Effect of Liposomes as a carrier on Pharmacokinetics of Cisplatin

 

Ramandeep Singh¹*, Ashutosh Upadhayay¹, M. K. Kale²

 

ABSTRACT:

Cisplatin is an alkylating agent which is used to treat a number of cancers. It is generally used in the treatment of advanced bladder cancers, liver cancer, metastatic ovarian cancers, and metastatic testicular cancer. It is also used in other cancers like, testicular, ovarian, bladder, head and neck, esophageal, small and non-small cell lung, breast, cervical, stomach and prostate cancers. However, apart from its usage in variety of cancers it has side effects too. The major ones are kidney toxicity and Ototoxicity (hearing loss). However, these side effects can be reduced by using novel carriers like liposomes, which may affect its kinetics and enhance its half-life too.

 

 

 

INTRODUCTION:

Cancer (medical term: malignant neoplasm) is a class of disease in which a group of cells display uncontrolled growth, invasion that intrudes upon and destroys adjacent tissues, and sometimes metastasis, or spreading to other locations in the body via lymph or blood. These three malignant properties of cancers differentiate them from benign tumors, which do not invade or metastasize.¹

 

Drug delivery remains a challenge in management of cancer. Approximately 12.5 million new cases of cancer are being diagnosed worldwide each year and considerable research is in progress for drug discovery for cancer. Cancer drug delivery is no longer simply wrapping up cancer drugs in new formulations 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 damage to normal tissues and prevent drug resistance.

 

Liposomes form excellent drug delivery system for anticancer drugs. Efficient encapsulation of active agent into liposome can enhance selectivity of drug and increase its therapeutic index through improved bioavailability, reduced systemic and organ toxicity or longer half time of circulation.²

 

Liposomes are composite structures made of phospholipids and may contain small amounts of other molecules. Liposomes are known to improve the therapeutic index of a variety of drugs. Intravenously administered liposomes generally undergo extensive opsonization and therefore get rapidly cleared by macrophages of the mononuclear phagocyte system (MPS), particularly Kupffer cells in the liver and spleen.[2-3] As a result, liposome targeting to pathological tissues becomes difficult. This drawback could be amended by coating liposomes with hydrophilic, neutral polymers such as polyethylene glycol (PEG). PEG increases the hydrophilicity of the liposome surface and provides a steric barrier against opsonization. The resulting long-circulating liposomes also referred to as ‘stealth’ or ‘sterically stabilized’ liposomes are still removed from the blood circulation however removal occurs at a much lower rate. Consequently, they circulate in the bloodstream for a prolonged period of time, enabling their extravasation into solid tumors and sites of inflammation by virtue of the presence of capillary discontinuities.[4-6] This so-called enhanced permeability and retention (EPR) effect allows for increased local drug concentrations in the target region.³

 

In the last two decades, many studies are focused on developing drug delivery systems to achieve controlled release or enable drug targeting to specific tumor sites. However, anticancer drugs accumulation in tumor tissue via Stealth Liposomes seems to be prerequisite but far from sufficient to guarantee a therapeutic improvement. PEG enables liposomes to accumulate in tumor tissue and creates a steric barrier that could cause a reduction in liposomes interaction with the target cells, leading to low uptake of the entrapped drugs via cell endocytosis or membrane fusion. In addition, various ligands or antibodies can be further attached to the surface-granted PEG chains, thus permitting them to be actively taken up by the target cells via receptor-mediated endocytosis.⁴

 

Cisplatin is a chemotherapeutic agent which was the first member of a class of platinum-containing anti-cancer drugs, which also includes carboplatin and oxaliplatin. These platinum complexes react in the body, binding to DNA and causing the DNA strands to crosslink, which ultimately triggers cells to die in a programmed way.⁵

 

Cisplatin was discovered in 1972. It is on the World Health Organization's List of Essential Medicines, the most important medications needed in a basic health system. Cisplatin is administered intravenously as short-term infusion in normal saline for treatment of solid malignancies. It is used to treat various types of cancers, including sarcomas, some carcinomas (e.g., small cell lung cancer, squamous cell carcinoma of the head and neck and ovarian cancer), lymphomas, bladder cancer, Liver cancer, cervical cancer, and germ cell tumors. Cisplatin is particularly effective against testicular cancer; the cure rate was improved from 10% to 85%.⁶

 

