Development of Liposomal Salbutamol Sulfate Dry Powder Inhaler Formulation

By

Wen-Hua HUANG, Zhi-Jun YANG,* Heng WU, Yuen-Fan WONG, Zhong-Zhen ZHAO, and Liang LIU

Summary by Sneha Subramanian

In this experiment, liposome formulation containing Salbutamol sulphate (SBS) is prepared in dry powdered form. This formulation is used in DPI for the treatment of asthma. Asthma is a pulmonary disease which can be treated with the drug SBS. It is widely used for the treatment of various pulmonary diseases like bronchial asthma, chronic bronchitis and emphysema. It is given in the form of injections, oral dose and aerosol. When taken orally or as injection, SBS is digested by the enzymes in the liver. These routes are not used these days. Inhalation is the best method as drug is directly deposited in the pulmonary track. By conventional delivery of SBS by earosols, it shows the effect in 5-15 minutes, but it is not prolonged. It needs to be repeated in every 4-6 hours. There is therefore a need to improve the dosage to show a prolonged effect and improve the treatment strategy

Delivery of SBS using liposome is more preferred due to its capacity of sustained release. Liposomes have no side effect and it effects the drug’s pharmacokinetics and pharmacodynamics. It enhances the drug uptake by delaying drug clearance. It’s surface viscosity prolongs the release of entrapped drug and decreases its clearance from the pulmonary track. Ry powdered inhaler (DPI) is used for the aerosol delivery of SBS.  In this study liposomal formulation is studied to treat asthma.

In this experiment the phospholipid gel was prepared using different mass ratios of SPC and aqueous SBS (160.1mg/ml) as 1:1 to 1:3. They were mixed together and allowed to swell in water bath at 60° for 2 hours.  This mixture was then stirred with a homogeniser until it formed a semisolid vesicular phospholipid gel (VPG) these VPGs were again allowed to swell in water bath at 60° for 2 hours.  These VPGs are hydrated with water and cryoprotectant solutions to form liposomal dispersions. The encapsulation efficiency of different concentration of SPC is shown in the graph below.

Encapsulation Efficiency of SBS in SPC VPG Liposome Suspensions, prepared Using Different SPC Concentrations

Different kinds of cryoprotectants like sucrose, mannitol, lactose and glucose were used for the lyophilisation of VPGs. Different ratios of cryoprotectants like 1:1 to 1:6 were used and frozed at -20°. These frozen mixture was then put in freeze dryer  for 48 hours to get liposomal powder. The temperatire in freeze dryer is -50° and vacuum was 133×10-3 mBar. The encapsulation od SBS using different ratios of cryoprotectant was calculated and shown in the table below.

After the preparation of different kind of liposomes using various ratios of cryoprotectants, their invitro deposition profile were studied. The formulation giving different entrapment of drug SBS according to table.1 was selected. All the cryoprotectants- lactose, sucrose, mannitol and trehalose were prepared in their optimal mass ratios of SPC. The resultant porous cake was sieved through 400 mesh sieves. These sieved liposome powder was then mixed with 1:5 ratio of lactose. The invitro deposition studies of these 4 formulation of liposome and cryoprotectant was studied using twin stage impinger. The fine particle fraction (FPF) was determneind for all 4 formulations. Lactose was selected as the most appropriate cryoprotectant among the 4 cryoprotectants used.

Further the effects of different amount of lactose was studied (63-106µm). The process of preparation was similar- 1:1 to 1:7 ratios of lactose and liposome powder was used. FPF of each formukation was determined.

In this experiment, Micron centrifugal filter device was used to determine encapsulation efficiecy. The liposome preparation was centrifuged for 15mins at 17400 x g, 6°C. UV spectrophotometer at 276nm was used to determine the concentration of SBS after being diluted with 1450µl of ethanol. Encapsulated drug in liposome was determined by destroying liposomal membrane by adding 1450µl ethanol to 50µg of liposomal suspensions. The following formule was used.

Encapsulation efficiency = ( total drug- encapsulation drug) x 100%

                             Total drug

Twin stage impinge was used for this purpose. 7ml and 30ml of capturing was used in upper and lower stage respectively. Aspinhaler was used as a delivery device with flow rate of 60/ml for SBS for 10 capsules. The SBS calculated after the washing of these chambers were used for the determination of SBS. HPLC was used for this purpose at 276nm. A 25cm x 4.6mm C18 column was used. Mobile phase consists of acetonitrile: water (10:90) with sodium dihydrogen phosphate 30mmol/l. The pH of mobile phase is 3.5 with 1.0 M phosphoric acid. The column temperature was 25°C. The amount of SBS collected in lower stage of impinge was calculated and the results were expressed in percentage of FPF of actual dose.

In this experiment, a series of SPC concentration from 250-500 mg?g was used to investigate its effect on encapsulation. It was seen that encapsulation efficiency increased with the increased concentrations of SPC. This maybe due to following reason- when VPG was hydrated with aqueous medium, the amount of drug entrapped in the control core remained entrapped while those between the vesicular layers was released. This encapsulation efficiency is determined by the ratio of core volume and not to overall aqueous space in VPG. It was also observed that after 400mg/g the encapsulation increases much as the vesicle would have reached the maximum of the total volume of all aqueous compartment. Hence, 400mg/g of SPC concentration was considered as optimum amount and was used for further studies like the inhaler test.

It was observed that the structure of liposome was best performed by 1:4 of SPC to lactose. The encapsulation efficiency of SBS was 80.71% before lyophilisation and 44.35 after dehydration rehydration. Hence, lactose was selected as cryoprotectant in the further studies. HPLC was used to determine SBS content before and after sieving and was found to be same. This indicated that drug was not lost during lyophilisation.

The optimum concentration of cryoprotectant was determined as it affected the structural and functional integrity of liposomes. It also effected the leakage of  SBS from liposomes. When 400mg/g was used and then hydrated by lactose solution of 1:4 of SPC: lactose ratio, the entrapment was found to be 80.71. this was found to be very much higher than just using deionised water. This concluded that cryoprotectants helped to preserve the structural and functional integrity of liposome during hydration. In in vitro release study, it was observed that SBS in liposomal formulation showed prolonged release. It was concluded that lipsome helped the drug to be released gradually and eventually.

It was concluded by the preparation of VPG SBS liposome with a high entrapment (more than 80%). This was also stabilised by lyophilisation using cryoprotectants and used in inhalers for ashtama. 4000mg/g of SPC content in VPG and 1:5 ratio of lactose as cryoprotectant carriers was concluded as the bet formulation. Sustained release of SBS due to liposome was also achieved.

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