TITLE OF THE INVENTION
A Gemcitabine based liposomal formulation for treatment of cancer and a process of preparation thereof.
FIELD OF THE INVENTION
The invention relates to a target delivery of a drug through a liposome formulation which is targeted to a pre-selected cell type. More specifically, the invention relates to a liposomal formulation for the treatment of cancer and a process for preparation thereof for target delivery of the drug whereby the drug is delivered to the targeted cancer cell.
BACKGROUND OF THE INVENTION
Cancer is a leading cause of death world-wide. The disease accounted for 7.4 million deaths (or around 13% of all deaths world-wide) in 2004. The most frequent type of cancer world-wide (in order of number of global deaths) are: among men lung, stomach, liver, colorectal, oesophagus and prostate; and among women breast, lung, stomach, colorectal and cervical.
The current therapy for cancer consists mainly of three approaches; radiotherapy, surgery and chemotherapy with anti-cancer drugs. The ideal prototype of an anticancer drug should display anti-tumor activity targeting and damaging cancer cells without causing adverse effects or toxicity to healthy cells. There are many potential barriers to the effective delivery of a drug in its active form to solid tumors. Most small-molecule chemotherapeutic agents have a large volume of distribution on i.v. distribution. The result of this is often a narrow therapeutic index due to a high level of toxicity in healthy tissues.
In the recent years, Gemcitabine has emerged as a very potent anti-cancer drug and it is currently used, alone or in combination, in the treatment of patients with different malignancies, including ovarian, pancreatic, colon, non-small cell lung and other cancers.
The cytotoxic activity of Gemcitabine in vivo is dose and dosage regimen dependent. This means the activity and the toxicity are related to the dose given and the dosage interval of the treatment. The problem with Gemcitabine is its short plasma t½ and its quick metabolism into 2'-deoxy-2',2'-difluorouridine (dFdU) followed by elimination from the body. Therefore high doses of 2',2'-difluorodeoxycytidine (dFdC) are required in order to achieve sufficient cytotoxic concentrations of dFdC triphosphate (dFdCTP). Due to the narrow therapeutic window, high administered doses increase the possibility of toxicities and concentration dependent side effects for patients.
The ideal therapeutic for cancer would be one that selectively targets a cellular pathway responsible for the tumor and would be nontoxic to normal cells. The cancer treatments involving target delivery of a drug through a novel liposomal formulation have substantial promise and can be considered as an efficient delivery of the drug to the site in the body where they are needed. Moreover, through encapsulation of drugs in a macromolecular carrier, such as the liposome the volume of distribution is significantly reduced and the concentration of drug in the tumor is increased.
Although liposomal or lipid formulations are known to possess aforesaid advantages, not all liposomal / lipid formulations exhibit same efficacy. Also the compatibility of different types of lipid formulations with every kind of anti-cancer drug has not been established. Therefore, there is a need in the art to provide an effective Gemcitabine based formulation that addresses the aforesaid disadvantages.
OBJECTS OF THE INVENTION
The object of the present invention is to provide a novel Gemcitabine based formulation exhibiting enhanced targeted delivery of Gemcitabine.
STATEMENT OF THE INVENTION
Accordingly, the present invention provides a Gemcitabine based liposomal formulation for targeted drug delivery, said formulation comprises a lipid mixture comprising 'X', Hydrogenated soy phosphatidylcholine (HSPC) and cholesterol and optionally a folate ligand and Gemcitabine, wherein X' is Digalactosyl diacylglycerol (DGDG) or Distearoylphosphatidylethanolamine (DSPE) and the ratio of Gemcitabine: lipid mixture is in the range of 1:4 to 1:6. Additionally, the present invention provides a process for preparing the Gemcitabine based liposomal formulation comprising the steps of: a) preparing the lipid mixture comprising 'X', Hydrogenated soy phosphatidylcholine, Cholesterol and optionally a folate ligand by conventional techniques; and b) loading of Gemcitabine into the lipid mixture thus obtained in step (a) by transmembrane ammonium sulphate gradient method. wherein. 'X' is Digalactosyl diacylglycerol or Distearoylphosphatidylethanolamine and the ratio of Gemcitabine : lipid mixture used in the process is in the range of 1:4 to 1:6.
SUMMARY OF THE INVENTION
In an embodiment of the invention, a Gemcitabine based liposomal formulation for treatment of cancer comprising: a lipid mixture comprising 'X', Hydrogenated soy phosphatidylcholine and cholesterol and optionally a folate ligand and Gemcitabine: wherein 'X' is Digalactosyl diacylglycerol or Distearoylphosphatidylethanolamine and the molar ratio of Gemcitabine: lipid mixture is in the range of 1:4 to 1:6.
In another embodiment of the invention, the lipid mixture comprises Digalactosyl diacylglycerol, Hydrogenated soy phosphatidylcholine and cholesterol.
In yet another embodiment of the invention, the ratio of Hydrogenated soy phosphatidylcholinexholesterol in the lipid mixture is in the range of 8:1 to 8:3.
In still another embodiment of the invention, the lipid mixture comprises 5 to 12% of Digalactosyl diacylglycerol, the % being based on total weight of lipid mixture.
In a further embodiment of the invention, Gemcitabine is present in the formulation as Gemcitabine Hydrochloride.
In a furthermore embodiment of the invention, the molar ratio of Digalactosyldiacylglycerol, Hydrogenated soy phosphatidylcholine and Cholesterol is about 1:9.5:2.75.
In one another embodiment of the invention, the lipid mixture comprises Folate-Polyethylene glycol - Distearoylphosphatidylethanolamine. Hydrogenated soy phosphatidylcholine and Cholesterol.
In yet another embodiment of the invention, the molar ratio of Folate- Polyethylene glycol - Distearoylphosphatidylethanolamine, Hydrogenated soyphosphatidylcholine and Cholesterol is about 0.25:7.25:2.0.
In still another embodiment, the invention provides a process for preparing Gemcitabine based liposomal formulation, comprising the steps of: a) preparing the lipid mixture comprising 'X', Hydrogenated soy phosphatidylcholine, Cholesterol and optionally a folate ligand by conventional techniques; and b) loading of Gemcitabine into the lipid mixture thus obtained in step (a) by transmembrane
ammonium sulphate gradient method; wherein. 'X', is Digalactosyldiacylglycerol or Distearoylphosphatidylethanolamine and the molar ratio of Gemcitabine : lipid mixture is in the range of 1:4 to 1:6.