Cisplatin entrapped in liposomes shows reduced non-specific toxicity and maintains or enhances anticancer effect. Surface grafted methoxy Polyethylene Glycol (mPEG) provides the hydrophilic stealth coating, which allows Cisplatin liposomes to circulate in the blood stream for prolonged periods. The lipid matrix and an internal buffer system together keep the Cisplatin encapsulated inside the liposome during its circulation in the body. This means that the drug is not free to exert its toxic effects. Liposome association alters the drug pharmacokinetics and therefore gives it a higher half-life too, whereas the free drug distributes to the tissues within a few minutes and is entirely cleared from circulation within minutes.⁷

 

The present investigation deals with the effect of liposomes “as a carrier” for Cisplatin and study the effect of the same on pharmacokinetics of Cisplatin.

 

MATERIAL AND METHODS:

Chemicals and reagents:

Cisplatin was purchased from Aptuit Laurus (now Laurus labs), Hyderabad. Hydrogenated Soy Phosphatidylcholine (HSPC), Dipalmitoyl Phophatidyl Glycerol Sodium (DPPG sodium), •   N – (Carbonyl-methoxypolyethylene glycol 2000)- 1,2– distearoyl-sn-glycero-3-phoshoethanolamine Sodium Salt  (mPEG-DSPE-2000) and Cholesterol were purchased from NOF, Japan. Sucrose was purchased from Ferro Pfanstiehl Laboratories (now Pfanstiehl), USA.  Histidine was purchased from Ajinomoto, Japan. All the excipients along with the API were of Injectable grade and other reagents used in the study are of analytical reagent grade.

 

Instrumentation:

The proposed work was carried out on HPLC system consists of a Shimadzu pump (LC-2010CHT), a Shimadzu UV detector (CHT HPLC system, EM: 220 nm), Rotavapor from Buchi, Switzerland, Extruder from Northern Lipids, Canada and Tangential Flow Filtration (TFF) system from Millipore.

 

Selection of Solvents:

On the basis of solubility study ethanol was selected as the solvent for dissolving phospholipids and cholesterol.

 

METHODS:

Preparation of Liposomes:

Empty liposomes were prepared by dissolving mPEG-DSPE-2000, HSPC and cholesterol in appropriate amount of ethanol. This lipid solution was then poured in round bottom flask and was vacuum evaporated using Rotavapor from Buchi, Switzerland to get a uniform film. The Cisplatin was dissolved separately in 30% ethanol solution in water to get a clear solution. DPPG sodium was added to the above solution to get a viscous dispersion. The viscous drug dispersion was then added in the rotavapor flask by adding glass beads to get a uniform liposomal dispersion. The size of liposomal dispersion was reduced using extrusion process with the help of polycarbonate membranes of 0.4 µm, 0.2 µm and 0.1 µm to get a liposomal dispersion having an average size of 100 nm. The free drug was removed using sucrose buffer using a TFF assembly using appropriate size of cassettes. The temperature of TFF was 2-8^oC. The histidine solution (pH adjusted to 7.0 using 0.1N HCl) is added to the above dispersion as a buffer to get a pH of product around 6.5-7.5.

Characterization of Liposome:

The mean particle size, polydispersity index (PDI) and Zeta potential of Cisplatin loaded liposomes dispersion was measured by dynamic light scattering on a Malvern Nano-ZS, detected at an angle of 173° to the laser beam.[25] The particle size of liposomes governs the Pharmacokinetics (PK) of the drug  as large particles tend to get excreted out of the body at a much faster rate than smaller ones, on the other hand, very small particles will move in the body for a longer period of time which may cause toxicity. The PDI gives information on the particle size distribution; and ranges from 0 to 1. The value towards 0 generally denotes that the formulation is a monodispersed system whereas values close to 1 suggest that the formulation is polydispersed. Zeta Potential of a liposomal preparation helps in the determining the stability of the liposomes. Liposomes in aqueous dispersions have a tendency to aggregate and subsequently fuse on storage if they have lower zeta potential values. The values below and above zero generally indicates a stable system.

 

Evaluation of Free Drug content:

The entrapment of Cisplatin was determined by calculating the amount of unentrapped drug in the liposome. For determining the entrapment efficiency of Cisplatin loaded liposome, 4 ml of dispersion was transferred in Amicon Ultra-filtration tube from Millipore, USA (Make: Ultracel-100K). The dispersion was centrifuged for 45 min at 6000 RPM using centrifuge from Hettich Lab technology, Germany. The filtrate was collected and analyzed for free drug content using High Performance Liquid Chromatrography.

