J.
MICROENCAPSULATION, 1991, VOL. 8, NO, 3, 295-300
Preparation and performance evaluation of plain proliposomal systems for cytoprotection
Journal of Microencapsulation Downloaded from informahealthcare.com by Nyu Medical Center on 01/07/15 For personal use only.
0. P. KATARE, S. P. VYAS and V. K . D I X I T Department of Pharmaceutical Sciences, Dr H. S. Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India
(Received 22 October 1990; accepted 13 November 1990) Plain liposomal systems composed of soyabean lecithin, cholesterol and stearylamine were formulated using various approaches. The prepared products were characterized and evaluated for their cytoprotective performance against the necrotizing NSAID’s (i.e. aspirin and phenylbutazone). Liposomes derived from proliposomes (effervescent granules based) demonstrated the best cytoprotective activity and physical and stability chracteristics. This system was shown to be superior. An increased availability of regular and small sized liposome born phospholipids to the damaged mucosal systems accounted for its better and enhanced performance.
Introduction Gastric ulceration has been the most common problem produced by various necrotizing agents and drugs. Prostaglandins (PG) and many other agents e.g. essential fatty acids, sucralfate, liquorice products, have been shown to provide protection to the gastric mucosal injury and are said to be cytoprotectants (Tarnawaski et al. 1987). Surface active phospholipids containing high proportions of unsaturated fatty acids have been postulated as cytoprotective agents by the possible mechanism of prostaglandin synthesis. Indomethacin is reported to produce gastric injury by P G synthesis inhibition and single administration of 16-1 6 dimethyl PGE, prevented indomethacin induced injury (Robert 1981). In view of the reported cytoprotective action of essential fatty acid, it was thought worthwhile to investigate the cytoprotective activity of lecithin-a natural phospholipid with high unsaturated fatty acid content. This study sets out to investigate the cytoprotective activity of lecithin (soyabean) and to look into the possibility of improving the activity in bi-molecular lipid membrane structures, i.e. liposomes. Various process and formulation variables have been studied to establish the suitability of the preparation. Proliposomes, the dry granular lipids carrier which forms liposomes on reconstitution, have been adopted (Payne et al. 1986). T h e improved method of preparation of proliposomes and the effervescent granular core material studies earlier have been used in this work (Katare et al. 1990). The cytoprotective activity of various formulations was measured by producing ulcers with the use of anti-inflammatory drugs, phenylbutazone and aspirin. T h e degree of ulceration calculated as ulcerogenic index exhibits the extent of cytoprotection generated by different formulations. 0265-2048/91 $3.00 Q 1991 Taylor & Francis Ltd.
296
0. P . Katare et al.
Journal of Microencapsulation Downloaded from informahealthcare.com by Nyu Medical Center on 01/07/15 For personal use only.
Materials and methods Proliposomes preparation Soyabean lecithin (Sigma Chemical Co. St. Louis MO), was dissolved in chloroform : methanol mixture (70 : 30). Cholesterol and stearylamine (Sigma) were also included in the lipid phase in order to observe the effect of it in the performance of products. The coating of lipids over the solid inert core material was undertaken in an earlier designed coating apparatus (Katare et al. 1990). T h e apparatus is based on a fluidized bed drying principle which provides uniform drying conditions under an inert nitrogen atmosphere. Lipids solution was sprayed over the core materials (sodium chloride, effervescent granules) in different aliquots. T h e fluidization of core material was effected by inlet nitrogen gas with a sufficient pressure. Drying temperature was maintained by an outside constant temperature bath. T h e operational and formulation parameters were studied to obtain the suitable formulations. T h e size of the core materials was kept within the range of 75 to 590pm. The prepared dry granular products were stored in sealed containers under the inert atmosphere of nitrogen in a dark place at 20°C. These products were reconstituted in distilled water just one hour prior to use.
Cast $film produced liposomes Liposomes were prepared using soyabean lecithin with or without cholesterol and stearylamine in different compositions. T h e lipids were dissolved in chloroform : methanol mixture (70 : 30) and dried in a round bottom flask under a stream of nitrogen. T h e dried lipid film was suspended in a hydrating medium (phosphate buffer saline, p H 7.4) for 24 hours, with intermittent shaking.
