High-dose liposomal aerosol pharmaceutical composition

 

(57) Abstract:

The invention can be used in medicine for treatment of various lung diseases. The composition contains 12-30 mg/ml cyclosporine a or budezonida and 130-375 mg/ml phospholipid in ultrapure water. The composition is placed in the tank of the sprayer. The second variant composition: up to about 21,3 mg/ml cyclosporin a, up to about 160 mg of dilauroyllecithin/ml initial concentration in the reservoir. Third option: up to about 12.5 mg/ml budezonida in to about 187.5 mg dilauroyllecithin/ml initial concentration in the reservoir. The size range of particles that liposomes measured by aerodynamic diameter of median weight, is from about 1.0 μm to about 3.0 mm. The invention allows to obtain a concentrated compositions with high doses of active substances, which ensures maximum performance obtained on the basis of aerosol products. 3 S. and 13 C.p. f-crystals, 13 ill., table 2.

The invention relates to the field of biochemical pharmacology and medicinal chemistry. More specifically, the present invention relates to ready preparative forms in the form of high-dose liposomal aerosols of different pharmaceutical preparations of aerosol delivery systems of medicines, used for their application directly to the lung area. For delivery of such developed by various devices (e.g., an inhaler with a measured dose and the inhaler for dry powder). Jet nebulizers have been used in clinical conditions for aerosol delivery of water soluble drugs and ultrafine suspensions, however, their use with water-insoluble hydrophobic compounds is limited.

Development of liposomal preparative forms compatible with aerosol delivery, allows the jet nozzle to deliver additional medicines. The use of liposomes for aerosol delivery has many advantages, including water compatibility, slow release in the lungs, which helps to maintain the level of therapeutic doses, and in addition, liposomes facilitate intracellular delivery, in particular, to alveolar macrophages.

The effectiveness of localized, local therapy with the use of aerosols is determined by the amount of drug delivered to the site of disease in lung volume, and there are several key parameters that determine the number of shipping such Claudia different designs and variants of nozzles, working conditions (e.g. flow rate) and the presence of auxiliary equipment (pipes, connections, devices for the mouth, on the face and the like). Thus, the efficiency of release of the aerosol can be improved by appropriate additions to a suitable sprayer. Inappropriate additional devices and/or imperfect parameters may affect the inhalation doses, parts delivery and to influence therapeutic result.

Drugs are also a critical factor in regulating the efficiency of release of the aerosol and the aerodynamic properties of complex drug-liposomes. Found that the efficiency of the output of the complex drug-liposomes can be improved by the use of liposomes obtained at low temperatures of the phase transition (see Waldrep et al., J. of Aerosol Med. 7:1994 (1994) and Waldrep et al., Int'l J. of Pharmaceutics 97:205-12 (1993)). An additional way to enhance the yield of aerosol complex drug-liposome consists in increasing concentrations of drugs and phospholipids in the tank. Spray some ready preparative forms with complex medication-liposomes with more than 50 mg/Mei up to 150 mg/ml (see Thomas, et al. Chest, 99:1268-70 (1991)). In addition, performance of the aerosol (output and particle size) influence of physico-chemical properties such as viscosity and surface tension. These variables affect the maximum concentration of the complex of the drug-liposome delivery spray through the jet nozzle.

Anti-inflammatory glucocorticoids are used to treat asthma and other severe inflammatory lung disease for more than forty years. Recently aerosol glucocorticoid therapy has been used as one way of introduction. Currently there are several different, although structurally similar, glucocorticoids with local activity - for example, beclomethasone, budesonide, flunisolide, triamcinolone acetonide and dexamethasone - suitable for use in spray metered dose and spray for dry powder in the aerosol treatment asthma and other inflammatory lung diseases. While systemic complications, such as suppression of the hypothalamic-pituitary system, cataract formation and inhibition of growth, are not frequent complications in the treatment of asthma using spray the glucocorticoid is additional devices. Currently in the United States does not exist glucocorticoid ready preparative forms approved for application by spraying, although ultra-thin suspension of beclomethasone and budezonida used in Europe and Canada.

The present invention relates to a concentrated aerosol formulations of cyclosporine-A-liposomes and budesonide-liposome with high doses that provide the maximum performance of aerosol particles with aerodynamic diameter in the range of 1-3 μm in median mass (MMAD).

The aim of the present invention is the creation of high-dose liposomal pharmaceutical aerosol composition comprising in a container of about 12-30 mg/ml pharmaceutical compounds and approximately 130-375 mg phospholipid/ml initial concentration.

One of the objects of the invention is an aerosol pharmaceutical composition connection liposomes containing about 12-30 mg/ml pharmaceutical compounds in up to about 130-375 mg of phospholipid/mg of the initial concentration in the reservoir, where the pharmaceutical compound is selected from the group of anti-inflammatory glucocorticoids, immunosuppressive compounds, antifungal compounds, antibiotics, Nikogosian liposomal aerosol composition of cyclosporine A (CsA), containing up to about 30 mg/ml cyclosporine A in up to about 225 mg of phospholipids/ml initial concentration in the tank.

According to the invention offers a high-dose aerosol composition budesonide-liposome (Bud), containing up to about 15 mg/ml budezonida to about 225 mg of phospholipids/ml initial concentration in the tank.