 

The HPLC system consists of a Shimadzu pump (LC-2010CHT), a Shimadzu UV detector (CHT HPLC system, EM: 220 nm) and Hypersil APS-2 Amino group analytical column (250 × 4.6 mm, 10 μm). Acetonitrile 90% (v/v) and Water 50% (v/v) was used as a mobile phase maintaining the flow rate of 1.5 ml/min. The volume of Injection was 100µl and column temperature was set at 25°C. Standard stock solutions was prepared by dissolving 50 mg of working standard of Cisplatin in 100 ml volumetric flask and added 5 ml of Water: Methanol (1:1) mixture to dissolve the working standard and then make up the volume using methanol.

 

Acute Toxicity study:[28]

Both male and female Swiss albino mice were selected for toxicological studies. The mice selected were having a weight of 40-45 gm.  Animals were maintained according to accredited procedures. The liposomal dispersion was tested to determine the acute intravenous toxicity. Groups of mice of each sex were administered the liposomal dispersion, as a single acute dose, by intravenous route and were observed for the incidence of mortality and signs of intoxication for 15 days thereafter.

 

RESULTS:

Physicochemical properties of the liposomes:

The physicochemical properties like Particle Size, PDI and Zeta Potential of Cisplatin liposomal dispersion were determined as shown in Table 1. The average size of the liposomes produced by extrusion phenomenon is shown in Figure 1. The PDI of the liposomes prepared by extrusion method was less indicating that the liposomes particles produced by this technique are more uniform. The Zeta Potential values were on negative side indicating that the liposomes have some charge which will help in keeping the individual liposomal particles segregated throughout the shelf life. The report of the zeta potential is shown in Fig. 2

 

Table 1: Physicochemical characteristics of Cisplatin loaded liposome

Method	Particle size Average (nm)	Particle size (nm)			PDI	Zeta Potential
		D10	D50	D90
Extrusion	109.3	80.4	112	178	0.113	-8.54

 

 

Fig. 1: Particle size report of Cisplatin Liposomes produced by extrusion technique determined using Malvern Zetasizer

 

 

Fig. 2: Zeta Potential report of Cisplatin Liposomes produced by extrusion technique determined using Malvern Zetasizer

Encapsulation Efficiency:

The encapsulation efficiency of Cisplatin loaded inside the liposomes was around 60%. The encapsulation efficiency of the liposomes was checked using ultra filtration tubes having two chambers. The chamber in which sample was placed has a filter which does not allow the liposomes to pass but allows the solubilized free drug to pass through it. Cisplatin being a water soluble drug will pass through the filter whereas the drug which is in complex will not pass. The free drug content of the liposome was around 40%.

Photostability studies:

Cisplatin is known to be a photo-labile drug. However, the liposomes have tendency to increase the photostability of the drug as they encapsulate the drug inside and it is less exposed to the environment. To study this effect both drug and drug loaded liposomes were exposed to light and the assay of the drug before and after light exposure was checked. The results of the same are provided in the table -2.

 

From the data of table -2 it can be concluded that the drug does exhibit photo-labile nature when exposed to light. However, the cisplatin liposomes don’t show this phenomenon indicating that the liposomes are effective in enhancing the stability of the drug during light exposure. The light exposure doesn’t alter the physicochemical properties of the liposomes.

 

Acute toxicity studies:

The treatment of mice with Cisplatin loaded Liposomes from Lot No. CPL 9 at dose of 12 mg/kg resulted in death of three male mice on 5^th day of the study while no deaths were observed in the treated females. No clinical abnormalities were observed in any of the treated mice. The LD₅₀ of that group was estimated to be more than 12 mg/kg body weight.

