Liposomic niflumic acid - new transdermal anti-inflammatory medicine

Medicilon's pharmacokinetics department offers the clients a broad spectrum of high quality of services in the areas of in vitro ADME, in vivo pharmacokinetics and bioanalysis services, ranging from small molecules to large molecules, such as protein and antibody. The animal species involved in our services are non-human primate, canine, mice, rat, rabbit and hamster. Meanwhile, non-human primate experimental platform and isotope platform for protein/antibody are certified by the Shanghai Government.

The present invention is referred to the encapsulation of the drug Niflumic acid in liposomes with the purpose to create a new transdermal non-steroidal anti-flammatory formulation. The Niflumic acid as well as its ester derivates, have been encapsulated into liposomes with different lipid formulations in order to achieve the increased effectiveness of the active compound. The liposome suspension, with or without the encapsulated drug has been formulated as a cream and a stability study of the final formulation has followed.

The encapsulation technology, of pharmaceutical molecules in liposomes, shows particular growth the last few years along with great scientific interest.

A variety of different molecules have been already encapsulated with success, resulting to a direct attenuation of toxicity, to an improvement of their pharmacokinetic properties and to an increase in their selectivity and specialization concerning the tissues and target organs.

In the international bibliography there is a wealth of references on liposomic pharmaceutical forms. Many of these are in the clinical study stage and some other have been already registered and marketed.

The present invention has as a main objective to prepare liposomes and to encapsulate into them the anti-inflammatory substance Niflumic Acid and its esters. The Niflumic Acid is a very well substantiated active ingredient which has been already released in various transdennal forms. It is aimed at the development of a new anti-inflammatory form with a leading liposomic form and advanced pharmacokinetic properties. The medicine will be applied to Arthropathies and similar situations in Traumatoloαv and Reumatoloαv.

The selection of lipids, among these which fulfil the criteria, will be made according to the results of the phamnacokinetic studies.

The lipid molecules which are going to be assessed are natural ones, like: HSPC (hydrogenated soy lipids), EPC (Mixture of saturated and unsaturated lipids from eggs) and some synthetic like : DSPC, DMPC and DPPC which are saturated esters of phosphatidylcholine. The techniques that are used for the preparation and the physicochemical characterisation of liposomes along with these for the system liposome- encapsulated medicine and also the methodologies for the pharmacokinetic studies are presented in the following section :

Scientific Methodology (full and analytical description)

/. Lipids and raw material supply, preparation of niflumic Acid Esters. The lipids which are going to be used for the preparation of liposomes are either natural ones like the HSPC (hydrogenated soya lipid), EPC or synthetic lipids like phospholipids DSPC, DMPC, DPPC. The aliphatic chains of the synthetic lipids are stearic or myristate or palmitic acid which differs in the number of carbons atoms, the length and the melting point of aliphatic chains, parameters crucial for the physicochemical characteristics of liposomes.

Liposomes will be prepared not only by combining different phospholipids but also by phospholipids with different levels of cholesterol. Cholesterol is regulating the stability of liposomes therefore essential for the controlled release of the medicine. Liposomes will also be prepared with composition similar to the skin's with ingredients like: ceramides, cholesterol, cholesterol sulfate and fatty acids. The aim is the preparation of products with greater possible biocompatibility.

For the implementation of the study, MLV (multilamellar vehicles), SUV (small unilamellar vehicles) and LUV (large unilamellar vehicles) liposomes will be prepared and assessed aiming the encapsulation, on one hand the hydrophilic part of the Niflumic acid into the hydratic part of the single layer liposomes and on the other hand the encapsulation of the lipophilic products of esters, into the lipid double-layers of the multilayer liposomes.

Physicochemical characterisation The physicochemical parameters of liposomes that must be assessed at this stage are :

Dimensions - dimensions homogeneity - number of double layers - morphology, thermodynamic stability (in connection with the temperature and the lipid sample concentration) existence of interactions in the sample, possibility of phase separation.

The physicochemical techniques that will be used are of great resolution potential, specified as quantitative and qualitative optical-thermal techniques.

Different kinds of liposomes will be used for the pharmaceutical molecules encapsulation.

a) Niflumic-acid encapsulation

The Niflumic-acid will be encapsulated in the internal part of liposomes following the method of active transfer due to different pH which is created inside and outside the liposomes. Because of the fact that the pharmaceutical molecule contains ionic groups, in the acidic internal environment of the liposome the molecule will receive protons and consequently it will be encapsulated. The encapsulation will take place at the transition temperature of the liposomes aliphatic chains. Percentage of encapsulation about 60%-80%. Ratio of niflumic acid to lipids 1 :53 (Theoretical value 1 :50). The separation of the non encapsulated Niflumic Acid will be succeeded by gel-chromatography, liposomes containing Niflumic acid will be received. The assay of the encapsulated Niflumic acid will be carried out with HPLC and UV-Vis.

The assessment of niflumic acid / lipids was carried out with UVΛ/is. For the encapsulation of niflumic acid MLV (multilamellar vehicles), SUV (small unilamellar vehicles) and LUV (large unilamellar vehicles) liposomes were used, the greatest possible internal value. The encapsulation into MLV liposome was greater than 90% whereas the ratio of niflumic acid to lipids was 1 :55 (theoretical value 1 :50).

b) Encapsulation of the lipophilic esters of the Niflumic Acid. The lipophilic esters of the Niflumic acid will be encapsulated into the lipid double-layers of liposomes. Multilayer liposomes will be used in order to increase the available volume (of the double-layers) for the encapsulation. The addition of the lipophilic molecules will take place during the evaporation of the organic solvent from the lipids for the preparation of the lipid film (see 2a). The separation of the non encapsulated medicine will be managed with gel-Chromatography along with the test of the medicine/lipids ratio. The assay of the medicine will be carried out with HPLC.

c) Physicochemical characterization of the medicine-liposome system. The physicochemical parameters are 1, 2, 3, 4 of pages 4, 5. In adition isothermic titration for the thermodynamic study of the encapsulation of the drug substances was applied.

At this point there is a great scientific interest especially concerning the physicochemical changes taking place in the liposome due to the encapsulation of the medicine.

The techniques which are used are similar to these on phase 2.

Additionally, the isothermal microcalorimetry titration technique will be applied for the thermodynamic study of the pharmaceutical molecules encapsulation. The suggested microscope upgrade will give the ability of a more detailed representation of the chemical composition of the liposomes surfaces under study. Especially for the case of the encapsulation into the lipid double layers it will be possible the <charting> of the active ingredients.

IV. Pharmacokinetic studies. The kinetic study of medicines includes the testing of their release in buffer solutions (in vitro) together with the testing in 50% plasma of rat (in vivo).

The kinetic study will be carried out in connection with time in order to study the stability of the medicine (Refrigerator temperature 2°C, room temperature 25°C) and to study the release of the medicine at a temperature of 37°C.

Hyperthermal studies will be carried out at 45°C and 55°C in order to test the gradual release of the medicines and also to avoid possible toxicity phenomena. In all the cases, by using gel-chromatography, the free pharmaceutical agent, towards the encapsulated one, will be under control and the assay will be carried out with UV-Vis. The ratio medicine/lipid will be also determined.