In a further embodiment of the invention, in the process step (b), loading of Gemcitabine into the lipid mixture is performed at temperature in the range of 50-65 degree Celsius and for time period in the range of 50-80 minutes.
In a furthermore embodiment of the invention, in the process step (a). 'X', Hydrogenated soy phosphatidylcholine and cholesterol are dissolved in ethanol.
In one another embodiment of the invention, the lipid mixture used in said process comprises Digalactosyldiacylglycerol, Hydrogenated soy phosphatidylcholine and cholesterol.
In yet another embodiment of the invention, the ratio of Hydrogenated soy phosphatidylcholinexholesterol in the lipid mixture used in said process is in the range of 8:1 to 8:3.
In still another embodiment of the invention, the lipid mixture used in said process comprises 5 to 12% of Digalactosyl diacylglycerol, the % being based on total weight of lipid mixture.
In a further embodiment of the invention, Gemcitabine is used in step (b) of the process as Gemcitabine Hydrochloride.
In a furthermore embodiment of the invention, the molar ratio of Digalactosyl diacylglycerol, Hydrogenated soy phosphatidylcholine and Cholesterol is about 1: 9.5 : 2.75.
In one another embodiment of the invention, the lipid mixture used in said process comprises Folate-Polyethylene glycol-Distearoylphosphatidylethanolamine, Hydrogenated soy phosphatidylcholine and Cholesterol.
In still another embodiment of the invention, the molar ratio of Folate- Polyethylene
glycol - Distearoylphosphatidylethanolamine, Hydrogenated soy
phosphatidylcholine and Cholesterol is about 0.25:7.25:2.0.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
In the drawings accompanying the specification.
Fig. 1 illustrates a flowchart for the preparation of GL liposomes.
Fig. 2 illustrates particle size analysis of GL liposomes.
Fig. 3 illustrates zeta potential of GL liposomes.
Fig. 4 illustrates Cryo-TEM image of GL liposomes.
Fig. 5 illustrates diffusion study of GL liposomes.
Fig 6 illustrates particle size analysis of the optimized batch of SL liposomes.
Fig 7 illustrates particle size analysis of the optimized batch of FL liposomes.
Fig 8 illustrates zeta potential analysis data of SL liposomes.
Fig 9 illustrates zeta potential analysis data of FL liposomes.
Fig 10 illustrates photography of multilamellar vesicles in Olympus microscopy.
Fig 11 illustrates Cryo-TEM images of the prepared small unilamellar vesicles.
Fig 12 illustrates in vitro release profile of Gemcitabine from CL. SL and FT
liposomes.
Fig 13 illustrates biodistribution of 99mTc labeled SL and FT liposomes in tumor
bearing mice.
Fig 14 illustrates tumor distribution of CL and FT in tumor bearing mice after intravenous
injection.
DETAILED DESCRIPTION
SECTION-I
In this section Gemcitabine based liposomal formulation comprising Digalactosyldiacylglycerol (DGDG), Hydrogenated Soy Phosphotidyl Choline (HSPC), and cholesterol as the lipid mixture, loaded with Gemcitabine is described in detail along with its process for preparation.
1. Preparation of Gemcitabine based liposomal formulation comprising Gemcitabine HC1, HSPC & Cholesterol (hereinafter referred to as GL liposomes and used as standard)
The Liposomes were prepared by ethanol injection method, described by RRC New -Liposome practical approach, inl990. Ethanol Injection method:
Ethanol solution of lipids is injected rapidly through a fine needle into a solution of saline or aqueous medium. This procedure gives high proportion of SUVs of 25-100nm approximate size. This method protects the sensitive lipids from degradation. Limit for using ethanol by this method is 7.5%v/v. The ethanol lipid solution is injected in aqueous medium maintained at 55-60°C with continuous stirring. Vaporization of solvent leads to the formation of single layer vesicle.
1(a) Active loading of Gemcitabine into GL liposomes via transmembrane ammonium sulphate gradient method
Active loading through a pH gradient is a technique based on the membrane permeability of the free base of a hydrophilic drug, whereas its charged, protonated form is membrane impermeable. The drop in pH is caused by an ammonium sulphate transmembrane gradient having liposomes with internal ammonium sulphate surrounded by an ammonium sulphate free medium. Encapsulated ammonium ions are in equilibrium with uncharged ammonia and protons. The capability to permeate the liposomal membrane is dependent on size and charge of the species according to the following relation: NH3>>> H+>> NH4+> SO42- > (NH4)2SO4. A shift in equilibrium to the right (Equation 1), thus a reduction in the pH within the liposomes, occurs when uncharged ammonia diffuses out of the vesicles, leaving the protons behind.
Equation 1: NH4+ - H+ + NH3
Simultaneously, the neutral form of the drug, in this case the free base of gemcitabine, is expected to diffuse into the vesicle where it becomes protonated. due to the low pH, and thus trapped. This decreases the proton concentration within the liposomes; however, more ammonia will subsequently be produced and diffuses out of the vesicle increasing the proton supply facilitating the drug uptake. In order to create the gradient, liposome formation is carried out in ammonium sulphate solution followed by removal of external ammonium sulphate. The better the removal of ammonium sulphate in the outer aqueous phase, the greater the gradient becomes. This was executed by size exclusion chromatography (SEC) using Sephadex G50 gel. 5% sucrose was used as hydration medium in the column since this concentration is expected to be iso osmotic with 120mM of ammonium sulphate when added to the liposome dispersion. Based on the amount of substance of encapsulated ammonium sulphate in the liposome fraction compared to the amount of substance in the initial liposome dispersion, the encapsulation efficiency (HE) can be determined as given by equation 2:
Equation 2:
(Equation Removed)

The gradient is determined by comparing the concentration of external ammonium
sulphate
([(NH4)?.S04] ext) in the liposome fraction after separation after SEC (see section
4.2.1 a) with the concentration of ammonium sulphate inside the vesicles
([(NH4)2.S04] int.). This gives the following equation:
Equation 3: (Equation Removed)

(i) Size exclusion chromatography (SEC) for generation of ammonium sulphate gradient
Gel filtration is a SEC method separating different particles or substances according to their size. A column is filled with a pre-swollen porous gel, completely packing the column. Particles diffuse in and out the pores of the gel and therefore pass through the column at different speed according to their size or MW. Larger particles pass through the column in a faster manner compared to smaller particles due to less diffusion into the matrix. As a result different fractions containing different particles can be collected subsequent to the separation. Packing of column:
The gel filtration column was prepared in-house using Sephadex G-50 as gel matrix. Sephadex G-50-powder was mixed with excess distilled water and placed in a waterbath B at 90 °C for one hour in order to swell. The gel filtration column was set in 2.5 ml Syringe. Degassed 5% sucrose was used as hydration medium to pack the column with Sephadex gel. Procedure:
Three times the column volume of 5% sucrose solution was run through the column before the sample was added. During separation continuous amounts of medium
were added in order to keep the column wetted. The fractions, containing the different particles, were collected and their volume was measured. 1 ml of liposome dispersion was transferred to the column and the fractions were collected. Liposomes pass through the column in a faster manner compared to ammonium sulphate giving ideally one fraction consisting of liposomes with an internal ammonium sulphate solution and 5% sucrose solution as the outer phase. The liposome fraction could easily be collected from the column since the fraction appeared turbid. After separation the column was washed with 5% sucrose solution in order to remove residual ammonium sulphate from the gel matrix. During longer breaks, the gel matrix was stored in 20% ethanol acting as a preservative to prevent growth of micro-organisms.