In a preferred embodiment according to the invention offers a high-dose liposomal aerosol composition of cyclosporine A, containing up to about 20 mg/ml cyclosporine A to about 150 mg of dilauroyllecithin (DLPC)/ml initial concentration in the tank.

In the most preferred embodiment according to the invention offers a high-dose liposomal aerosol composition of cyclosporine A, containing up to about 21,3 mg/ml cyclosporine A to about 160 mg of dilauroyllecithin (DLPC)/ml initial concentration in the tank.

According to the invention offers a high-dose aerosol composition budesonide-liposome containing up to about 15 mg/ml budezonida to about 225 mg of dilauroyllecithin (DLPC)/ml initial concentration in the tank.

In the most preferred embodiment according izobreteniya in to about 200 mg of dilauroyllecithin (DLPC)/ml initial concentration in the reservoir. Other phospholipids in the finished formulation of complex budesonide-liposome can be replaced by DLPC.

Thus, the present invention offers a high-dose aerosol composition of the complex anti-inflammatory glucocorticoid-, immunosuppressive connection, an antifungal compound, antibiotic, antiviral compound and anticancer compound-liposomes containing about 12-30 mg/ml pharmaceutical compounds in up to about 130-375 mg phospholipid/ml initial concentration in the tank.

Other additional features and advantages of the present invention will be apparent from the following description of the present preferred embodiments of the invention, given with the purpose of disclosure.

For a clearer understanding in the description of the drawings. It should be noted that these drawings illustrate the preferred variants of the invention and are not limiting of the claimed scope of the invention.

Fig. 1 illustrates the distribution of liposomal aerosol drug high and low doses of cyclosporine A-DLPC sprayed using a spray gun Aerotech II at a flow rate of 10 liters per minute, by definition, cast content fraction of the total number of cyclosporine A, selected at each stage of impact impact device with an associated maximum size in μm (n=3 analysis). Range of aerodynamic diameter of median weight (MMAD) and geometric standard deviation calculated on the logarithmic plot of the probability.

Fig. 2 illustrates cyclosporine A, sprayed from the spray Aerotech II high-and low-dose cyclosporine A-DLPC liposomes at a flow rate of 10 liters per minute, as defined on the model, simulating the human lung. Values represent cyclosporine A, collected at different time spraying aerosol samples on filters attached to the Respirator Harward, brought to a total volume (TV) in 500 ml and at a speed of 15 breaths per minute (BPM).

Fig. 3 represents the distribution profile of aerosolized liposomal drug forms: Budesonide-DLPC with high and low doses, sprayed with spray Aerotech II at a flow rate of 10 liters per minute, as determined using a cascade probe shock Andersen. The data obtained (average standard deviation) represent the percentage of fractions relative to the total number of cyclosporine A, selected on camadian mass (MMAD) and geometric standard deviation (GSD) were calculated on a logarithmic graph of probability.

Fig. 4 shows Budesonide inhaled in the form of liposomal drugs forms Budesonide-DLPC with doses from low to high, spray with spray Aerotech II at a flow rate of 10 liters per minute, as defined on the model, simulating the lungs of a person with total volume (TV) 500 ml and 15 breaths per minute (BPM). Values represent Budesonide collected at different time spraying aerosol samples on filters attached to the Respirator Harward, brought to a total volume (TV) in 500 ml and at a speed of 15 breaths per minute (BPM).

Fig. 5 illustrates the time dependence CsA concentrations, inhaled through liposomal spray and crematoria finished products. The graph shows the complexes CsA-Cremophor (50 mg/ml; circles), CsA-DLPC (5 mg/ml; filled triangles) and CsA-DLPC (20 mg/ml; diamonds).

Fig. 6 illustrates the concentration of pulmonary CsA during inhalation in ICR mice (35 g) after inhalation of the powdered complex CsA-DLPC (20 mg/ml).

Fig. 7 illustrates the anti-inflammatory effect of the complex Bud-DLPC with high vines on leukocytes when conducting pulmonary bronchioalveolar lavage (BAL) in response to LPS (endotoxin) stimulus.

Fig. 8 illustrates the analysis of parallelogrammatic empty DLPC, CsA-DLPC and Bud-DLPC with increasing concentrations. Aerosols were obtained using water tested and standardized spray Aerotech II (initial starting volume of 4 ml; flow rate 10 l/min) and paired samples were collected in the cutter AGI-4 at 4-5 and 6-7 minutes of spraying. The concentration of DLPC was determined using HPLC analysis. The data presented are typical of finished products are tested at each specified concentration, and plotted on the graph depending on the initial content of DLPC (mg/ml) in the liposomes.

Fig. 10 illustrates the distribution of mass (mg/min) sputtered ready preparative forms with empty DLPC, CsA-DLPC and Bud-DLPC with increasing concentrations. Aerosols were obtained using water tested and standardized spray Aerotech II (initial starting volume of 5 ml; flow rate 10 l/min) and the output mass was determined using an analytical balance after 10 minutes of spraying. The data presented are typical ready preparative forms tested at each of the indicated concentration, and plotted on the graph depending on the initial content of DLPC (mg/ml) in the liposomes.

