KR100497564B1 - High Dose Liposome Aerosols - Google Patents

Abstract

The present invention provides a high dose pharmaceutical liposome aerosol composition comprising about 12-30 mg / ml of the pharmaceutical compound and about 130-375 mg of phospholipid per ml of starting reservoir concentration. Specifically, the present invention relates to anti-inflammatory glucocorticoids, immunosuppressive compounds, antifungal compounds, antibiotic compounds, anti-viral compounds and anticancer compounds carried in high dose liposome aerosol compositions in phospholipids. More particularly, the present invention provides high dose cyclosporin A liposome aerosol compositions comprising up to about 30 mg / ml cyclosporin A in up to about 225 mg of phospholipid per ml of starting reservoir concentration. Also provided are high dose budesonide liposome aerosol compositions comprising up to about 15 mg / ml of budesonide in up to about 225 mg of phospholipid per ml of starting reservoir concentration.

Description

High Dose Liposome Aerosol Formulations

The present invention relates generally to the fields of biochemical pharmacology and pharmaceutical chemistry. More specifically, the present invention relates to high dose liposome aerosol formulations of various agents, including cyclosporin A and budesonide.

Many different lung diseases have been successfully treated with the aerosol delivery system used to deposit drugs directly on the pulmonary artery surface. For transportation in this manner, various devices have been developed (eg metered dose inhalers and dry powder inhalers). Jet-atomizers for aerosol delivery of water soluble drugs and particulate suspensions have been used clinically; The use of these atomizers for water insoluble hydrophobic compounds has been a limitation.

By developing liposome formulations that are compatible with aerosol delivery, these jet nebulizers can deliver additional drugs. The use of liposomes for aerosol delivery has several advantages, for example, suitable for use in the aqueous state; Delaying pulmonary artery release to maintain therapeutic drug levels and liposomes in particular facilitate intracellular delivery to alveolar macrophages.

Local local therapeutic efficacy via aerosol is determined by the amount of drug delivered to the disease site in the lung; There are several different key parameters that determine the therapeutic efficacy of an aerosol formulation by determining this amount of delivery. For example, nebulizer design and versatility, operating conditions (eg flow rates) and the presence of auxiliary equipment (tubes, connectors, mouthpieces, face masks, etc.) are important variables. Thus, the aerosol emission efficiency can be increased by properly implementing a suitable nebulizer device. Improper implementation of the device and / or incomplete parameters may affect the dose to be inhaled and the site of delivery and thus affect the outcome of the treatment.

Drug formulations are also important factors controlling aerosol emission efficiency and the aerodynamic properties of drug-liposomes. It has been found that drug-liposomal emission efficiency can be increased by using liposomes formulated at low phase transition temperatures. 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 increase aerosol drug-liposomal emissions is to increase drug and phospholipid reservoir concentrations. Spraying some drug-liposomal formulations at concentrations above 50 mg / ml will result in poor operation of the nebulizer jet; Empty liposome formulations up to 150 mg / ml have been successfully sprayed (Thomas, et al., Chest 99: 1268-70 (1991)). In addition, aerosol performance (emission volume and particle size) is in part affected by physicochemical properties such as viscosity and surface tension. These variables affect the maximum drug-liposomal concentrations matched to aerosol delivery through jet nebulizers.

Anti-inflammatory glucocorticoids have been used for the past 50 years to treat asthma and other serious inflammatory lung diseases. More recently, the use of aerosol glucocorticoid therapy as a route of administration is increasing. Currently, several different glucocorticoids, although structurally similar, have local activity, such as beclomethasone, budesonide, flunisolide, triamcinolone acetonide, and dexamethasone (all of which are asthma and other inflammatory agents) Commercially available as metered dose inhalers or dry powder inhalers for aerosol treatment of lung diseases. Asthma patients treated with glucocorticoid inhalation often have systemic complications such as hypothalamic-pituitary axis suppression, cataract formation and growth inhibition, but because of the more common local side effects of candidiasis and negative disorders, secondary spacers The use of the device is necessary. Currently there are no approved glucocorticoid preparations for spraying in the United States, but beclomethasone and budesonide in the form of particulate suspensions are used in Europe and Canada.

The present invention is directed to concentrated high dose cyclosporine-A-liposomes and budenoside-liposomal aerosol formulations that provide maximum aerosol emissions in a particle size range within an optimal range of 1-3 micron mass center aerodynamic diameter (MMAD). . There is no such liposome aerosol formulation in the prior art. The present invention satisfies the need for 'long term persistence' desired in the art.

Summary of the Invention

It is an object of the present invention to provide a high dose pharmaceutical liposome aerosol composition comprising about 12-30 mg / ml of the pharmaceutical compound and about 130-375 mg of phospholipid per ml of starting reservoir concentration.

