INTRODUCTION:

Ketorolac is a non-steroidal anti-inflammatory drug with a potent analgesic and anti inflammatory activity due to prostaglandin related inhibitory effect of drug¹.It is a non selective cyclo oxygenase (COX) inhibitor. The drug is currently used orally and intra muscularly in multiple divided doses clinically for the management of cancer pain, post surgical pain and in the treatment of migraine pain^{1, 2, 3}. Ketorolac tromethamine is also used in the treatment of arthritis. An open label, uncontrolled ED study was conducted by shrestha et al in 1995 proved that intra muscularly administered ketorolac and oral indomethacin have similar effects on the pain of acute gouty arthiritis.⁴ So IM ketorolac is an alternate to the oral indomethacin if a parenteral medication is required.⁴ Ketorolac is one of the NSAIDs which is approved for parenteral administration.⁵

Another study showed that ketorolac is 36 times more potent than phenyl butazone, approximately twice as potential as indomethacin and three times more potent than naproxen in suppressing carrageenan induced paw edema in rat.⁶ It has been found through previous studies that analgesic efficacy of ketorolac is greater than that of other NSAID’s and that of Morphine in acute pain models.^{7, 8, 9} So ketorolac has certain advantages in comparison with opioid analgesic drugs that it is non sedating, it is not associated with pruritis, nausea, respiratory depression, urinary retention and it has opioid sparing qualities. Since the drug is a non selective cyclo oxygenase inhibitor of arachidonic acid with no increase of lepoxygenase pathway, the adverse effects associated with this is very much severe, and the drug is implicated as a contributing cause of increased post operative bleeding¹⁰, renal failure and gastritis.⁵The severity of these side effects is dose related.^{11, 12}ketorolac has short biological half life from 4- 6 hours, which necessitates frequent dosing to retain the action¹³.The frequent occurrence of gastro intestinal bleeding, perforation, peptic ulceration and renal failure lead to the development of other drug delivery strategies for the appropriate delivery of ketorolac. The ideal solution would be to target the drug only to the cells or tissues affected by the disease.¹⁴ Selected carriers such as liposomes, niosomes, micro and nanospheres, erythrocytes and polymeric and reverse micelles were studied, but by far the most widely studied approach makes use of liposomes.¹⁴ Their attraction presents in their composition, which makes them biodegradable and biocompatible.¹⁵ Liposomes consists of an aqueous core enveloped by one or more bilayers of natural or synthetic phospholipids.¹⁵ These are the carriers which are suitable for encapsulation of drugs with different lipophilicities such as strongly lipophillic drugs, strongly hydrophilic drugs and drugs with intermediate log P.¹⁵ Liposomes can protect the encapsulated drug or drugs and can target the organ or tissue passively.¹⁵

In the present investigation our main was to develop a simple vesicular delivery system for ketorolac tromethamine which can deliver the drug at a lower concentration over the prolonged period of time to the target site and thereby reducing the potential dose related side effects.

Method of preparation of CXB liposomes:^16,
17

KT multilamellar liposomal vesicles were prepared by using thin film hydration technique. Nine formulations were prepared by using SPC as a lipid component with or without cholesterol. Accurately weighed quantities of drug, SPC with or without cholesterol were transferred to 250ml round bottom flask and dissolved in solvent mixture of chloroform and methanol (2:1, v/v). Thin layer of lipid film was formed by evaporating the solvent system under reduced pressure using rotary evaporator (HS-3001 NS). During this process, the conditions of the instrument such as temperature (45±2ºC) and speed (150rpm) were kept constant. Residual solvents were removed by storing the thin film overnight in vacuum desiccator. Then the thin lipid film was hydrated with phosphate buffer saline [PBS] pH7.4 using vortex mixture about 2min to form MLVs. The suspension was allowed to stand at room temperature for an optimized period of 2h to achieve the complete swelling of the lipid film and to obtain the liposomal suspension. Then the suspension obtained was sonicated for 3min in ultrasonic homogenizer (ultrasonic 3000).

Incorporation of charged species:

Inclusion of negatively charged lipids such as dicetyl phosphate or positively charged lipid such as stearyl amine tend to increase the interlamellar distances between the successive bilayers in the MLV swelling the structure with the greatest proportion of the aqueous phase. These effects lead to greater overall entrapped volume. For this purpose two batches of liposomes were prepared to find out the influence of DCP and its content on %EE.