Fig. 11 illustrates the output of the CsA and Bud (mg/min) in sputtered aerosoling water and standardized spray Aerotech II (initial starting volume of 5 ml; the flow rate of 10 l/min) and paired samples were collected in the cutter AGI-4 at 4-5 and 6-7 minutes of spraying. The concentration of DLPC was determined using HPLC analysis of aliquot samples are also analysed for the content of DLPC (Fig. 1). The data presented are typical ready preparative forms tested at each of the indicated concentration, and plotted on the graph depending on the initial concentration of drug (mg/ml) in the liposomes.

Fig. 12 illustrates the viscosity (centipoise) liposomal ready preparative forms with empty DLPC, CsA-DLPC and Bud-DLPC with increasing concentrations (initial starting volume of 10 ml at room ambient temperature). Data represent the average of 10 observations for each of the tested ready preparative forms for each of the above concentrations and plotted in dependence of the initial content of DLPC (mg/ml) in the liposomes.

Fig. 13 illustrates the analysis of spatial tension (Dina/cm) liposomal preparations with empty DLPC, CsA-DLPC and Bud-DLPC with increasing concentrations (initial starting volume of 7 ml; at room ambient temperature). The data presented represent the average of 10 there is a chart depending on the initial content of DLPC (mg/ml) in the liposomes. Samples were also tested for viscosity.

The aim of the present invention is to improve the efficiency of delivery of high-dose pharmaceutical aerosol compositions consisting of a complex compound-liposome. For example, the present invention describes an improved efficiency of delivery of liposomal aerosol with cyclosporine A. In a series of experiments determined that the yield of aerosol drugs can be improved by the use of liposomal ready preparative forms with low temperature phase changes, such as DLPC (containing 12 carbon atoms, fatty acids with saturated side chains). It was also determined that certain nozzles increase the output of aerosolized liposomal drugs when the desired size of the aerodynamic diameter in the range of 1-3 μm in median mass (MMAD). The concentration of cyclosporine A, used in these early studies was 1.0 mg 7.5 mg DLPC per ml of the original solution in the tank.

In 1993 came the need to increase the yield of aerosol liposomal cyclosporine A by increasing the production of the drug. This could be done in various ways, such as choosing more than effem Aerotech II (ATII) (CIS-USA, Bedford, Mass). ATII gives an approximate 50% increase of the aerosol output compared to first Puritan Bennett 1600sj.

The second way to enhance the yield of aerosol liposomal drug was to increase the concentration of drugs and phospholipids in the liquid in the tank of the sprayer. The concentration of liposomal cyclosporine A-DLPC 5 mg of cyclosporine A/37.5 mg per ml was successfully increased to achieve the desired release of the aerosol with an aerodynamic diameter in the range of 1-3 μm in median mass (MMAD). When using the model of the human lung data analysis of aerosols showed that approximately 3.2 mg of cyclosporine A could theoretically be in the lungs after a single 15-minute inhalation. Studying at the University of Pittsburgh groups of patients with lung allograft receiving treatment aerosol cyclosporine A (dissolved in ethanol or propylene glycol), demonstrated clinical improvement (cancel transplant rejection upon delivery 20 mg of cyclosporine A in the lungs. Using the appropriate liposomal systems cyclosporine A-DLPC to achieve this number takes approximately 2 hours inhalation aerosol. This prolongirovtsa, ought to increase the concentration of cyclosporine A-DLPC in the tank. However, it is well known that it is impossible to spray the liposomes at a concentration greater than 50 mg/ml, as large concentrations lead to the bonding of the sprayed particles.

In the present invention has been achieved in the concentration of cyclosporine A 20-30 mg/ml: 140-225 mg DLPC/ml initial concentration in the reservoir. The particle size was increased marginal, while moving up MMAD of the aerosol to 2.0 μm, 1.6 μm, as shown with cyclosporine A-DLPC (5 mg/37.5 mg), without changes in GSP (Fig. 1). Aerosol output "high doses" of 20-30 mg of cyclosporine A-DLPC was significantly higher than 5 mg of cyclosporine A - DLPC.

As shown in Fig. 2 on models that mimic the human lung, 15 minutes of cyclosporine A-DLPC with high doses, the time required to deliver the required therapeutic doses to patients with lung transplant, would be approximately 45 minutes or less. Of course, the basis for determining this time period was based on the results of the study doses by other researchers using other aerosol cyclosporine A. liposomal cyclosporine A is therapeutically more effective at low doses and m is of the cyclosporine A-DLPC above, than about 30 mg of cyclosporine A-225 mg DLPC ineffective.

The present invention demonstrates the suitability of high-dose liposomal aerosol cyclosporine A-DLPC in the range of 20-25 mg of cyclosporine A/150-200 mg per ml DLPC, although the number, up to 30 mg/ml, are also included in the concept of high-dose. Other phospholipids in high-dose aerosol composition cyclosporin A liposome can be replaced by DLPC. Typical examples of suitable phospholipids include phosphatidylcholine egg yolk, hydrogenated phosphatidylcholine soybeans, dimyristoyl hold, dioleoyl dipalmitoyl the phosphatidyl hold and dipalmitoylphosphatidylcholine.

High-dose aerosol cyclosporin A liposome is suitable for various immunologic lung diseases, such as graft rejection, bronchodilators obliteration, allergies, hypersensitivity and asthma, and using different spraying systems suitable for use in Pediatrics, in the treatment of adolescents and adults. In the treatment of various these diseases require different time inhaling.

