrapped drug predicts how a delivery system might function and gives
valuable insight into its in vivo behaviour. All the five formulations of Etoposide aquasomes
were subjected to in vitro release studies. These studies were carried out using dialysis cassettes
(Slide-A-Lyzer®3.5K, Pierce, U.S.A) in phosphate buffer saline pH 7.2. The release data
obtained for formulations Fl, F2, F3, F4 and F5 are tabulated in Table 4. Cumulative percent
drug released after 120 hours was 56.73%, 57.43%, 58.46%, 72.64% and 79.19%for Fl, F2, F3,
F4 and F5 respectively. The results obtained of in vitro release studies were attempted to fit into
various mathematical models as follows:
1. Cumulative percent drug released Vs. Time (Zero order rate kinetics).
2. Log Cumulative percent drug retained Vs. Time (First order rate kinetics).

3. Cumulative percent released Vs. VT [Higuchi's classical diffusion equation (Higuchi
matrix)].
4. Log of cumulative percent drug released Vs. Log Time (Peppas exponential equation).
The regression coefficient (r) and 'n' values for formulations (F-l toF-5) of zero order, first order, Higuchi matrix, Peppas are tabulated in Table 5. The regression coefficients for formulations Fl to F5 of zero order plots were found to be 0.9855, 0.9844, 0.9857, 0.9945, and 0.9945 respectively. The regression coefficients for formulations Fl to F5 of first order plot were found to be 0.9851, 0.9859, 0.9945, 0.9757, and 0.9757 respectively. These results indicate that zero order plots were not linear for all formulations and the first order plots were almost linear for all formulations. This Higuchi's plot was non linear with regression co-efficient values of 0.9880, 0.9900, 0.9890, 0.9860 and 0.9860 for formulations Fl to F5. The non-linearity suggests that the release of etoposide from aquasomes was not diffusion controlled.
Peppas - Korsmeyer Equation is given as: R = Ktn & Log % R = log K + n log t, Where R = drug release, k = constant, n = slope & t = time.
This model is widely used when the release mechanism is not well known or when more than one type of release phenomenon could be involved. The 'n' value could be used to characterize different release mechanisms. The 4n' values for Fl to F5 were 0.7773, 0.7713, 0.7722, 0.7638 and 0.7638 respectively, which is more than 0.5. This indicates that the release approximates Anomalous transport mechanism.

In vivo drug targeting studies: This study was carried out to compare the targeting efficiency of drug loaded aquasomes with that of free drug in terms of percentage increase in targeting to various organs of reticuloendothelial system like liver, lungs, spleen and kidneys. Experiments

were performed on Wistar rats of 180-240 g weight. All the experiments were carried out in
accordance with the protocols approved by the Institutional animal ethics committee.
Dose Calculation: Dose of Etoposide to be administered in rats was calculated according to
body surface area ratio of rats to human being.
Dose (mg/200 gin of mice) = Human dose (mg) x Surface area ratio; Dose = 50 x 0.018; For 200
gm mice = 0.9 mg/ 200 gm mice; Dose = 4.5 mg/kg.
Nine healthy adult rats weighing 180-240 gm were selected, a constant day and night cycle was maintained and they were fasted for 12 hrs. The animals were divided into 3 groups, each containing 3 rats. Group I received aquasomes equivalent to 4.5 mg of Etoposide intravenously in the tail vein after redispersing them in sterile phosphate buffer saline solution. Aquasomes from F5 batch were selected for the study. Group-II received 4.5 mg of pure Etoposide intravenously. Group-Ill mice were treated as solvent control and were injected intravenously with sterile phosphate buffer saline solution. After 3 hrs the mice were sacrificed and their liver, lungs, spleen and kidneys were isolated. The
individual organs of each mice were homogenized separately by using a tissue homogenizer with
5ml of methanol and the homogenate was centrifuged at 15,000 rpm for 30 minutes. The
supernatant was collected and filtered through 0.45 jam filters and analyzed
spectrophotometrically after dilution with phosphate buffer saline at the wavelength of 286 nm.
Formulation F5 with optimal particle size, high encapsulation efficiency and satisfactory in vitro release was selected for in vivo drug targeting studies. The comparison between the amount of drug targeted from aquasomes and free drug in various organs is presented in Table 6. The average targeting efficiency of drug loaded aquasomes was found to be 42.54% of the injected dose in liver, 12.22% in lungs, 25.12%o in spleen and 4.14% in kidney, whereas accumulation of pure drug was 21.33% in liver, 5.90% in lungs, 11.22%, in spleen and 2.68% in kidneys of the injected dose. These results reveal that the drug loaded aquasomes showed preferential drug targeting to liver followed by spleen, lungs and kidneys. It was also revealed that as compared to pure drug, higher concentration of drug was targeted to the organs after administering the dose in the form of aquasomes. Higher drug targeting in liver and spleen as compared to lungs and kidneys may be attributed to high macrophages load. After opsonization by the plasma proteins

the macrophages residing in liver and spleen (the mononuclear phagocyte system [MPS]) rapidly uptake the aquasomes.