Liposome size distribution Liposomes obtained either from proliposomes or from cast film method, were studied for their homogeneity in size. T h e liposomal suspension was wet mounted on a hemocytometer and photographed by photomicroscope. T h e negatives were projected on a piece of paper using an enlarger with a magnification adjusted to 1000 diameters. Diameters of 800-1000 liposomes were noted for each preparation and assigned to the nearest one micrometer size group.
Cytoprotective evaluation Prepared products were evaluated for their cytoprotective activity by measuring ulcerogenicity produced by anti-inflammatory drugs in rats. T h e degree of ulceration was measured as an ulcerogenic index according to the method of Dhawan and Srimal(l973). Albino male rats of wistar origin, weighing 1 50-200 g were taken and divided in groups of six rats each. T h e drugs (phenylbutazone, 30mg/kg and aspirin 100 mg/kg), plain lecithin (50-250 mg/kg) and liposomal preparation (50 mg/kg equivalent to lipids) were fed daily for six days, water was given ad libitum. Rats were killed on the seventh day, their stomachs were removed, cut along the curvature, and the gastric contents washed with saline. T h e mucosa of each stomach was examined under the low power (20 x ), dissecting microscope. T h e degree of single ulceration was determined for each stomach and scored according to the following scale.
Plain proliposomal systems for cytoprotection
297
Degree 0.0 : Absence of any detectable lesion 0.5 : Small haemorrhagic effusion 1.0 : Haemorrhagic effusion 1.5 : Mucosal ulcerations of limited diffusion involving not more than of the whole surface 2.0 : Mucosal ulcerations of limited diffusion involving not more than 3 of the whole surface 2.5 : Mucosal ulceration of generalized diffusion 3.0 :Deep ulceration of limited diffusion 3.5 : Deep ulceration of generalized diffusion 4.0 : Perforated ulcers
Journal of Microencapsulation Downloaded from informahealthcare.com by Nyu Medical Center on 01/07/15 For personal use only.
4
Ulcerogenic Index (UI) was calculated using the formula ADU x per cent RU/100 ADU : Average degree of ulceration per cent RU : Percentage of rats with ulcers
Results and discussion Preparation of liposomes Liposomes of different characteristics occurred when different methods, using various lipids composition, were adapted (table 1). T h e multilamellar vasicular preparation, with heterogeneous size distribution, was obtained when cast film method was used. T h e uniformity in distribution of small sized liposomes could be produced by adopting proliposomal systems. Proliposomes, based on effervescent granules were shown to be quite effective in preparing the liposomes of desired narrow size distribution, with other soluble inert core material for proliposome preparation, i.e. sodium chloride, the same results could not be achieved. Inclusion of stearylamine, a positive charged fatty acid, was helpful in preventing the aggregation and agglomeration of lipid vesicles during the rehydration of proliposomal products.
Ulcerogenic Index in rats ( a ) Effect of lecithin. T h e results (figure 1) reveal the effectiveness of soyabean lecithin in reducing the ulcerogenicity caused by anti-inflammatory drugs, phenylbutazone and aspirin. T h e reduction in ulcerogenic index was found to be dose related. Table 1. Liposomes :composition and characterization. Codes
Lipids composition (molar ratio)
Method used
Type of preparation
CL-A CL-B CL-C PL-A PL-B PL-C PL-D
SL SL: CH (7:3) SL : SA (9 : 1) SL : SA (9 : 1) SL : SA (9 : 1) SL: CH (7:3) SL : CH : SA (7 : 2 : 1)
Cast film method Cast film method Cast film method Proliposomes (S) Proliposomes (E.G.) Proliposomes (E.G.) Proliposomes (E.G.)
MLV (aggregated) MLV (aggregated) MLV (heterogeneous) MLV (heterogeneous) MLV (homogeneous) MLV (heterogeneous) MLV (homogeneous)
SL = Soyabean lecithin, CH = Cholesterol, SA= Stearylamine, S = Sodium choloride, E.G. =Effervescent granules, MLV = Multilamellar vesicles.
0. P . Katare et al.
298
a
2.0
Aspirin
induced U.I.
Phenyl butazone induced U.
L.
I .6
I .2 k
3
Journal of Microencapsulation Downloaded from informahealthcare.com by Nyu Medical Center on 01/07/15 For personal use only.
0.8
0.4
0.0 00.0
50.0
100.0
150.0 200.0 2500
DOSE ( SOYABEAN LECITHIN )in Mg/Kg/ body weight.