The creation of high doses of cyclosporine A-DLPC is necessary because aerostich studies patients were treated by spraying cyclosporine A-cremophor (50 mg cyclosporine A/ml). As shown in Fig. 5, the output of the aerosol complex cyclosporine A-liposomes (5 mg/ml and 20 mg/ml) is much higher. Cyclosporine A-cremophor is very annoying, but this spray gives some clinical benefit. Thus, the complex of cyclosporin A liposome will be even better at the same time to test for similar patients. Cyclosporine A-DLPC liposomal spray is also effective in the treatment of asthma, as described for oral cyclosporine A.

The authors of this invention have also determined that with the glucocorticoid budesonide is possible to obtain stable liposomes, which can be sprayed effectively and get the aerosols in the range of 1-3 μm MMAD. At a concentration in the tank for delivery daily dose in the treatment of asthma requires the usual time of spraying, and spray ATII it will be approximately 15 minutes. It is clinically feasible and practicable.

Company Boehringer-ingelheim was investigated glucocorticoid liposomes in apparatus for spraying. The design of the apparatus is designed to deliver 100-200 µg of glucocorticoid 20 ml working volume. A simple mathematical transformation shows that the required 5000 - 10000 ág/ml in the reservoir device experiments, to achieve the necessary concentration in the liposomal preparation was obtained concentrated and viscous suspension. In previous experiments with Budesonide was used a ratio of 1: 25 (Budesonide to DLPC by weight). Based on the required high concentrations DLPC, tested different ratios Budesonide:DLPC, and the ratio of 1: 15 was identified as the most suitable. The drug is then concentrated first to 5 mg Budezonida: 75 mg DLPC per ml, and finally to 10 mg Budezonida: 150 mg DLPC on ml Concentration with other corticosteroids (beclomethasone dipropionate or flunisolide) was more difficult due to the instability ready preparative forms. Ready preparative form containing 10 mg Budezonida 150 mg DLPC was stable and could be effectively sprayed using a spray gun ATII.

Fig. 3 shows that increasing the concentration leads to the increase of aerosol particles, increasing MMAD from 1.2 μm to 1.0 μm with complex Budesonide-DLPC with high doses. Fig. 4 shows that after one 15-minute inhalation of the drug Budezonida with high doses, will be inhaled approximately 6 mg Budezonida or 6-fold higher clinical daily dose. The affinity between the output of the aerosol osonovanny" Budesonide-DLPC liposomal aerosol is at about 12.5 mg Budezonida/225 mg DLPC on ml Other phospholipids in liposomal drug Budezonida with high doses can be replaced by DLPC. This liposomal aerosol drug complex Budesonide-DLPC clinically suitable for the treatment of certain lung diseases, such as asthma and interstitial fibrosis, as well as immunological diseases rejection, lung transplant, bronchodilators obliteration, allergies and hypersensitivity. It is suitable for the treatment of children, adolescents and adults using a variety of sputtering systems.

The following examples are given to illustrate various embodiments of the invention and in no way limit the present invention.

Example 1

Liposomal preparation: Receive high-dose complex drug/liposome

According to the invention was carried out, the process of freeze-drying for optimum medication of various complexes of the drug/liposome. It was found that the optimal ratio by weight between cyclosporine A and DLPC is 1:7,5. To determine the maximum concentration that is compatible with the spray, ready formulation designed for aerosol delivery of drugs, was received with seeeduino, what preparations containing 21,3 mg of cyclosporine A: 160 mg DLPC, are the best, judging by the aerosol yield and particle size for inhalation. For optimization of complex cyclosporin A liposome with high doses 100 mg of cyclosporine A (Sandoz Pharmaceuticals or Chemical Company) is mixed with 750 mg of synthetic alpha-lecithin; 1,2-Dilauroyl-sn-glycero-3 - phosphocholine (DLPC from Avanti Polar Lipids). Working at 37oC in a warm room, the complex drug/DLPC mixed in 20 ml of tertiary butanol with stirring as described Waldrep et al. , Int'l J. of Pharmaceutics 97:205-12 (1993). After mixing, the mixture of drug/lipid transfer pipette in a glass bubble, quickly frozen, then lyophilizer during the night to remove the tert-butanol, leaving a powdery mixture. Multilayer liposomes obtained by adding 10 ml of ultrapure water above the phase transition temperature (Tc) at 25oC to achieve a final standard concentration of the medicine 1-30 mg of cyclosporine A: a 7.5-225 mg DLPC in ml. of the Mixture is incubated for 30 minutes at room temperature with periodic mixing to obtain a multilayer vesicular liposomes. As an alternative, these are ready preparative forms can policital HPLC. This simple method of obtaining liposomes chosen due to the fact that it can be easily scaled to produce large quantities.

After swelling a number of prepared liposomes check on the size and the presence of crystals of a medicinal product by microscopy, visually, both before and after spraying. The Association of the drug-lipid (encapsulation efficiency) is determined using analysis of gradient paralleli, as described by O'riordan et al., J. of Aerosol Med., in press (1996). There is no need to reduce the size of the drug layered vesicular cyclosporine A-DLPC drug-liposome prior to spraying, as the drug-liposome (heterogeneous starting mixture of 2.2 to 11.6 μm after swelling) reduced in size later during spraying and long-lasting reflux) due to the forces generated during the extrusion outlet of the dispenser. The size of these liposomes in the droplets of the aerosol is 271-555 nm. Fluid particles in the aerosol containing one or more liposomes. The diameter of the liposomes is less than the aqueous aerosol particles in which they are transferred (see Waldrep et al., Ink l J. of Pharmaceutics 97:205-12 (1993)). After swelling is ready which should be stored for months at room temperature or in the refrigerator.