(ii) Loading of Gemcitabine HC1
According to Fenske, Maurer and Cullis, a transmembrane ammonium sulphate gradient is ideal for drugs supplied as an HC1 salt. Like gemcitabine, these drugs form an equilibrium existing either in their water soluble salt form or uncharged as a free base. This equilibrium is a function of their pKa value, which is expressed by equation 4:

(Equation Removed)

with B as the free base and BET as the cation of the salt form.
Gemcitabine HO has a pKa of 3.58. According to the Henderson-Hasselbalch equation, the amount of salt form equals the amount of basic form when pH equals pKa equation 5.
Equation 5: (Equation Removed)

A solution of 38mg/ml gemcitabine has a theoretical pH of 2.2 and is protonated to 95.7 %. Accounting for the dilution with incubation, the pH of the outer phase was calculated to 4.88 for the sample loaded with 60ul of Gemcitabine HC1. At this pH, 94.9% of gemcitabine is unprotonated and therefore able to penetrate the liposome membranes. Once inside the liposomes, the acidic environment with pH -2.1 leads to an 85.7% protonation of the gemcitabine base, resulting in the entrapment.
1(b) Optimization of Process Parameters
Optimization of various formulation process parameters like stirring speed, time for process, volume of organic phase and aqueous phase were carried out. The criterions taken for the optimum formulation are discussed below.
(i) Stirring speed and process time
Series of trials were taken for optimization of process parameters - Stirring speed and process time. The complete dispersion formation with convenient injection rate was observed for selecting the optimum stirring speed.
Table 1: Optimization of stirring speed
(Table Removed)
Process time was optimized by injecting the lipid solution in ammonium sulphate solution at temperature range 60~65°C for 30 to 90min.
Table 2: Optimization of process time
(Table Removed)
(ii) Volume of organic phase and aqueous phase.
Volume of organic phase was optimized by taking aqueous phase as constant
parameter. Maximum limit for ethanol used in preparation of liposomes by ethanol
injection method is 0.75% v/v. Fixed quantity of lipid concentration was taken and
dissolved in 0.4ml, 0.6ml and 0.75ml ethanol at 60-65°C in hot water bath. Lipid
solutions were injected separately in 10 ml of 120mM ammonium sulphate solution
at 60-65°C.
Table 3: Optimization of volume of organic phase and aqueous phase.
(Table Removed)
(iii) Drug incubation time and temperature
Placebo liposome was incubated with Gemcitabine HC1 at temperature range of 40°C
to 60°C for the predetermined time of 30min, 60min and 90min.
Thereafter, entrapment efficiency was calculated by the formula given below:
(Equation Removed)
• Separation of Free Drug From Drug Entrapped In Liposomes
For separation of free drug from liposomes, the Liposomal suspension was centrifuged at 25000 rpm for 30min, at 2-4°C. Supernatant was collected by decantation. This supernatant solution was analyzed for the free drug.
• Estimation of Gemcitabine HC1 from Liposomes
Liposomal fraction present in sedimented pellet was dissolved in methanol: chloroform (2:1) mixture and transferred to 10 ml volumetric flask and volume made up to 10 ml with methanol: chloroform (2:1). The sample was then analyzed for Gemcitabine HC1 content using UV-spectroscopic method.
It was observed that the drug incubation time and temperature should be 60min and 60°C respectively for better drug entrapment.
1(C) Optimization of Formulation Parameters
Liposomes were prepared with varying ratio of HSPC: Cholesterol. More particularly. HSPC: cholesterol ratio was kept as 7:3, 8:2, 9:1. Also different ratio of drug : lipid were tested for drug entrapment. More particularly, drug:lipid was kept as 1:10, 1:5, 1:4, 1:3.3 as given in table 4.
Table 4: Optimization of Formulation parameters
(Table Removed)
* Molar ratio
An effect of lipid (phospholipids) to cholesterol ratio was evaluated with respect to entrapment efficiency. Entrapment efficiency of liposomes prepared using different lipid to cholesterol ratio was determined and results shows that at 8:2 lipid to cholesterol ratio gives maximum entrapment and beyond this ratio shows decrease in entrapment efficiency. Cholesterol concentration is important because it gives the stability (rigidity) to the liposomes which provide stability during their blood circulation and prevent the major leakage of the entrapped drug from the liposomes.
It was observed that the Batch GL 8 prepared with 1:4 (drug to lipid molar ratio) and 8:2 (HSPC to cholesterol molar ratio) had a highest percentage of drug entrapment and hence was considered as optimized batch.
2. Preparation of Gemcitabine based liposomal formulation comprising
Gemcitabine HCl, HSPC, Cholesterol and DGDG (hereinafter referred to as GDL liposomes)
The Liposomes were prepared by ethanol injection method, as described earlier.
2a) Optimization of Process and Formulation Parameters of preparing GDL
liposomes
Inferring from section 1 (i.e. the optimum temperature being 60°C, optimum time period for drug incubation being 60 min. the optimum drug:lipid ratio being 4:1 and the optimum ratio between HSPC: Cholesterol in the lipid being 8:2). Gemcitabine
based liposomal formulation comprising gemcitabine, DGDG, HSPC & cholesterol is prepared by adopting the process as shown in Figure 1.