Instruments: The HPLC system consisted of a Thermoquest Spectra system P1500 isocratic pump coupled with a Spectra System UV 6000 LP photodiode array detection system, A Spectra System AS 3000 auto sampler, a SCM 1000 vacuum membrane degasser, a SN 4000 system controller. The detector was set to scan from 200 to 500 nm.
Chromatographic conditions: Cg reverse-phase column (Millipore, o.d. 6.35 mm and length 25 cm) jim was used as analytical column. The column temperature was 30 C. The control of the HPLC system and data collection was done by a computer equipped with spinchrome software. The flow rate was 1.5 ml/min and an injection volume of 20 uL was used. A modified isocratic mobile phase consisted of acetonitrile-acetic acid-water (34:1:65), pH 4.0. The eluents were monitored at 230 nm. 8-Methoxypsoralen was used as the internal standard. Plasma sample preparation procedure: A standard stock solution containing Etoposide (1 mg/ml) was prepared in acetonitrile-acetic acid-water (34:1:65) and stored at 4 °C. Working standard stock solutions were prepared from the stock solution by sequential dilution with acetonitrile-acetic acid-water (34:1:65) to yield final concentration range 1 to 100 jig/ml. Then

plasma standard solutions were prepared by spiking into drug-free rat plasma with different working standard solutions to give final concentrations between 1, 5, 10, 20, 40 and 50 jig/ml for calibration curve. 200 ul aliquot of each plasma sample was transferred to a 1.5 ml polypropylene tube. Then 15 µl of 1 mg/ml internal standard solution and 400 ul cold methanol were added. After a brief vertex mixing, the tubes were centrifuged (14000 rpm at 4 °C for 15 min). 100 ul aliquot of the supernatant was transferred to the injection vials and 20 uJ were injected into the chromatographic system.
Application of the method: The central animal facility of the institute provided white male rats with mean weight of 180-240 gm. The study was conducted as per guidelines prescribed by institutional animal ethics committee under the supervision of registered veterinarian. Animals were issued 6 days prior to experimentation for acclimatization and were kept on standard pellet diet and water ad libitum. Food was stopped to all animals 8-10 h prior to experimentation. Food and water was not given to animals till 2 h after the start of the study. Each rabbit was dosed with specific dose of Etoposide aquasomes without taking weight of the rabbit into consideration. For each study blood samples (1ml) were withdrawn from the jugular vein of rat using a 21G needle. Samples were withdrawn before dosing and 0, 2, 5, 10, 15, 20, 25 and 30 hrs post dosing. The collected blood was harvested for 45 min at ambient temperature and centrifuged at 5000 rpm for 20 mins. The clear supernatant serum layer was collected and stored at -20 °C until analysis. The in vivo evaluation of Etoposide loaded Aquasomes was conducted in one group of rats. Rats have been chosen as a model for study because the blood volume of the rabbit is sufficiently large to permit frequent blood sampling and allow a full characterization of the absorption and determination of the pharmacokinetic profile of the drug. Table 7 shows the plasma concentration of the drug at each sampling interval for formulations F-5.
Data Analysis: The maximum plasma concentration (Cmax) and time (Tniax) of it occurrence were directly computed from the plasma concentration vs. time plot.The elimination rate constant (Kcl)

was determined from the terminal phase of the log plasma concentration vs. time profile and was calculated as Kel = 2.303 x slope.The elimination half life was calculated using the formula 0.693/ Kel.-The area under the curve (AUC) was calculated from the plasma concentration vs. time profile by trapezoidal method and was statistically analysed by applying one-way ANOVA. The Cmax of formulation F-5 was found to be 3.87 ug/ml and the corresponding Tmax was 2 hrs. The pharmacokinetic parameters of the formulations F-5 were estimated and are given in Table 7.

Stability studies: According to ICH guidelines, Etoposide containing nanoparticles were stored at elevated temperature and relative humidity (25±2°C/60%±5% RH, 40±2°C/75%±5% RH) in a stability analysis chamber over a period of 3 months.52 Freshly prepared nanoparticles were stored at 5±3°C used as control. Samples were kept for 90 days for stability analysis and after 90 days, drug loading of nanoparticles were compared with those of the control formulations. The optimized batch of Etoposide loaded aquasome (F-5) was subjected to stability studies at refrigeration temperature (4°C), room temperature (25°C) and body temperature (37°C) for a period of 90 days. A batch of aquasome dispersions (F-5) was observed for any signs of sedimentation or creaming, changes in particle size, polydispersity index, zeta potential and encapsulation efficiency. The observations are recorded in Table 8. Formulation F-5 shows no

signs of sedimentation at 4°C whereas slight sedimentation was observed at room temperature and 37°C after 90 days of storage. There was no significant change in the particle size, ZP and EE even after 90 days of storage at 4°C. Reduction in Particle size, ZP and EE was observed following storage at 37°C. F-5 showed slight changes in Particle size and ZP. From the above studies it was confirmed that aquasomes remained more stable at refrigeration temperature (4°C). The maximum instability of aquasomes was observed at 37°C.