For ready preparative forms the optimal ratio between the drug and the lipid is determined by testing various preparative forms with a ratio of Budesonide-DLPC from 1:1 to 1:20. The ratio of 1: 15 (by weight) is selected as optimal for the finished high-dose formulations of Budesonide-DLPC. This drug is produced by mixing 10-150 mg Budezonida from 150 to 2250 mg DLPC (as described above for cyclosporine A-DLPC). Working at 37oC at room temperature, drug/DLPC mixed in 20 ml of tertiary butanol with stirring. After mixing, the mixture of drug/lipid transfer pipette in a glass bubble, quickly frozen and then lyophilizer during the night to remove the tert-butanol, and remains powdery mixture. Multilayer liposomes obtained by adding 10 ml of ultrapure water at a temperature above the phase transition temperature (Tc) at 25oC for delivery to the final standard concentration of the drug in 1-15 mg Budezonida: 15-225 mg DLPC per ml of solution. The mixture is incubated for 30 minutes at room temperature with periodic mixing to obtain a multilayer vesicular liposomes. For measuring mnie ready preparative forms can be obtained by using rotary evaporation. Fig. 8 shows the analysis of the gradient percolatin Bud-DLPC liposomes (see O'riordan et al., J. of Aerosol Med., in press (1996)). Nabokov once, multi-vesicular Budesonide-DLPC liposomes remain stable for several weeks at room temperature. Sterile prepared liposomes are stable for months. The benzalkonium chloride (10 mg/l) may be added as a preservative.

Example 2

Liposomal aerosols: Treatment with aerosol medication-liposomes

To work with aerosol drug liposome used spray Aerotech II (CIS-USA, Bedford USA), although other commercial sprays can also be used. ATII has a high output, efficient spray shows receiving liposomal aerosols with an optimum particle size of 1-3 microns MMAD for delivery in the peripheral lungs (see Vidgren et al., Int'l J. of Pharmaceutics 115:209-16 (1994)). The source of dry air is delivered to the atomizer and internal absorption of dry air is regulated by the flow regulator and is 10 liters per minute. The initial tank volume is 5 ml and sufficient for 15-20 minutes steps aerosol. Longer intervals of treatment require re-filling the tank.

Example 4

Evaluation of the inhaled dose

For determination of respirable dose Bec-DLPC liposomes sprayed samples collected in a system that simulates the human lung, as described Smaldone et al. , Am. Rev. Respir. Dis 143:727-37 (1991). Using a Harvard respirator, aerosol samples from the spray ATII (flow rate 10 l/min) collected in the filters Whatman GF/F at 15 breaths per minute with one inhalation of 500 ml. of This, the main volume of one inhalation, consisting of 500 men (450 for women), defined by the nomogram, adapted to account for the respiration rate, weight and sex. Aerosol samples were collected over a fifteen minute period of spraying. The number of cyclosporine A or Budezonida trapped on the filters was determined after extraction with HPLC.

Example 5

Analyses of lung cyclosporine A: Solid-phase extraction

Carried out YSI. Add internal standard CSD 10 μg (1 μl of 1/mg/ml initial solution). The homogenized tissue or in the mixer, or in test tubes Wig-L-Bug (using 4-5 balls per tube).

2. Gomogenizirovannogo tissue is extracted in 1 ml of ultrapure water for 1-2 minutes. This volume is used for a single tissue sample, and by combining more than one sample, it is diluted.

3. Add 2 ml of 98% acetonitrile/2% methanol and the samples vigorously shaken.

4. The samples are centrifuged at full speed for 20 minutes; the supernatant transferred to a clean tube and centrifuged for 10 minutes at full speed.

5. The collect supernatant and add 5 ml of ultrapure water to each 1 ml of tissue extract.

6. Prepare column Sep-Pak C18 (Waters Sep-Pak Light for a single mouse tissue) and washed with 5 ml of 95% ethanol and 5 ml of ultrapure water. Samples add slowly and washed with 5 ml ultrapure water and 5 ml of 50% acetonitrile.

7. The eluate is transferred into the Assembly tube and elute 1 ml methanol, and then 0.5 ml of water.

8. The dirt can be removed by washing of the eluate twice with 1.5 ml of hexane and discard the top layer.

9. Extragere isalnum air flow.

10. The restoration carried out in 0.3 ml of mobile phase CSA and the sample in HPLC.

Example 6

The analysis of pharmaceuticals by high performance liquid chromatography (HPLC): analysis budezonida

A study using HPLC is used with different objectives to determine: content Budezonida in liposomal ready preparative form, the efficiency of encapsulation, content Budezonida in aerosol samples, obtained on the model of the lungs. The concentration Budezonida determined by HPLC analysis using avtozagruzochnyh device Waters WISP 717 and column Waters Nova-Pak C18 (3.9 x 150 mm) at room temperature. The peak recorded at 238 nm using a detector operating at different wavelengths in the UV and visible part of the spectrum with the quantitative evaluation Version 2.15 Millenium 2010 Chromatography Manager of the firm's Waters. The mobile phase used in these studies is 50:50 ethanol/ methanol at a flow rate of 0.6 ml / min (see Anderson & Ryrfeldt, J. Pharm. Pharmacol. 36: 763-65 (1984)). Samples for analysis of dissolved directly in ethanol to dissolve the liposomes). Standards of medicines prepared from the original solution of ethanol, stored at -80oC.