Fourier transform, infrared (FTIR) study:

All the excipents such as SPC, cholesterol individually, physical mixture of excipients, pure drug KT, physical mixture of excipients and drug were mixed separately with infrared (IR) grade KBr in the ratio of 1:100 and corresponding pellets were prepared by applying 15000 lb of pressure in a hydraulic press. The pellets were scanned in an inert atmosphere over a wave number range of 4000-400 cm^-1 in Magna IR 750 series II (Nicolet, USA) FTIR instrument.

Determination of percentage encapsulation efficiency:¹⁸

10ml of liposomal suspension were placed in centrifuge tubes and they were centrifuged at 4500rpm after balancing the tubes on the other side with equivalent weight. The centrifugation was continued for 10min. The supernatant was removed and the drug content of supernatant was analyzed. The pellet resulted was dissolved in methanol and it was diluted suitably and analyzed for the drug content.

Percentage encapsulation efficiency (EE %) = Amount of drug in pellet/ Total drug x 100

(EE%) was calculated for 3 formulations of each formulation code and average was tabulated. (Tab. 1)

Vesicle size distribution profile & microscopy:^{19, 20}

All the batches of KT loaded liposomes were examined for their morphological attributes using binocular compound microscope (optics) at suitable magnification. The batches containing non dispersed lipid film, drug precipitate or aggregates were detected and discarded. Scanning electron microscopic analysis was carried out on selected formulations for their morphology.

Stability analysis:¹⁷

The behavior of the liposome to retain the drug was studied by storing the liposome at 4 different temperature conditions, i.e., 4 - 8⁰ (refrigerator RF), 25±2⁰ (room temperature RT), 37±2⁰ and 45±2⁰ for a period of 1 month. The liposomal preparations were kept in sealed vials. Periodically samples were withdrawn and analyzed for the drug content following the same method described in % drug encapsulation efficiency. (Table 1)

In vitro drug release:²¹

Modified USP XXI dissolution rate model was used for the determination of drug release from liposomic preparation. This model consists of a beaker (250ml) and a plastic tube of diameter 17.5mm opened from both the ends. Sigma membrane (sigma 12000 MW cutoff) was tied at one end of the tube & the other end left free. This assembly was dipped in to the beaker containing 100ml of dissolution medium. The temperature was maintained at 37±1ºC. 10ml of liposomal suspension was added in to the tube and a paddle type stirrer was placed in the center of the beaker. The speed of the stirrer was maintained at 100rpm. Dissolution sample of 1ml was withdrawn periodically every one hour up to 24 hours and analyzed spectrophotometrically at 323nm. With the help of the standard curve prepared earlier, drug concentration was measured.

Preparation of ketorolac liposomes:

Various factors that influence the product such as vaccum, speed of rotation , hydration medium, and hydration time were studied in order to prepare liposome encapsulated CXB with desired qualities. Thickness and uniformity of lipid film were found to be influenced by rotational speed of the flask. The speed of 150rpm was found to be optimum, since the same resulted the uniform, thin film on the flask and responded the homogenous lipid vesicles after hydration. The lipid film was kept under vacuum overnight to remove the presence of residual solvents if any and to attain complete drying. Further this may avoid formation of emulsion which may result due to the presence of solvent residuals during hydration. Hydration of the lipid film was achieved in two minutes vortexing, as this was found to be optimum in obtaining the liposomes free from aggregation. Further it was found that percent drug entrapment was not affected by the process of vortexing, confirmed by drug entrapment studies done before and after vortexing. Nine formulations were prepared using varying drug – lipid ratio, with or without cholesterol and including charge inducing agents.

First four formulations KL1 to KL4 were prepared without cholesterol to study the effect of drug - lipid ratio on percent drug entrapment of the vesicles. It was found that encapsulation efficiency % (EE%) of liposomes was dependent on the drug - lipid ratio used in the preparation of the vesicles. Increasing amount of the KT from 5mg to 10mg increases the EE% considerably from 50.83 to 68.37 However the PDE value was found to be decreased with further increase in quantity of the drug used. In all the four formulations the amount of the lipid used was fixed i.e. 100mg and drug - lipid ratio of 1:10% w/w showed the greater drug entrapment. (Table1). Various techniques may be used to optimize the drug loading and this is very important in industrial settings. One method used to maximize the drug loading is to increase the cholesterol content in to vesicles. Three formulations were prepared to find out the influence of cholesterol on the EE% as well as on drug release. It was found that increasing amount of cholesterol increased the EE% form 70.13 – 75.84% where as further increase in cholesterol from 4:1(%w/w of SPC and cholesterol) to 2:1(%w/w of SPC and cholesterol) decreased the EE%. Presence of cholesterol was known to influence vesicle stability positively and permeability reversely.^{22, 23}This may be due to increased bilayer stability, while cholesterol content is increased. On the other hand, increasing cholesterol content above a certain limit decreases the drug entrapment. This may be due to the fact that the cholesterol beyond a certain limit may disrupt the regular linear structure of vesicular membrane. ²⁴ Thus the EE% of ketorolac is dependent on the cholesterol content of the liposomes.