In one embodiment of the invention, a group consisting of an anti-inflammatory glucocorticoid, an immunosuppressive compound, an anti-fungal compound, an antibiotic compound, an anti-viral compound and an anticancer compound in about 130 to 375 mg of phospholipid per ml of starting reservoir concentration A high dose pharmaceutical compound-liposomal aerosol composition is provided comprising about 12 to 30 mg / ml of a pharmaceutical compound selected from among them.

In one aspect of the invention there is provided a high dose cyclosporin A (CsA) liposome aerosol composition comprising up to about 30 mg / ml cyclosporin A in about 225 mg of phospholipid per ml of starting reservoir concentration.

In another aspect of the present invention there is provided a high dose of budenoside-liposome (Bud) aerosol composition comprising up to about 15 mg / ml of budenoside in up to about 225 mg of phospholipid per ml of starting reservoir concentration.

In a preferred embodiment of the present invention there is provided a high dose cyclosporin A liposome aerosol composition comprising up to about 20 mg / ml cyclosporin A in about 150 mg or less of dilauroylphosphatidylcholine (DLPC) per ml of starting reservoir concentration.

In a most preferred embodiment of the invention, there is provided a high dose cyclosporine A liposome aerosol composition comprising up to about 21.3 mg / ml cyclosporin A in about 160 mg or less of dilauroylphosphatidylcholine (DLPC) per ml of starting reservoir concentration. In these high doses of CSA-liposomal preparations, other phospholipids may replace DLPC.

In another aspect of the present invention there is provided a high dose of budenoside-liposome aerosol composition comprising up to about 15 mg / ml of budenoside in about 225 mg of dilauroylphosphatidylcholine (DLPC) per ml of starting reservoir concentration.

In a more preferred embodiment of the invention, a high dose of budenoside-liposome aerosol composition is provided comprising up to about 12.5 mg / ml of budenoside in about 200 mg of dilauroylphosphatidylcholine (DLPC) per ml of starting reservoir concentration. . Other phospholipids may replace DLPC in these high dose budenoside-liposomal preparations.

Accordingly, the present invention provides a high dose of anti-inflammatory glucocorticoid-, immunosuppressive compound-, anti-fungal compound-, comprising about 12-30 mg / ml of pharmaceutical compound in about 130-375 mg of phospholipid per ml of starting reservoir concentration. , Antibiotic compounds-, anti-viral compounds- and anticancer compounds-liposomal aerosol compositions.

Other and further aspects, aspects, and advantages of the invention will be apparent from the following description of the preferred embodiments of the invention, presented for purposes of illustration only.

Since the above-mentioned aspects, advantages and objects of the present invention can be understood in detail, reference may be made to the specific embodiments illustrated in the accompanying drawings in which the more specific technical content briefly summarized above. These drawings form part of this specification. It should be appreciated, however, that the appended drawings illustrate only preferred embodiments of the invention and thus do not limit the scope of the invention.

1 shows the aerosol distribution profile of high and low dose cyclosporine A-DLPC liposome formulations sprayed using an Aerotech II nebulizer at a flow rate of 10 l / min, as measured by the Andersen Cascade Impactor. It is. The data (mean ± standard error) represent the percent split of total cyclosporin A recovered at each stage of the impactor using the associated size cut-off value (μm) (n = 3 analysis). Center of mass aerodynamic diameter (MMAD) and geometric standard deviation (GSD) are calculated on log-probability plots.

FIG. 2 shows cyclosporin A inhaled from high and low dose cyclosporine A-DLPC liposome formulations sprayed using an Aerotech II nebulizer at a flow rate of 10 l / min, as measured in the human lung simulation model. Values represent cyclosporin A collected at different spray times from aerosol samples by a filter attached to a Harvard respirator adjusted to 500 ml of tidal volume (TV) and 15 breaths per minute (BPM).

FIG. 3 shows the aerosol distribution profile of high and low doses of budesonide-DLPC liposome formulations sprayed using an Aerotech II nebulizer at a flow rate of 10 l / min, as measured by the Andersen Cascade Amplifier. The data (mean ± standard error) represent the total cyclosporine A fraction in percent recovered at each stage of the impactor using the associated size cut-off value (μm) (n = 3 analysis). Center of mass aerodynamic diameter (MMAD) and geometric standard deviation (GSD) are calculated on log-probability plots.

FIG. 4 shows the high and low volume sprayed using an Aerotech II nebulizer at a flow rate of 10 l / min, as measured in a human lung simulation model with 500 ml of tidal volume (TV) and 15 breaths per minute (BPM). Budesonide inhaled from a budesonide-DLPC liposome formulation is shown. Values represent budesonide collected at different spray times from aerosol samples by a filter attached to a Harvard respirator adjusted to 500 ml of tidal volume (TV) and 15 breaths per minute (BPM).