Figure 1. Effect of soyabean lecithin on the ulcerogenic index in rats. 2.0
D
MICROENCAPSULATION, 1991, VOL. 8, NO, 3, 295-300
Preparation and performance evaluation of plain proliposomal systems for cytoprotection
Journal of Microencapsulation Downloaded from informahealthcare.com by Nyu Medical Center on 01/07/15 For personal use only.
0. P. KATARE, S. P. VYAS and V. K . D I X I T Department of Pharmaceutical Sciences, Dr H. S. Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India
(Received 22 October 1990; accepted 13 November 1990) Plain liposomal systems composed of soyabean lecithin, cholesterol and stearylamine were formulated using various approaches. The prepared products were characterized and evaluated for their cytoprotective performance against the necrotizing NSAID’s (i.e. aspirin and phenylbutazone). Liposomes derived from proliposomes (effervescent granules based) demonstrated the best cytoprotective activity and physical and stability chracteristics. This system was shown to be superior. An increased availability of regular and small sized liposome born phospholipids to the damaged mucosal systems accounted for its better and enhanced performance.
Introduction Gastric ulceration has been the most common problem produced by various necrotizing agents and drugs. Prostaglandins (PG) and many other agents e.g. essential fatty acids, sucralfate, liquorice products, have been shown to provide protection to the gastric mucosal injury and are said to be cytoprotectants (Tarnawaski et al. 1987). Surface active phospholipids containing high proportions of unsaturated fatty acids have been postulated as cytoprotective agents by the possible mechanism of prostaglandin synthesis. Indomethacin is reported to produce gastric injury by P G synthesis inhibition and single administration of 16-1 6 dimethyl PGE, prevented indomethacin induced injury (Robert 1981). In view of the reported cytoprotective action of essential fatty acid, it was thought worthwhile to investigate the cytoprotective activity of lecithin-a natural phospholipid with high unsaturated fatty acid content. This study sets out to investigate the cytoprotective activity of lecithin (soyabean) and to look into the possibility of improving the activity in bi-molecular lipid membrane structures, i.e. liposomes. Various process and formulation variables have been studied to establish the suitability of the preparation. Proliposomes, the dry granular lipids carrier which forms liposomes on reconstitution, have been adopted (Payne et al. 1986). T h e improved method of preparation of proliposomes and the effervescent granular core material studies earlier have been used in this work (Katare et al. 1990). The cytoprotective activity of various formulations was measured by producing ulcers with the use of anti-inflammatory drugs, phenylbutazone and aspirin. T h e degree of ulceration calculated as ulcerogenic index exhibits the extent of cytoprotection generated by different formulations. 0265-2048/91 $3.00 Q 1991 Taylor & Francis Ltd.
296
0. P . Katare et al.
Journal of Microencapsulation Downloaded from informahealthcare.com by Nyu Medical Center on 01/07/15 For personal use only.
Materials and methods Proliposomes preparation Soyabean lecithin (Sigma Chemical Co. St. Louis MO), was dissolved in chloroform : methanol mixture (70 : 30). Cholesterol and stearylamine (Sigma) were also included in the lipid phase in order to observe the effect of it in the performance of products. The coating of lipids over the solid inert core material was undertaken in an earlier designed coating apparatus (Katare et al. 1990). T h e apparatus is based on a fluidized bed drying principle which provides uniform drying conditions under an inert nitrogen atmosphere. Lipids solution was sprayed over the core materials (sodium chloride, effervescent granules) in different aliquots. T h e fluidization of core material was effected by inlet nitrogen gas with a sufficient pressure. Drying temperature was maintained by an outside constant temperature bath. T h e operational and formulation parameters were studied to obtain the suitable formulations. T h e size of the core materials was kept within the range of 75 to 590pm. The prepared dry granular products were stored in sealed containers under the inert atmosphere of nitrogen in a dark place at 20°C. These products were reconstituted in distilled water just one hour prior to use.
Cast $film produced liposomes Liposomes were prepared using soyabean lecithin with or without cholesterol and stearylamine in different compositions. T h e lipids were dissolved in chloroform : methanol mixture (70 : 30) and dried in a round bottom flask under a stream of nitrogen. T h e dried lipid film was suspended in a hydrating medium (phosphate buffer saline, p H 7.4) for 24 hours, with intermittent shaking.