 

Study design:

Groups of mice of each sex were administered CPL 9 formulation, as a single dose, by intravenous route and were observed for the incidence of mortality and signs of intoxication for 15 days thereafter. Based upon the finding of this comparative acute toxicity study in mice, it was observed that formulation CPL 9 induced slight loss of body weight in the surviving mice. The data of the same is provided in the table-3:

 

Pharmacokinetic studies in animals:

To study the effect of liposomes on kinetics of Cisplatin the formulation was injected in Sprague Dawley rats at a dose of 3 mg/ml. For comparison purpose, Cisplatin dissolved in water was also injected at same concentration. The blood samples were collected from retro-orbital veins of rats and were analyzed for drug concentration. The graph of the same is provided in fig.-3:

 

 

Fig 3: Pharmacokinetic behaviour of Cisplatin liposomes in rats

 

From the above graph it can be seen that the drug exhibited two compartmental body model in both free form and encapsulated form. The drug in liposomal form had higher residence time as compared to free form indicating that the use of mPEG-DSPE 2000 in the formulation enhances the residence time of the drug vis-a-vis free drug which may ultimately decrease the dosing as well as the toxicity of the drug.

 

 

 

 

Table 2: Photostability data of Cisplatin Liposomes

Parameter	Initial	Cisplatin		Cisplatin Liposomes
		15 days control	15 days photo-exposed	15 days control	15 days photo-exposed
Assay (%)	100.2%	99.8%	89.6%	99.7%	100.1%
Particle Size (nm)	-	-	-	107	106
PDI	-	-	-	0.124	0.110
Zeta potential (mv)	-	-	-	-8.75	-6.90

 

Table 3:  Acute toxicity data in mice

Formulation	Dose (mg/kg)	Mortality
		Male		Female		Male and Female (pooled)
		Absolute	%	Absolute	%	Absolute	%
CPL 9	12	3/5	30	0/5	0	3/10	30

 

 

Organ toxicity Studies:

The organ toxicity studies were also carried out in rats to determine the concentration of drug in Spleen and liver. Cisplatin is used to treat liver cancer, however, the drug is found to have lesser penetration in liver and is found more in other organs. To verify the same the drug and liposomal formulation was administered at concentration of 3mg/kg. The graph highlighting the drug concentration is provided below:

 

Fig 4: Organ toxicity of Cisplatin liposomes in Liver and spleen

 

 

From the above graph it can be deduced that the drug has better penetration in Liver when administered in liposomal form as compared to Cisplatin alone, whereas, it is having lesser concentration in other organ, i.e., spleen which indicated that liposomal Cisplatin has more targeted drug delivery as compared to plain drug.  

 

DISCUSSION:

The average size of the liposomes prepared using extrusion is around 100 nm. The lower PDI value indicates that more uniform particles have been produced and it will directly affect the in-vivo performance of the drug as uniform particles will have longer residence time inside the body. The zeta potential value is on negative side indicating that the liposome particles will tend to repel each other during storage and therefore the formulation will remain stable during shelf-life. Cisplatin is known to be a light sensitive API and the same was highlighted when the photostability studies were conducted.  The drug encapsulated inside the liposomes was photostable which corroborates that the liposomes tend to affect the light sensitivity of the API thereby making it more stable to light exposure.

The entrapment efficiency of liposomes produced was close to 60% which will help in maintaining the plasma levels of the drug once the 40% of free drug provides the loading dose. The same was highlighted in PK studies too indicating that due to encapsulation the drug had higher residence time in rats as compared to drug alone.

LD₅₀ of Cisplatin liposomes was estimated to be greater than 12 mg/kg. This is another study which suggests that the Cisplatin encapsulated inside the liposome has lower toxicity as compared to Cisplatin API and therefore it might decrease the toxic effects of the drug and the same was highlighted in organ toxicity studies too.

 

CONCLUSIONS:

Cisplatin is an established and anticancer drug which is used to treat a variety of cancerous conditions. Unfortunately, the therapeutic application of the drug is severely restricted by its toxicity, especially kidney toxicity and ototoxiciy. The inclusion of this drug inside surface modified liposomes i.e. coated with polymers like PEG will help in decreasing the toxicity and will increase its circulation time thereby reducing its dosing intervals. The method of manufacture of these liposomes is extremely important as this can also significantly impact on performance of drug inside the body. In the above study liposomes were produced using film formation method and then size reduction was perfumed using extrusion process which ultimately affected the pharmacokinetics of the API. Therefore the particles produced will have better circulation time inside the body and will be less toxic.  The organ toxicity studies also highlights that the drug has more targeted delivery as compared to plain drug.

 

DECLARATION OF INTEREST:

The authors report no declarations of interest.

 

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Accepted on 20.10.2018           © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(11): 5073-5077.