Influence of charged species:

Surface potential plays an important role in the behavior of liposomes in vivo and in vitro. In general charged liposomes were found to be more stable against aggregation and fusion than uncharged liposomes as well as it increases the EE% since it tends to increase the interlamellar distances between the successive bilayers in the MLV, swelling the structure with the greatest proportion of the aqueous phase and hence lead to the greater overall entrapped volume. Two formulations were prepared to find out the influence of charged species such as dicetyl phosphate and stearyl amine on the EE% of ketorolac in to liposomes. It was observed that incorporation of DCP increased the PDE considerably from 75.84-78.24%,where as incorporation of stearylamine increased the PDE from 78.24-84.46%

Fourier transform, infrared (FTIR) study:

Drug, excipient interaction was studied before developing the formulation by using FTIR-spectroscopy, which is one of the most important analysis to describe about the stability of formulation, presence of drug & drug release.

Figure 1. FTIR Spectrum of Pure SPC

Figure 2. FTIR Spectrum of Pure cholesterol.

Figure 3. FTIR Spectrum of physical mixture of excipients.

Figure 4. FTIR Spectrum of Pure Ketorolac

Table 1: Composition and physicochemical properties of Ketorolac liposomes

S. No	Drug mg	SPC	CHOL	DCP	SA	Encapsulation Efficiency %	Drug Release %	Vesicle Size (µ)
KL1	5	100	---	---	---	50.83	85.12	3.83±0.3
KL2	7.5	100	---	---	---	58.43	86.88	4.32±0.4
KL3	10	100	---	---	---	68.37	88.64	4.45±0.3
KL4	15	100	---	---	---	45.49	88.25	4.17±0.8
KL5	10	100	12	---	---	70.13	78.24	4.52±0.7
KL6	10	100	24	---	---	75.84	76.48	4.69±0.5
KL7	10	100	50	---	---	68.8	84.37	4.84±0.6
KL8	10	100	24	17	---	78.24	65.6	4.95±0.5
KL9	10	100	24	---	17	84.46	63.32	5.21±0.7

Abbreviations used:SPC-Soy phosphatidyl choline, CHOL-Cholesterol, DCP-Dicetyl phosphate, SA-stearyl amine

Figure 5. FTIR Spectrum of Physical mixture of drug and excipients

Fig. 3 shows minor shifting of some peaks compared with individual excipients (Fig. 1 and Fig. 2), like aliphatic alcoholic O-H stretch (3422.91 to 3421.68), C-H stretch (2920.16 to 2918.71), carbonylic C=O stretch of ester (1738.98 to 1739.88), C-O stretch of ester (1237.74 to 1237.60). Minor shifts were observed when the fig. 5 compared with spectrum of pure drug (Fig. 4) and excipients (Fig. 1 and Fig. 2) like, aliphatic O-H stretch (3352.28-3379.55), aromatic C-H stretch (2873.88-2820.23), aromatic C=C stretch (1539.01-1559.03), O-H stretch of acid (1275.53-1243.91). These shifts observed may be due to the formation of hydrogen bonds, vanderwalls attractive forces or dipole moment which are weak forces seen in the polar functional groups of drug and excipients. The frequency of absorption due to the carbonyl group depends mainly on the force constant which in turn depends upon inductive effect, conjugative effect, field effect, stearic effects. The shifts seen due to the above mentioned interaction may however support the formation of favorable vesicle shape, structure with good stability and sustained drug release

Microscopy and Vesicle size distribution profile:

The vesicle size of the liposome was found to be in the range of 3.83±0.3 - 5.21±0.7µm with 90% population of the liposomes equal or below 4.81µm. Most of the vesicle was found to be spherical in shape. Log-size distribution curve confirms the normal size distribution of the vesicles. The liposomes were photographed using scanning electron microscope. Size analysis was repeated for 3 formulations of each formulation code and vesicle size data was compared. Data was found to be highly reproducible every time. The figure 6 shows SEM photograph of liposomal formulation KL9. (Figure 6), (Table 1).