Example 7

Analia

Cyclosporine A in liposomal ready formulation (for determination of cyclosporine A and the efficiency of encapsulation) and aerosol samples was determined using HPLC. The study used an automatic sample injector Waters (Milford, MA) WISP and column Supelco LC-1, heated to 75oC. the Mobile phase consisted of 50% acetonitrile, 20% methanol and 30% water (see Charles et al., Ther. Drug Monitor. 10: 97-100 (1988)). The peak recorded at 214 nm using a detector operating at different wavelengths, and quantify in Version 2.15 Millenium 2010 Chromatography Manager of the firm's Waters. Samples for analysis of dissolved directly in methanol (dilution of liposomes). Standards of medicines produced from the source solution of methanol, stored at -80oC.

Example 8

The analysis of pharmaceuticals by high performance liquid chromatography (HPLC): analysis of DLPC

Was used modification schemes of the HPLC Grit and Commelin, Chem. & Phys. of Lipids 62:113-22 (1992). Used automatic sample injector Waters 717 WISP and amino column Sperisorb S5 (0.25 cm x 4.6 mm, 5 μm) mobile phase: acetonitrile, methanol and 10 mm ammonium/triperoxonane acid, pH of 4.8 (64:28:8 by volume). PI is 5 Millenium 2010 Chromatography Manager of the firm's Waters. Samples for analysis of dissolved directly in ethanol or methanol (for dissolution of liposomes).

Example 9

The lung model for testing drugs in mice: investigation of acute inflammation: (LPS) technique bronchiolar lavage

The wall lipopolysaccharide of gram-negative cells (LPS) has been used for healing induction of acute pulmonary inflammation in mice. the 10-minute exposure of E. coli 055:B5 LPS (Sigma) aerosol released from the spray PBsj 1600 (concentration in the tank 100 µg/ml; delivered dose of 60 ng), has aroused strong floristicheskii response that is defined by the accumulation of PMN in the alveoli in response to the production of chemotactic cytokines (definable at 3 o'clock; the peak response at 6 hours after the stimulus). At different times after application of aerosol LPS mice were scored under methoxyflurane anesthesia and were bled via the abdominal aorta. In the trachea surgically inserted cannula with PE50 tube (outer diameter 0,965 mm Clay Adams). Using a balanced salt solution in Hanks in the total volume of 2.0 ml (HBSS; Ca/Mg free with EDTA), light watered 5 minutes with a volume of approximately 1.0 ml Output is normally accounted for 85 percent extraction lavagno fluid. Receiving asifali. According to various estimates, the effect of the drug is noted to reduce the number of white cells and to reduce PNM and/or myeloperoxidase positive cells relatively constant macrophages and/or myeloperoxidase negative cells. This study is used as a standard to test the mode of action of aerosols containing the drug/liposomes, biological activity by reducing acute inflammatory cell proliferation of the respiratory tract. Fig. 7 shows anti-inflammatory effect of high doses of Bud-DLPC on lung bronchoalveolar lavage (BAL) leukocytes in response to the challenge of LPS (endotoxin).

Example 10

Cytology: lung lavage

Cell preparations (lavage, thymocytes, lymph nodes or splenocytes) were counted on hemocytometer were cytocentrifugation on the slides (using Miles Cyto-Tek) and stained with Wright-Giemsa. May-Grunwald-Giemsa or leukocyte peroxidase directly depends on the method of preparation. The differential count was performed by microscopic observation with oil immersion. The biological effect of mode of application of an aerosol complex drug-liposonix cells relative to normal macrophages.

Example 11

Inhibition of antigen/mitogen-induced lymphocytic blastogenesis in vitro using CsA, isolated from the lung tissue of the mouse, after aerosol delivery CsA-DLPC liposomes (see tab.A)

The biological activity of CsA delivered to the lungs with liposomal aerosol

Primary immune response to the test was called in associated with bronchi lymphoid tissue and the related light mediastinal lymph nodes. After local intranasal immunization of mice of Balb/c alum precipitated egg albumin (AP-OVA (80 µg), supplemented by Bordetella pertussis vaccine) mice were scored after 7 days, mediastinal tissue was removed and isolated lymphocytes for in vitro assays. The study of cell proliferation is the change in the stimulation of lymphocytes after activation sensitizing antigen egg white or non-specific T-cell mitogen, Con a plus combined with culture CsA isolated from the lung tissue of the mouse with the solid-phase extraction for HPLC. Absorption3[H]-TdR into DNA was determined in 48-72 hours. Inhibition of specific or non-specific activation of lymphocytes was demonstrated by the destruction or reduction of antigen-specific, pyrogenation the C:

Spatial tension and viscosity: Spatial tension (Dina/cm) is measured using Tensiomat Model 21, Fisher Scientific, Indiana, PA). A platinum-iridium ring of known size was raised from the surface of the fluid for testing under carefully controlled conditions. "Apparent" value from the display of the device multiplied by the correction factor, including the size of the measuring ring, the liquid density and other parameters (in accordance with the manufacturer's instructions). The viscosity measurement is carried out with the use of the viscometer Gilmont Falling Ball (Gilmont Instruments, Barrington, IL). Viscosity in centipoise determine at room ambient temperature.