CONCLUSION: In present work the Formulation and evaluation of etoposide loaded Aquasomes was successfully carried out. From the reproducible results of the executed experiments, it can be concluded that-Etoposide was successfully immobilized onto hydroxapetite core. The particle size analysis revealed that aquasomes formulated were in nanometer range. Particle size for F-4 and F-5 were smallest. High positive and negative zeta potential values for formulations (F-l to F-5) indicate the stability of aquasome suspension. Formulation F-5 showed higest encapsulation efficiency with 88.41% of drug content. Formulation F-5 showed maximum cumulative percent drug release. It was observed that all the formulations Fl - F5 followed the First order kinetics suggesting drug release by Anomalous transport mechanism. The 'n' value obtained from Peppas plot was greater than '0.7' indicating that the all formulations Fl - F5 did not follow Fickian controlled release mechanism. On the basis of particle size, encapsulation efficiency, in-vitro release and satisfactory release kinetics, formulation F5 was selected as an optimum formulation for in vivo studies. The order of

targeting was found to be liver > spleen > lungs > kidneys. Present study shows that the targeting efficiency of drug-loaded aquasomes over free drug is higher, which may provide increased therapeutic efficacy. Moreover higher concentration of drug targeted to various organs may help in reduction of dose required for the therapy and thereby dose related side effects could be minimized. The in-vivo studies were carried out by HPLC method to assess the pharmacokinetic parameters like Cma.\. tmax. AUC, ti/2. Formulation F5 was selected for this study and it showed Cmax value of 1.48 ug/ml, tmax of 2 hrs, AUC was 20.40ug/ml and a Xm of 13.5 hrs. Stability studies of selected formulation F-5 showed closest encapsulation efficiency, particle size, and zeta potential to initial data at 4°C storage. Thus it can be concluded that 4°C is the most suitable temperature for storage of Etoposide aquasomes. Present work was a satisfactory preliminary study in immobilizing Etoposide onto hydroxyapetite core. Further detailed investigations needed towards the optimization of immobilization of Etoposide onto hydroxyapetite core. An in vitro -in vivo correlation need to be established to guarantee the efficacy and bioavailability of the formulation.
BRIEF DESCRIPTION OF DRAWINGS:
Figure 1: FT-IR spectra of Etoposide, pure Lactose and physical mixture.
Figure 2: DSC of Etoposide, pure Lactose and Etoposide loaded aquasome formulations
Figure 3: X-ray diffraction patterns of Etoposide, pure Lactose and Etoposide loaded aquasome
formulations
Figure 4: A) Scanning electron micrograph of Formulation Fl
B) Scanning electron micrograph of Formulation F2
C) Scanning electron micrograph of Formulation F3
D) Scanning electron micrograph of Formulation F4
E) Scanning electron micrograph of Formulation F5

We Claim,
1. Aquasome formulation for cancer therapy comprising:
a. therapeutically effective amount of anticancer agent;
b. oligomer and
c. inorganic core.
2. Aquasome formulation as claimed in claim 1, wherein the anticancer agent is antineoplastic.
3. Aquasome formulation as claimed in claim 2, wherein the antineoplastic is etoposide.
4. Aquasome formulation as claimed in claim 1, wherein the oligomer is lactose.
5. Aquasome formulation as claimed in claim 1, wherein the inorganic core is hydrated calcium phosphate (brushite).
6. Aquasome formulation as claimed in any of the preceding claims wherein the effective amount of is etoposide ranges from 0.01 to 0.1% (w/v).
7. Aquasome formulation as claimed in any of the preceding claims wherein the amount of lactose ranges from 10 to 90% (w/v).
8. Aquasome formulation as claimed in any of the preceding claims wherein the amount of hydrated calcium phosphate ranges from 0.0005 to 0.002%) (w/v).
9. Aquasome formulation as claimed in any of the preceding claims wherein the particle size is less than 250 nm.
10. Process for preparation of etoposide loaded aquasome by lyophilization method comprising:
a. coating of lactose by resuspending the inorganic core into distilled water and mixing
with lactose with continuous mechanical agitation to form the polyhydroxylated core;
b. loading of etoposide dissolved in acetone at a dispersion of lmg/ml of the
polyhydroxylated core;
c. filtering and lyophilization of formed nanoparticles.