In the process as illustrated in Figure 1, DGDG was added at different concentrations as given in Table 5.
Table 5: Optimization of Formulation parameters for preparing GDL liposomes
(Table Removed)
GDL2 prepared with 10% DGDG by weight of the total lipids. Its drug entrapment was found 61.2 ± 2.1 % and Liposomal size of 167.0 ± 3.7 nm was considered as optimized batch after comparisons with results of other batches.
3. Characterization Of Liposomes
Physical and chemical characterization of liposomes is very important for a
meaningful comparison of different liposome preparation or different batches prepared according to the same protocols. Liposomes characterization should be performed immediately after preparation. One should also ensure that no major changes occur on storage so that a well characterized product is injected and the liposomes dispersion warrants optimal reproducibility of clinical effects. Characterization
The prepared GDL liposomes containing Gemcitabine IIC1 was characterized for following attributes.
3a) Size
The mean particle size of the prepared liposomes was obtained by using Zeta Sizer ZS, Malvern UK. Diluted liposome suspension was observed for the average particle size. The mean particle size of GDL liposome was 167nm. The same is illustrated in Figure 2.
3b) Zeta potential
Zeta potential was measured with a Zeta-nano particle electrophoresis analyzer setup equipped with a 5-mV He-Ne laser (633nm). The sample was suitably diluted 5 times with filtered distilled water and placed in a small disposable zeta cell. Zeta limits ranged from -200 to +200mV. The electrophoretic mobility (µm/sec) was converted to zeta potential by in-built software using Helmholtz-Smoluchowski equation. Average of 30 measurement of the sample was used to derive average zeta potential. The zeta potential was observed to be 24.5. The same is illustrated in Figure 3.
3c) Morphology and Lamellarity
Morphology and lamellarity of the liposomes was determined using Cryo-Transmission Electron Microscopy (Tecnai G2 Spirit BioTwin -120KV, FEI Company).
Cryo-TEM images were taken at magnification of 49000 at observation temperature of-175 ± 2°C with electron source from Tungsten filament. The images show clearly the single lamely of liposomes. Lamely size was found 4-6nm. Dark circular structures appeared in the image was ice. Circular structured liposomes were observed.
3d) % Drug Entrapment
The optimized liposomal formulations were analyzed for % drug entrapped using the method elaborated in section 1(c). The % drug entrapment of GDL liposomes was found to be 61.2%.
4a) Diffusion study characteristics (In-Vitro Drug release)
Studies of drug release / diffusion from any type of system are directed towards issues that are relevant to the in vivo as well as to the non in vivo arenas. The in vivo arena, the drug release studies are expected to yield data and understanding that will lead to:
1. Minimizing the loss of encapsulated drug on route from the site of
administration to the site of drug action.
2. The ability to match the rate of release to the requirements of therapy.
The objectives of drug release studies that concern the non in vivo arena are
1. Various aspects of the system optimization such as the selection of liposome type, lipid composition and parameters of shelf life.
2. Criteria for quality assurance.
3. Physiological characterization of the systems.
• Reagents
Disodium hydrogen phosphate, Potassium dihydrogen phosphate and Sodium chloride of analytical reagent grade procured from S.D fine chemicals Ltd.. Boiser.
• Solutions
Phosphate buffer saline, pH 6.8 (PBS) was prepared as per procedure given in the Indian Pharmacopoeia 1996.
• Apparatus
Magnetic stirrer (Remi scientific equipments, Mumbai); Teflon coated bar magnet; open ended dialysis tubing made of cellulose (retains proteins with molecular weight greater than 12,000 Daltons) having flat width 35 mm and inflated diameter 21 mm (Sigma Diagnostics, USA).
• Preparation of dialysis sac and dialysis set up
A 10 cm long portion of the dialysis tubing was made into a dialysis sac by folding and tying up one end of the tubing with thread, taking care to ensure that there would be no leakage of the contents from the sac. The sac was then soaked overnight in PBS (pH-6.8). The wet sac was gently opened and washed copiously with PBS (pH-6.8). Then it was filled with PBS and examined for leaks. The sac was then emptied and 1 ml of liposomes solutions containing drug to be investigated was accurately transferred into the sac, which thus became the donor compartment. The sac was once again examined for any leaks and then was suspended in a glass beaker containing 200 ml of PBS (pH-6.8), which acted as a receptor compartment. The contents of the beaker were stirred using Teflon coated bar magnet and the beaker was closed with the aluminium foil to prevent any evaporative losses during the experiment run.
• Sampling
At predetermined intervals of time, 5 ml aliquots were withdrawn from the receptor compartment and subjected to analysis. Fresh buffer was used to replenish the receptor compartment. Analysis was carried out immediately after withdrawal. Study was carried out for 6 hr. A comparative diffusion analysis between GL and GDL liposomes is illustrated in Table 6. Table 6 Diffusion study for liposomes containing Gemcitabine HCI
(Table Removed)
4b) Stability study characteristics
A comparative analysis regarding stability was performed between GL and GDL liposomes.
The GL and GDL liposomes suspension was diluted with distilled water containing suitable amount of sucrose. This suspension was then freezed using nitrogen and lyophili/ed for 24 hours. The lipid to cryoprotectant ratio was optimized based on the drug retention capacity of the rehydrated lyophilized powder (1:3 is the optimized ratio). The lyophilized samples were then subjected to stability study in triplicate, at the conditions according to ICH guidelines i.e. 2-8°C with ambient humidity and 30±2°C/60± 5% RH after storing in a sealed glass vials. The lyophilized samples were withdrawn from the vials at the interval of one month for the period three months, rehydrated for the analysis of the size distribution and % drug loss. The results are shown in Table 7 and 8.
Table 7; Percentage drug loss from the rehydrated lyophilized liposomal formulation after storing at different temperatures for a period of 3 months.
(Table Removed)
Table 8: Particle size of the rehydrated lyophilized liposomal formulation after storing at different temperatures for a period of 3 months.
(Table Removed)
4c) In vivo study data
• Experimental design
Entire in-vivo study was carried out by dividing the animals into two groups of 3 male rats each. The rats were injected with GL and GDL liposomes. After 1 hr, 5 hrs and 24 hours animals were sacrificed and the organs collected for analysis:
• Animal's group distribution
Group 1: Animals injected with GL liposome. Group 2: Animals injected with GDL liposome.