The measurement of the size of the complex drug-liposomes: the particle size of the complex drug-liposomes was measured with a Nicomp Model 370, Submicron Particle Sizer (Program Version 5,0 Nicomp Particle Sizing Systems, Santa Barbara, CA). The samples of the complex drug-liposome, dispersed in water, were analyzed in accordance with the manufacturer's instructions and data were expressed as the amount of suspended vesicles. Complex drug-liposome implies that the particle diameter is measured from tank samples in the initial period after 10 minutes of spraying and of Aeros is="ptx2">

The results in Fig. 9 (graph built for the concentration of DLPC) demonstrate that there is an increase of the output aerosol DLPC liposomes to 170 mg/ml with reduced output at higher concentrations. The dissemination of these data to CsA-DLPC liposomes gives similar results with maximum liposomal aerosol output from 21.3 mg CsA: 160 mg DLPC/ml (Fig. 9). For liposomes Bud-DLPC maximum output DLPC aerosol was demonstrated with ready preparative form, consisting of 12.5 mg Bud:187.5 mg DLPC/ml analysis of violations of the spray liquid indifferent substances, shown in Fig. 10 (graph built for DLPC), shows dependent on the concentration reduction of the yield, as determined by mass converted into an aerosol in a minute.

With increasing concentration of the liposomes is related to a similar increase in the yield of aerosol up to the critical point (Fig. 11) (graph built for the concentration of the drug). The measurement of the output aerosol drugs CsA and Bud when HPLC analysis shows the maximum concentration for spraying (Fig. 11). For CsA-DLPC liposomes maximum output amounted to 21.3 mg CsA: 160 mg/ml For Bud-DLPC was up 12.5 mg Bud:187.5 mg DLPC. Physico - chemical analysis of these concentratie DLPC) (Fig. 12). Results for DLPC, Bud-DLPC and CsA-DLPC were the same. The viscosity of the finished preparative forms Bud-DLPC was about 20 percent less than for the empty DLPC. Viscosity CsA-DLPC was steadfastly very low and ranged between 16 mg CsA/120 mg DLPC and 24 mg CsA/180 mg DLPC/ml These results suggest that for each finished formulation there is a maximum viscosity that is compatible with the aerosol spray; above these values do not exist incremental output to the high concentration of complex drug-liposomes.

The results in Fig. 13 (graph built for the concentration of DLPC) demonstrate that the addition of CsA and Bud to the DLPC liposomes causes a decrease in the spatial tension of the finished formulation. The decrease of spatial tension depends on the concentration, reaching a plateau at about 100 mg DLPC/ml. there is No particular relationship between aerosol release of liposomal ready preparative forms and spatial tension. However, with increasing concentration of the liposomal ready preparative forms observed inverse relationship between spatial tension and viscosity measurements.

Analyses of finished products of complex drug-liposome Assoc approximately 2.2 to 11.6 μm (or about the upper precision limit Nicomp 370). After spraying a defined minimum difference between any ready preparative forms. The particle size of liposomes inside the tank for spraying was 294-502 nm and the aerosol samples were collected using the web clipper AGI - 4, in the range from 271 to 555 nm.

Ready high-dose drugs, the drug-liposome consisting of 10 mg Bud:150 mg DLPC and CsA 20 mg:150 mg DLPC selected for further aerosol study. Analyses with cascading meter shock Andersen show a value of 2.0 μm MMAD/GSD of 1.5 for Bud-DLPC and 2.0 μm/1.8 for sA-DLPC (table 1). Analyses of these ready preparative forms on models that mimic the human lung at 15 BPM and 500 ml total volume demonstrate that 3 minutes of inhalation can breathe 1000 µg daily dose of Bud in liposomes, and 12 minutes up to 5000 ág (table 1). The results of the inhalation CsA-DLPC on the model of the human lung show that with high doses of CsA-DLPC requires 4 minutes for inhalation 5000 mcg spray CsA in liposomes; 11.5 minutes is required for inhalation 15000 μg CsA (table 1). These results demonstrate the ability of liposomes for aerosol drug delivery.

The present invention relates to high-dose liposomal aerosol is the concentration in the tank. Preferably, the liposomal aerosol composition contains up to about 21,3 mg/ml cyclosporine A to about 160 mg of phospholipid/ml initial concentration in the reservoir. Usually according to the invention, liposomal aerosol compositions with cyclosporine A particle size, as measured by the method of the aerodynamic diameter of the median mass is at a level of from about 1.0 μm to about 3.0 mm. In addition, cyclosporine A liposomal aerosol composition ratio of cyclosporine A and phospholipid is from about 1 to about 7.5. Preferably, the phospholipid is selected from the group consisting of phosphatidylcholine egg whites, phosphatidylcholine hydrogenated soybean dimyristoylphosphatidylcholine, dioleoylglycerol phosphatidylcholine and dipalmitoyl phosphatidylcholine. In General, liposomal aerosol composition with cyclosporine A according to the invention can be used to treat immunologic lung diseases. Preferably, such immunologic lung diseases selected from the group consisting of reaction, transplant rejection, bronchodilators obliteration, allergies, hypersensitivity and asthma.