Liposome size distribution Liposomes obtained either from proliposomes or from cast film method, were studied for their homogeneity in size. T h e liposomal suspension was wet mounted on a hemocytometer and photographed by photomicroscope. T h e negatives were projected on a piece of paper using an enlarger with a magnification adjusted to 1000 diameters. Diameters of 800-1000 liposomes were noted for each preparation and assigned to the nearest one micrometer size group.
Cytoprotective evaluation Prepared products were evaluated for their cytoprotective activity by measuring ulcerogenicity produced by anti-inflammatory drugs in rats. T h e degree of ulceration was measured as an ulcerogenic index according to the method of Dhawan and Srimal(l973). Albino male rats of wistar origin, weighing 1 50-200 g were taken and divided in groups of six rats each. T h e drugs (phenylbutazone, 30mg/kg and aspirin 100 mg/kg), plain lecithin (50-250 mg/kg) and liposomal preparation (50 mg/kg equivalent to lipids) were fed daily for six days, water was given ad libitum. Rats were killed on the seventh day, their stomachs were removed, cut along the curvature, and the gastric contents washed with saline. T h e mucosa of each stomach was examined under the low power (20 x ), dissecting microscope. T h e degree of single ulceration was determined for each stomach and scored according to the following scale.
Plain proliposomal systems for cytoprotection
297
Degree 0.0 : Absence of any detectable lesion 0.5 : Small haemorrhagic effusion 1.0 : Haemorrhagic effusion 1.5 : Mucosal ulcerations of limited diffusion involving not more than of the whole surface 2.0 : Mucosal ulcerations of limited diffusion involving not more than 3 of the whole surface 2.5 : Mucosal ulceration of generalized diffusion 3.0 :Deep ulceration of limited diffusion 3.5 : Deep ulceration of generalized diffusion 4.0 : Perforated ulcers
Journal of Microencapsulation Downloaded from informahealthcare.com by Nyu Medical Center on 01/07/15 For personal use only.
4
Ulcerogenic Index (UI) was calculated using the formula ADU x per cent RU/100 ADU : Average degree of ulceration per cent RU : Percentage of rats with ulcers
Results and discussion Preparation of liposomes Liposomes of different characteristics occurred when different methods, using various lipids composition, were adapted (table 1). T h e multilamellar vasicular preparation, with heterogeneous size distribution, was obtained when cast film method was used. T h e uniformity in distribution of small sized liposomes could be produced by adopting proliposomal systems. Proliposomes, based on effervescent granules were shown to be quite effective in preparing the liposomes of desired narrow size distribution, with other soluble inert core material for proliposome preparation, i.e. sodium chloride, the same results could not be achieved. Inclusion of stearylamine, a positive charged fatty acid, was helpful in preventing the aggregation and agglomeration of lipid vesicles during the rehydration of proliposomal products.
Ulcerogenic Index in rats ( a ) Effect of lecithin. T h e results (figure 1) reveal the effectiveness of soyabean lecithin in reducing the ulcerogenicity caused by anti-inflammatory drugs, phenylbutazone and aspirin. T h e reduction in ulcerogenic index was found to be dose related. Table 1. Liposomes :composition and characterization. Codes
Lipids composition (molar ratio)
Method used
Type of preparation
CL-A CL-B CL-C PL-A PL-B PL-C PL-D
SL SL: CH (7:3) SL : SA (9 : 1) SL : SA (9 : 1) SL : SA (9 : 1) SL: CH (7:3) SL : CH : SA (7 : 2 : 1)
Cast film method Cast film method Cast film method Proliposomes (S) Proliposomes (E.G.) Proliposomes (E.G.) Proliposomes (E.G.)
MLV (aggregated) MLV (aggregated) MLV (heterogeneous) MLV (heterogeneous) MLV (homogeneous) MLV (heterogeneous) MLV (homogeneous)
SL = Soyabean lecithin, CH = Cholesterol, SA= Stearylamine, S = Sodium choloride, E.G. =Effervescent granules, MLV = Multilamellar vesicles.
0. P . Katare et al.
298
a
2.0
Aspirin
induced U.I.
Phenyl butazone induced U.
L.
I .6
I .2 k
3
Journal of Microencapsulation Downloaded from informahealthcare.com by Nyu Medical Center on 01/07/15 For personal use only.
0.8
0.4
0.0 00.0
50.0
100.0
150.0 200.0 2500
DOSE ( SOYABEAN LECITHIN )in Mg/Kg/ body weight.
Figure 1. Effect of soyabean lecithin on the ulcerogenic index in rats. 2.0
D