• Administration of formulation
Both GL and GDL liposomal formulation equivalent to 2.5 mg of drug were administered. Liposomal formulation was prepared by dispersing liposomes in
phosphate buffer saline. Before administration of formulations, they were sterilized by filtration sterilization. Liposomal formulation was first filtered through 0.45 urn filter (Pall filters, NY, USA). The filtrate obtained was further filtered through 0.22 µm membrane filter. It was then filled in aseptic vial and used for further purpose.
Intravenous administration was done by tail vein method wherein the 0.5 ml drug solution (5mg/ml) was injected intravenously into the tail vein of the rats. Subsequently animals were sacrificed by cervical dislocation at different time intervals (lhr, 5hrs and 24hrs) and their organs (Kidney, spleen, liver) were removed and homogenized in TRIS-HC1 buffer. The homogenate produced was then centrifuged at 4000 rpm for 15 minutes at 4°C. The supernatant was then taken for analysis. 20 uL of sample was injected into HPLC. The organ distribution of GL and GDL liposomes after 1, 5 and 24 hrs of intravenous injection in rat are shown in fable 9. Various organs / tissues like liver, spleen and kidney were removed and analyzed for drug content.
Table 9: %Drug present in liver, kidney, and spleen after intravenous administration of drug solution and liposomal formulations (Dose-2.5 mg)
(Table Removed)
5. Results and Discussion
In the present invention an attempt was made to prepare DGDG containing liposomes of Gemcitabine HC1 for hepatocyte targeting. The liposomes were prepared by Ethanol Injection technique using various parameters like Drug to Lipid ratio, IISPC: Cholesterol ratio. Hydration volume, Hydration time, stirring speed, drug incubation time and temperature, as a means for increasing the efficacy. The liposomes were characterized for parameters like morphology and lamallarity. size, % drug entrapment, stability etc. The liposomes were spherical in shape confirmed by Cryo-TEM image and having a uni-lamallarity.
Comparative Diffusion study results
The diffusion study of both GL and GDL liposomes was performed in PBS (pH-6.8), and results show that there was burst release in the Gemcitabine release profile of both the formulations during the first 30 min. This finding was probably due to a rapid desorption of Gemcitabine from external liposomal bilayers. The significant higher burst release was observed for the GL Liposome, which were due to more permeability to Gemcitabine than the GDL liposomes. The burst release of drug depends on the two factors:
• Interaction between the drug and lipids - Higher the interaction, lower the burst release.
• Fluidity of the bilayers - higher the Fluidity , greater and rapid drug leakage from the liposomes
Thus, from the above discussion we conclude that the Gemcitabine is better interacted with GDL Liposomal composition and gives less fluidity to the bilayers. A slow release from DGDG liposomes as compare to phosphatidylcholine liposomes is because liposomes made from DGDG is more stable - both physically and chemically, than made from phospholipids. This is because of: 1) Well balanced
hydrophilic- lipophilic property of DGDG. 2) Also P=O group phosphatidylcholine show strong interactions (H-bonding) between sugar OH groups of DGDG.
Comparative Stability study results
The stability study of lyophilized liposomes were carried out at conditions as per ICH guidelines. The liposomes were stable for 3 months which was confirmed by observing particle size and % drug entrapment of liposomes at predetermined intervals. The GL liposome was found to be stable at 2-8°C with liposomal size 170.1nm. The GDL liposome was found to be stable at 2-8(C with liposomal size 180.2nm. At 30+ 2 C and 60+5% RH results shows that moderate increase in particle size of 179.9nm and 189.6nm for GL and GDL liposomes, respectively. % drug entrapment was found decreasing by 5-7% at 30+2°C at 60+5 °C RH storage condition.
Comparative Invivo study results
The comparative invivo study results show that liver and spleen accumulated a major portion of the administered dose as they are the two major organs of reticuloendothelial system (RES) which are known to accumulate and metabolize liposomes. The biodistribution data reveals higher initial rapid uptake by liver, which was 41.6%) and 59.7% for GL and GDL after lhr of post injection respectively. GDL higher uptake was due to the liposome composition, as these liposomes were composed of mixture of phospholipids containing DGDG as one of the phospholipid. Drug content in liver after 5 and 24 hrs was 36.8% and 29.3% for GL and 55.2% and 43.6% for GDL indicating higher retention of GDL in liver. The possible reason for higher retention of GDL would be because of higher cellular uptake of these DGDG liposomes. Also, higher accumulation of GDL liposomes in liver after 24 hrs makes it more effective for site specific (hepatocytes) delivery and thus will show better therapeutic effect.
Irrespective of the correctness of the possible reasons given above, the higher value of drug in liver with GDL liposomes thus observed is a clear-cut indication of synergistic unexpected improved efficacy.
SECTION-II
In this section the Gemcitabine liposomal formulation comprising Monomethoxy polyethylene glycol 2000-distearoylphosphatidylethanolamine (mPEG-DSPE) / folate- polyethylene glycol- distearoylphosphatidylethanolamine (F-PEG-DSPE), Hydrogenated Soy Phosphotidyl Choline (HSPC), cholesterol and folic acid as the lipid mixture loaded with Gemcitabine is described in detail along with its process for preparation.
1. Preparation of gemcitabine based liposomal formulation comprising gemcitabine HC1, HSPC and Cholesterol (hereinafter referred to as conventional or CL liposomes).
Liposomes were prepared by thin film hydration method. The lipid compositions comprising gemcitabine HC1, HSPC and Cholesterol were dissolved in CHCl3: methanol (2:1) and dried into a thin film by rotary evaporation and then further dried under vacuum. The lipid film was hydrated with 200mM ammonium sulfate ((NH4)2S04) for 50 min at 60°C with vortex mixing. The liposomal suspension was then probesonicated (2x2minx0.6cyclex80amplitude) using a serotorius probsonicator to produce small unilamellar vesicles (SUVs). The un-entrapped (NH4)2S04 outside of the liposomes was removed by centrifugation method by using HEPES buffer at 25000RPM 4°C at 30 mm, 3 cycles. The mean diameter of the liposomes was determined by dynamic light scattering using malvern mastersizer. Gemcitabine HC1 was loaded into the liposomes by a transmembrane pH gradient method (as described in 1(a) of section I).