The present invention also relates to high-dose b is IDA/ml initial concentration in the reservoir. Preferably, the budesonide-liposomal aerosol composition contains up to about 15 mg/ml budezonida to about 225 mg of phospholipid/ml starting concentration in the tank. For budesonide-liposomal aerosol compositions according to the invention the particle size, as measured by the range of aerodynamic diameter of the median mass is at a level of from about 1.0 μm to 2.0 μm. In General, budesonide-liposomal aerosol composition has a ratio of budezonida to phospholipid is from about 1 to about 15. Typical examples of phospholipids are given above. Usually budesonide - liposomal aerosol composition can be used for the treatment of immunological and inflammatory lung diseases.

Typical examples of immunological and inflammatory lung diseases listed above. Cyclosporine A is indirectly inhibits inflammation by blocking the immune response. Budesonide inhibits immune responses and inflammation. These pulmonary diseases have both.

The present invention also is directed to a method of treating patients with immunologic lung disease, comprising the step of assigning the patient a pharmacologically acceptable dose of cyclosporine A. On Alemania, including the step of assigning the named patient a pharmacologically acceptable dose of aerosol composition budesonide-liposome. Obtain a suitable pharmaceutical compositions and concentrations for use in the methods described here are obvious for the skilled professionals.

The present invention is also directed to liposomal aerosol composition with cyclosporine A, containing up to about 30 mg/ml cyclosporine A in up to about 225 mg of dilauroyllecithin/ml initial concentration in the reservoir. In addition, we offer high-dose aerosol budesonide-liposomal composition containing up to about 15 mg/ml budezonida to about 225 mg of dilauroyllecithin/ml initial concentration in the tank.

The present invention relates generally to high-dose liposomal aerosol compositions containing about 12-30 mg/ml pharmaceutical compounds and approximately 130-375 mg phospholipid/ml initial concentration in the reservoir. For example, the present invention relates to anti-inflammatory glucocorticoids, immunosuppressive compounds, antifungal compounds, compounds, antibiotics, antiviral compounds and anticancer compounds, patency and publications mentioned in this description illustrate only the level of technology in the field of the invention. These patents and publications is incorporated herein by reference.

These examples of the methods, techniques, treatment, and specific compounds are given as preferred embodiments and do not limit the scope of the claimed invention.

1. High-dose liposomal aerosol composition containing 12-30 mg/ml cyclosporine a or budezonida and 130-375 mg/ml of phospholipid in the ultrapure water held in the tank of the sprayer.

2. The composition according to p. 1, characterized in that it contains up to about 30 mg/ml cyclosporine a and up to about 225 mg of phospholipid/ml initial concentration in the tank.

3. The composition according to p. 1, characterized in that it contains up to about 21,3 mg/ml cyclosporine a to about 160 mg of phospholipid/ml initial concentration in the tank.

4. The composition according to p. 1, characterized in that the size of particles called liposomes is in the range from about 1 μm to about 3.0 mm.

5. The composition according to p. 1, characterized in that the ratio of cyclosporine And to phospholipid is from about 1 to about 7.5.

6. The composition according to p. 1, wherein the phospholipid is selected from outstationed, dilauroyllecithin, dioleoylglycerol and dipalmitoyl phosphatidylcholine.

7. The composition according to p. 1, characterized in that it is used for the treatment of immunologic lung diseases.

8. The composition according to p. 7, characterized in that the immunologic lung diseases are a group consisting of transplant rejection, bronchodilators obliteration, allergies, hypersensitivity and asthma.

9. The composition according to p. 1, characterized in that it contains up to about 15 mg/ml budezonida and up to about 225 mg of phospholipid/ml initial concentration in the reservoir, and the size of particles called liposomes when changing the aerodynamic diameter of the median mass is in the range from about 1.0 μm to about 3.0 mm.

10. The composition according to p. 9, characterized in that it contains up to about 12.5 mg/ml budezonida in to about 187.5 mg phospholipid/ml initial concentration in the tank.

11. The composition according to p. 9, characterized in that the ratio budezonida to phospholipid is from about 1 to about 15.

12. The composition according to p. 9, wherein the phospholipid is selected from the group consisting of phosphatidylcholine egg yolk, phosphatidylcholine hypostatically and dipalmitoyl phosphatidylcholine.

13. The composition according to p. 9, characterized in that it is used for the treatment of immunological and inflammatory lung diseases.

14. The composition according to p. 13, characterized in that the aforementioned immunological and inflammatory lung diseases are a group consisting of transplant rejection, bronchodilators obliteration, allergies, hypersensitivity and asthma.

15. High-dose liposomal aerosol composition with cyclosporine And containing up to about 21,3 mg/ml of cyclosporine And to about 160 mg of dilauroyllecithin/ml initial concentration in the tank.

16. High-dose liposomal aerosol composition with budesonide containing up to about 12.5 mg/ml budezonida in to about 187.5 mg dilauroyllecithin/ml initial concentration in the reservoir, wherein the size range of particles called liposomes, as measured by the aerodynamic diameter of the median weight is from about 1.0 μm to about 3.0 mm.

 

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