Figure 6. SEM Photograph of KL9

Stability Profile:

There is no evident for aggregation, fusion or disruption of the vesicles during the studied period and it was found that the prepared formulations were able to retain their multilamellar nature and shape uniformity to an appreciable extent. The bar diagram shows the % drug leakage from the lipid vesicles over the period of 30 days at different storage temperature. It was found that samples stored at elevated storage temperatures, i.e. 37ºC ± 2ºC and 45ºC ± 2ºC showed the % drug leakage of the samples varied from 7%-12%. On the other hand liposomes stored at lower temperatures i.e. room temperature (RT) and refrigerated temperature (RF) showed that they could retain 96%-97% of the encapsulated drug respectively. (Figure 7)

Figure 7. Extent of drug leakage from KL9 liposomes at different storage temperatures.

Drug release:

Results of an invitro release study of ketorolac from liposomes are shown in figure 8 and figure 9. Release profile of ketorolac from liposomes of different cholesterol content were apparently biphasic release processes. Rapid drug leakage was observed during the initial phase exactly in the first hour where about 10 – 25% of the drug was released from various formations during its release in 100ml phosphate buffer pH7.4, followed by the release up to 55% were observed in subsequent 7hrs. However during the following 24hrs a slow release occurred in which 20 - 40% of drug was released from different liposomes. This could be because the drug is mainly incorporated between the fatty acid chains in the lipid bilayer of the vesicles which leads to rapid ionization and release upon dispersing liposomes of large buffer pH7.4 volumes until reacting equilibrium. Also it has been reported that a highly ordered lipid particles cannot accommodate large amount of drug and is the reason for drug expulsion.²⁵

Figure 8. Plots of in vitro cumulative percentage drug released vs. time for different Ketorolac liposomes of KL1-KL4.

Figure 9. Plots of in vitro cumulative percentage drug released vs. time for different Ketorolac liposomes of KL5-KL9.

Among the formulations prepared, KL3 containing 1:10% w/w of the drug, SPC and zero cholesterol could release the highest amount of drug in 24h, i.e 88.64% followed by KL7, KL5 and KL6 and containing2:1, 8:1and 4:1 %w/w of SPC and cholesterol respectively. Thus the presence of cholesterol restricts the drug release. When the percentages retained in liposomes were normalized to the lipid content of each sample, SPC only liposomes were seen to retain remarkably lower, where as other liposomes could retain the drug at higher percentages, supporting the result that highest extent of drug release occurred in cholesterol free formulations. Thus addition of cholesterol to a liposomal formulation composed of SPC/Cholesterol group of 8:1%w/w and 4;1%w/w decreased the drug release in comparision with cholesterol free formulations. This could be due to decreased leakage and permeability of the formulation at this weight ratio in presence of cholesterol. It has been reported that cholesterol increases the hydrophobicity which decreases the formation of transient hydrophilic holes by decreasing membrane fluidity, responsible for drug release through the bilayers.²⁶On the other hand further further increase in cholesterol amounts to2;1%w/w could increase the drug release. This result is again due to increasing cholesterol beyond a certain concentration can disrupt the regular linear structure of the membrane and increase the drug release.^{24, 27} Drug release from all the prepared formulation followed zero order kinetics and release mechanism was of diffusion. This was confirmed by the regression values of the respective plots. (Table 2)

Table 2: Mathematical model showing order and mechanism of drug release

Formulation	Zero order		First order	Higuchi’s plot
Formulation	Regression	slope % hour	Regression	Regression
FL1	0.872	2.956	0.437	0.980
FL2	0.846	2.596	0.371	0.975
FL3	0.893	2.641	0.386	0.981
FL4	0.881	2.637	0.379	0.976
FL5	0.821	2.380	0.401	0.950
FL6	0.905	2.469	0.449	0.984
FL7	0.877	2.034	0.382	0.979
FL8	0.784	1.933	0.390	0.932
FL9	0.861	1.838	0.417	0.976

CONCLUSION:

Concluding the above said results, EE% of the ketorolac in to liposomes prepared by the thin film hydration technique was a function of formulation and processing variables such as drug lipid ratio, cholesterol content, vacuum, speed of rotation, hydration medium and hydration time. The electrostatically induced change in bilayer packing and electrostatic interaction between drug and charged head groups of SA and DCP could influence the incorporation ketorolac in to liposomes. Liposomes could enhance the charecteristics of ketorolac but to a maximum limit after which increase in drug concentration may lead to drug precipitation. It is clear that liposomes containing 4:1%w/w SPC and cholesterol are the most stable among other tested formulations. However further increase in cholesterol in to liposomes could increase the drug release and display much lower stability.

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Received on 17.08.2011 Modified on 29.08.2011

Research J. Pharm. and Tech. 4(11): Nov. 2011; Page 1766-1771