HSPC and Cholesterol at different molar ratios were used for preparing lipid mixture and upon the lipid mixture thus obtained, gemcitabine at different (gemcitabine:lipid) ratio was loaded. It was observed that when the gemcitabine : lipid ratio is 1:4 and when the lipid mixture comprises HSPC, Cholesterol at a ratio of 8:2, maximum drug loading occurs as illustrated in Table 10.
Table 10 Optimization of drug : lipid and lipid: cholesterol ratio in CL
(Table Removed)
* Mean±SD (No. 3)
2. Preparation of gemcitabine based liposomal formulation comprising gemcitabine HC1, HSPC, Cholesterol and m-PEG DSPE (hereinafter referred to as SL liposomes)
Liposomes were prepared by thin film hydration method. The lipid compositions comprising gemcitabine HCI, HSPC, Cholesterol and m-PEG DSPE were dissolved in CHCl3: methanol (2:1) and dried into a thin film by rotary evaporation and then further dried under vacuum. The lipid film was hydrated with 200mM ammonium sulfate ((NH4)2SO4) for 50 min at 60°C with vortex mixing. The liposomal suspension was then probesonicated (2×2minx0.6cycle×80amplitude) using a
serotorius probsonicator to produce small unilamellar vesicles (SUVs). The un-entrapped (NH4)2S04 outside of the liposomes was removed by centrifugation method by using HEPES buffer at 25000RPM, 4°C at 30 min, 3 cycles. The mean diameter of the liposomes was determined by dynamic light scattering using malvern mastersizer. Gemcitabine HC1 was loaded into the liposomes by a transmembrane pH gradient method (as described in 1(a) of section I).
mPEG-DSPE, HSPC, Cholesterol at different molar ratios were used for preparing lipid mixture and upon the lipid mixture thus obtained gemcitabine at different (gemcitabine:lipid) ratio was loaded. It was observed that when the gemcitabine : lipid ratio is 1:4 and when the lipid mixture comprises mPEG-DSPE, HSPC, Cholesterol at a ratio of 7.5:2:0.5, maximum drug loading occurs as illustrated in Table 11.
Table 11 Optimization of drug : lipid and lipid mix ratio in SL
(Table Removed)
* Mean±SD (No. 3)
3. Preparation of gemcitabine based liposomal formulation comprising gemcitabine HC1, HSPC, Cholesterol and Folate-PEG DSPE (hereinafter referred to as folate-targeted or FT liposomes)
(i) Synthesis of folate-polyethylene glycol-distearoylphosphatidylethanolamine (F-PEG-DSPE)
Firstly, NHS ester of FA, folate-PEG-amine and N-Succinyl DSPE were synthesized by methods described previously by Stephenson and Kempen. This was followed by the synthesis of folate-PEG-DSPE by reacting folate-PEG-amine with N-Succinyl DSPE. Briefly, for synthesis of NHS ester of FA. FA is dissolved in DMSO. 2.5mL of triethylamine. A 1.1 molar excess of NHS (2.6g) and DCC (4.7g) is added the mixture is stirred overnight at room temperature in dark. Folate-PEG-bis-amine. PEG-bis-amine (500 mg) were dissolved in 2mL DMSO with 1.1M excess of NHS folate (88.3mg) and the reaction was allowed to proceed overnight at room temperature. The product folate-PEG-amine was then purified by Sephadex G-25 gel-filtration chromatography. For synthesis of Second, to synthesize an N-Succinyl 100mg DSPE dissolved in anhydrous 5 mL chloroform (CHC13). 10µl. pyridine was reacted with 1.1M excess of 14.7mg of succinic anhydride the mixture was incubated overnight at room temperature. Finally, to synthesize folate-PEG-DSPE, the carboxyl group of N-succinyl DSPE is activated by reacting with 1M equivalent of DCC for 4 hrs at RT. An equimolar quantity of folate-PEG-amine dissolved in CHC13 is added and the mixture is allowed to react overnight at RT. The solvent was then removed on a rotary evaporator and the product is washed twice with cold acetone. The identity of the product was confirmed by thin-layer chromatography (TLC) and FTIR Studies.
(ii) Preparation of gemcitabine loaded FT liposome
Lipid composition comprising Folate-PEG-DSPE, HSPC, Cholesterol at different molar ratios were used for preparing liposomes using thin film hydration method. Upon the prepared liposome, Gemcitabine HCl was loaded by transmembrane pH gradient method (as described in 1(a) of section I). It was observed that when the gemcitabine : lipid ratio is 1:4 and when the lipid mixture comprises folate-PEG-DSPE, HSPC, Cholesterol at a ratio of 7.75:2:0.25, maximum drug loading occurs as illustrated in Table 12.
Table 12 Optimization of drug:lipid and lipid mixture ratio in folate targeted liposomes (FT)
(Table Removed)
Mean±SD (No. 3)
4. Optimization of Process parameters
For preparing CL, SL and FT liposomes, (as described in sub-sections 1, 2 & 3) following process parameters were optimized.
a) Rotation Speed and vacuum
The effect of rpm and vacuum on the quality of film formed was evaluated by determining quality of film formed at different rpm and vacuum conditions. These parameters were optimized for formation of smooth film with complete removal of the solvent residue. The presence of residual solvent may lead to physical destabilization of liposomes by interfering with the co-operative hydrophobic interactions among the phospholipids methylene groups, which hold the structure together. The optimization chart is shown in table 13. Table 13 Optimization of RPM, vacuum, time
(Table Removed)
b) Hydration Time
The film was hydrated for different time intervals between 40 to 80 min, and was evaluated for complete hydration of lipid film with maximum entrapment and uniform size. The results reveal that after 40 minutes the film was not properly hydrated, some portion of lipid was still remained unhydrated on the surface of the RBF; at 60 minutes the film was completely hydrated and gives homogenous suspension of liposome with optimized entrapment efficiency. Optimized hydration time is shown in table 14. Table 14 Optimization of Hydration Time
(Table Removed)
c) Sonication cycles
Sonication is important to convert multilamellar vesicles (MLVs) to small lamellar vesicles (SUVs) for drug delivery system, by using probesonicator the size will be optimized by using different parameters like amplitude, time cycle etc. the optimization chart is shown in table 15.
Table 15 Optimization of Sonication
(Table Removed)
Mean±SD (No. 3)
5. Characterization of liposomes
(a) Particle size analysis
The particle size (z-average) and polydispersity index (PDI) of the liposomes was analyzed by photon correlation spectroscopy (PCS) using a Malvern Zetasizer Nano (Malvern Instruments; UK). 0.2 mL of liposomes suspension was diluted to 1.0 mL with DW and measured after an equilibration time of 2 minutes. The particle size analysis results of liposomal formulation are shown in figure 6 & 7 and table 16.
(b) /eta potential analysis
The zeta potential (q potential) of the various liposome suspension prepared was measured by microelectrophoresis using Malvern Zetasizer Nano ZS (Malvern. Instrument, U.K.). Zeta potential of the liposome was measured after separation of the free drug from the liposome. 0.2 mL of liposome was diluted to 1 mL of DW. The determination of the zeta potential was realized at 25°C after injecting 1 mL of
the sample into a standard sample cell. The zeta potential data results of formulations
are shown in figure 8 & 9 and table 16.
Table 16 Particle size and Zeta potential analysis of CL, SL and FT formulations
(Table Removed)
* Mean±SD (No. 3)
(c) Morphology
Morphological evaluation was conducted using Optical microscope with polarizer BX 40. Olympus Optical Co. Ltd., at a magnification of 40X. The photographs of MLVs are shown in figure 10.
(d) TEM
TEM images were taken using Cryo-Transmission Electron Microscope. 5 µL of dilute liposome dispersion was placed on a 200-mesh formvar copper grid (TAAB Laboratories Equipment, Berks, UK), allowed to absorb, and the surplus was removed by filter paper , then stained in 2.5 % uranyl acetate for 30 seconds and dried. Then the surplus was removed, and the sample was dried at room conditions before imaging the liposome with a transmission electron microscope operating at an acceleration voltage of 200 KV. The TEM images of the prepared SUVs are shown in figure 11.
6. In vitro diffusion studies
Phosphate buffer solution (PBS) at pH 7.4 was selected as the release medium. Liposomal suspension transferred into a dialysis membrane (MW cut-off: 15000 Da). The dialysis bag was placed in 50mL PBS (pH 7.4). The release study was
performed at 37°C in magnetic stirrer. At selected time intervals. 5mL buffered solution from the receptor compartment was removed and replaced with 5mL fresh buffer solution. Gemcitabine concentration was estimated in the sample by UV-visible spectrophotometry method based on the absorbance intensity at 268 nm. In vitro diffusion profile is given in figure 12.
Reduction in release from the FT and SL liposomes in comparison to CL liposome occurred because the linkers incorporated in previous formulations retained drug in the bilayer by making it more rigid. The slower and continuous release may be attributed to slow trans-layer permeation kinetics and diffusion from the interior. It has been shown that the aqueous medium slowly penetrates the internal structure of the particles and causes progressive degradation of the polymer chains.
7. In vivo studies
Radiolabeled liposome have been successfully used for preclinical evaluation of pharmacokinetic parameters of liposomal delivery system as this can be administered to animals by different routes and its uptake in various organs can be estimated with time.
Liposomes can be labeled using various isotopes among them Technetium 99m (99mTc) is used due to its easy availability from Molybdenum-99 (99Mo) -99mTc generator and its favorable physical characteristics such as convenient short half life of (6 hrs), 140KeV gamma energy which is ideal for detection with current instruments.
• Biodistribution Studies
The Social Justice and Empowerment Committee. Ministry of Government of India, approved all animal experiments were conducted for the purpose of control and supervision on animals and experiments. Swiss mice (aged 4 to 5 months), weighing
between 20 to 25 g. Swiss mice bearing desire size tumor (~1 to 1.5cm), and without tumor were selected for the study. Three mice for each formulation per time point were used in this study. Radiolabeled complex of 99mTc-formulations of 100 µL of radiolabeled complex of 99mTc-solution was injected through tail vein of the mice. Blood was withdrawn by cardiac puncture after different time interval and the mice were sacrificed by cervical dislocation. The blood was weighed and the radioactivity present in the whole blood was calculated by keeping 7.3% of the body weight as the total blood weight. Major organs (heart, liver, spleen, kidney, lungs, tumor, intestine, stomach, brain and carcass) were isolated weighed and radioactivity present in each tissue/organ was measured using shielded well-type gamma scintillation counter. Radiopharmaceutical uptake per gram in each tissue/organ was calculated as a fraction of administered dose using equation:
(Equation Removed)
The pharmacokinetic parameters, like half life, Area under the curve (AUC). liver /blood ratio, liver/tumor, tumor/carcass in all time points were calculated. The area under the % radio activity per gram of tissue vs. time curve from zero to 24 hour (AUC) was calculated by standard trapezoidal rule. Terminal elimination rate constant ((3) for drug following intravenous administration was obtained by linear regression analysis of the terminal log-linear portion of % radio activity per gram of tissue vs. time curve. The corresponding half life of drug was calculated using the relationship 0.693/(3. The 0th time concentration followed by IV route was calculated by interpolation of terminal elimination curve to the Y axis. Blood pharmacokinetic parameters of CL, SL and FT are compared in table 17.
Table 17 Plasma Pharmacokinetics of 99mTc - CL/FT in Swiss mice
(Table Removed)
99mTc labeled CL and FT was injected via tail vein in tumor bearing Swiss mice. Animals were sacrificed 1 and 24 hrs post injection, all major organs and tumor was excised, rinsed in saline, wiped dry, weighed. Blood was withdrawn just before sacrificing the animal. Radioactivity associated with organs as well blood was measured in solid scintillation counter. Percentage of injected dose associated with per gram of tissue or organ was calculated. The values represented in Figure 13 are the mean of three values for each time point with ± standard deviation. Results of bio-distribution studies of FT were compared with that of plain liposomes in order to assess the safety of FT with respect to the general toxicity of CL on RES organs (Table 18 and Figure 13).
Table 18 Biodistribution of 99mTc labeled SL and FT Liposomes in tumor bearing mice
(Table Removed)
• Biodistribution in Tumor
Uptake of radio labeled CL and FT liposomes were compared in tumor (Ehrlich Carcinoma) induced mice after 1 and 24 hrs of injection. The relevance of folate receptor as a useful target for tumor-specific drug delivery is supported by findings indicating up-regulation (higher expression) in many human cancers including those of the ovary, brain, kidney, lung, breast, and myeloid cells. In addition, aggressive or undifferentiated tumors with advanced stage or grade appear to have an increased folate receptor density suggesting that folate receptor mediated delivery may be a broad approach in targeting tumor cells. Tumor distribution of CL and FT in tumor bearing mice after intravenous injection is represented in figure 14. Tumor was isolated after 1 and 24 hrs of post injection and estimated for the radioactivity. Radioactivity is expressed as percent of administered dose per gram of tissue or organ. The values represented in figure 14 are mean of three mice with iSD.
8. Results and Discussion
Microscopic study revealed that the prepared liposomes by TFH method were spherical in nature and were MLVs before sonication. Sonication produced SUVs as depicted by TF.M images. The % drug entrapment efficiency of optimized SL liposome is lesser than the optimized FT liposomes.
Reduction in the release of gemcitabine from the FT and SL in comparison to CL liposomes occurred because the linkers incorporated in previous formulations retained drug in the bilayer by making it more rigid. In addition to the above, it can also be noticed from the invitro study that the drug release is more sustained with FT liposomes. The slower and continuous release may be attributed to slow trans-layer permeation kinetics and diffusion from the interior. It has been shown that the aqueous medium slowly penetrates the internal structure of the liposomes and causes progressive degradation of the polymer chains.
In vivo studies
No significant difference was observed in the blood pharmacokinetic parameters between CL and SL while increase in all parameters (particularly MRT) was observed in case of FT confirming over all increase in efficacy. Compared to CL liposome, Folate targeted liposome (FT) showed much lower distribution in vital organs like lungs, liver and kidney confirming the reduced toxicity by folate-PEG conjugation. This is due to synergistic effect of decreased uptake by RES as a result of PEGylation and tumor targeting due to enhanced permeability and retention (EPR) effect as well as folate receptor mediated tumor targeting. These results are further confirmed by drug tumor level studies where, the tumor uptake of FT was 2.49 and 0.28 % and for CL was 0.25 and 0.18 % of the administered dose after 1 and 24 hrs respectively. Tumor uptake of FT was higher compared to CL at 1 hr and remained higher even up to 24 hrs.

CLAIMS
We Claim:
1. A Gemcitabine based liposomal formulation for treatment of cancer, said
formulation comprising:
a) a lipid mixture comprising 'X\ Hydrogenated soy phosphatidylcholine and Cholesterol and optionally a folate ligand; and
b) Gemcitabine;
characterized in that:
• 'X' is Digalactosyl diacylglycerol
or Distearoylphosphatidylethanolamine;
• the molar ratio of Gemcitabine: lipid mixture is in the range of 1:4 to 1:6.

2. The formulation as claimed in claim 1, wherein lipid mixture comprises Digalactosyl diacylglycerol, Hydrogenated soy phosphatidylcholine and cholesterol.
3. The formulation as claimed in claim 2, wherein molar ratio of Hydrogenated soy phosphatidylcholine : cholesterol in the lipid mixture is in the range of 8 :
1 to 8 : 3.
4. The formulation as claimed in claim 2, wherein the lipid mixture comprises 5 to 12% of Digalactosyl diacylglycerol, the % being based on total weight of lipid mixture.
5. The formulation as claimed in any of the preceeding claims, wherein the molar ratio of Digalactosyl diacylglycerol, Hydrogenated soy phosphatidylcholine and Cholesterol is about 1 : 9.5 : 2.75.
6. The formulation as claimed in claim 1, wherein Gemcitabine is present in the formulation as Gemcitabine Hydrochloride.
7. The formulation as claimed in claim 1, wherein the lipid mixture comprises Folate Polyethylene glycol - Distearoylphosphatidylethanolamine, Hydrogenated soy phosphatidylcholine and Cholesterol.
8. The formulation as claimed in claim 7. wherein the molar ratio of Folate-Polyethylene glycol - Distearoylphosphatidylethanolamine. Hydrogenated soy phosphatidylcholine and Cholesterol is about 0.25:7.25:2.0.
9. A process for preparing Gemcitabine based liposomal formulation as claimed in claim 1, said process comprising the steps of:

(a) preparing the lipid mixture comprising 'X’ Hydrogenated soy phosphatidylcholine, Cholesterol and optionally a folate ligand by conventional techniques; and
(b) loading of Gemcitabine into the lipid mixture thus obtained in step (a) by transmembrane ammonium sulphate gradient method;
characterized in that:
• 'X' is Digalactosyl diacylglycerol or
Distearoylphosphatidylethanolamine; and
• the molar ratio of Gemcitabine : lipid mixture is in the range of 1 : 4 to
1:6.
10. The process as claimed in claim 9. wherein in step (b), loading of Gemcitabine into the lipid mixture was performed at temperature in the range of 50-65 degree Celsius and for time period in the range of 50-80 minutes.
11. The process as claimed in claim 9, wherein in step (a), 'X', Hydrogenated soy phosphatidylcholine and cholesterol are dissolved in ethanol.
12. The process as claimed in claim 9, wherein lipid mixture comprises Digalactosyl diacylglycerol, Hydrogenated soy phosphatidylcholine and cholesterol.
13. The process as claimed in claim 9, wherein molar ratio of Hydrogenated soy phosphatidylcholine : cholesterol in the lipid mixture is in the range of 8 : 1 to 8: 3.
14. The process as claimed in claim 9, wherein the lipid mixture comprises 5 to 12% of Digalactosyl diacylglycerol, the % being based on total weight of lipid mixture.
15. The process as claimed in any of claims 9 to 14. wherein the molar ratio of Digalactosyl diacylglycerol, Hydrogenated soy phosphatidylcholine and Cholesterol is about 1 : 9.5 : 2.75.
16. The process as claimed in claim 9, wherein Gemcitabine is present in the formulation as Gemcitabine Hydrochloride.
17. The process as claimed in claim 9, wherein the lipid mixture comprises folate- Polyethylene glycol - Distearoylphosphatidylethanolamine, Hydrogenated soy phosphatidylcholine and Cholesterol.
18. The process as claimed in claim 9, wherein the molar ratio of Folate-Polyethylene glycol - Distearoylphosphatidylethanolamine. Hydrogenated soy phosphatidylcholine and Cholesterol is about 0.25:7.25:2.0
19. A Gemcitabine based liposomal formulation for treatment of cancer, substantially as herein described with regard to the foregoing detailed description and drawings.
20. A process for preparing Gemcitabine based liposomal formulation for
treatment of cancer, substantially as herein described with regard to the
foregoing detailed description and drawings.