FIG. 5 depicts time zones of simulated CsA concentrations inhaled from nebulized liposomes and cremophor formulations. CsA-Cremophore (50 mg / ml; round), CsA-DLPC (5 mg / ml; black triangle) and CsA-DLPC (20 mg / ml; diamond) are plotted.

Figure 6 shows the concentration of pulmonary artery CsA with inhalation time in ICR mice (35 g) after inhalation of nebulized high dose of CsA-DLPC (20 mg / ml).

FIG. 7 shows the anti-inflammatory effect of high doses of Bud-DLPC on pulmonary bronchoalveolar intestinal (BAL) leukocytes in response to LPS (endotoxin) inoculated immunoassay.

8 depicts percol gradient analysis of Bud-DLPC liposomes.

FIG. 9 shows aerosol DLPC emissions (mg / min) of sprayed DLPC alone, CsA-DLPC and Bud-DLPC liposome formulations with increasing concentration. Aerosols were produced using a water-tested, standardized Aerotech II nebulizer (initial starting volume 5 ml; flow rate 10 l / min) and paired samples in an AGI-4 impinger at 4-5 minutes and 6-7 minutes after spraying. Collect it. DLPC concentration is determined by HPLC analysis. Data presented are representative of the formulations tested at each concentration indicated and plotted by initial liposome DLPC content (mg / ml).

FIG. 10 shows the mass release (g / min) of sprayed DLPC alone, CsA-DLPC and Bud-DLPC liposome formulations with increasing concentration. Aerosols were produced using a standardized, water-tested Aerotech II nebulizer (initial starting volume 5 ml; flow rate 10 l / min) and 10 minutes after spraying was used to determine mass emissions using analytical balance. Data presented are representative values of the formulation tested at each concentration indicated and plotted by initial liposome DLPC content (mg / ml).

FIG. 11 shows aerosol CsA and Bud emissions (mg / min) of nebulized CsA-DLPC and Bud-DLPC liposome formulations with increasing concentration. Aerosols were produced using a water-tested, standardized Aerotech II nebulizer (initial starting volume 5 ml; flow rate 10 l / min) and paired samples in an AGI-4 impinger at 4-5 minutes and 6-7 minutes after spraying. Collect it. Drug concentrations are determined by HPLC analysis from aliquots of samples that have also been analyzed for DLPC content (FIG. 1). Data presented are representative values of the formulation tested at each concentration indicated and plotted by initial liposome drug content (mg / ml).

FIG. 12 depicts viscosity (centipoise) analysis of DLPC alone, CsA-DLPC and Bud-DLPC liposome formulations (initial starting volume 10 ml; ambient room temperature) with increasing concentration. Data presented are the average of the values observed 10 times for each formulation tested at each concentration indicated and plotted by initial liposome DLPC content (mg / ml).

FIG. 13 shows surface tension (dynes / cm) analysis of DLPC alone, CsA-DLPC and Bud-DLPC liposome preparations (initial starting volume 7 ml; ambient room temperature) with increasing concentration. Data shown are the average of the values observed 10 times for each formulation tested at each concentration indicated and plotted by initial liposome DLPC content (mg / ml). Samples are also analyzed for viscosity.

One object of the present invention is to improve the delivery efficiency of high dose pharmaceutical compound-liposomal aerosol compositions. For example, the present invention relates to improved delivery efficiency of cyclosporin A-liposomal aerosols. A series of experiments has shown that aerosol drug emissions can be enhanced by using liposomes with low phase transition temperatures, for example, formulated with DLPC (containing 12 carbon atoms and saturated fatty acid side chains). It has also been shown that certain nebulizers increase aerosol drug-liposomal emissions in the size range of 1 to 3 μm mass center aerodynamic diameter (MMAD). The cyclosporin A concentration used in these initial studies was 1.0 mg per ml of starting solution in the reservoir when using 7.5 mg DLPC.

In 1993, a need was recognized to increase cyclosporine A-liposomal aerosol emissions by increasing the proportion of the formulation. This could be done in different ways such as more efficient nebulizer selection. Cyclosporine A-liposomal aerosol emissions were increased in stages by implementing an Aerotech II (ATII) nebulizer (CIS-USA, Bedford, Mass). ATII increased aerosol emissions by about 50% compared to the previously used Puritan Bennett 1600sj.

A second method of increasing aerosol drug-liposomal emissions is to increase the reservoir concentration of the drug and phospholipids in the nebulizer reservoir liquid. Cyclosporin A-DLPC liposome concentrations of 5 mg cyclosporine A / 37.5 mg / ml were successfully increased while achieving the desired aerosol emissions in the range of 1-3 micron mass center aerodynamic diameter (MMAD). Using a human lung deposition model, the results of analyzing this aerosol showed that approximately 3.2 mg of cyclosporin A could accumulate in the lung 15 minutes after inhalation in theory. According to a Pittsburgh University study in a group of pulmonary allografts treated with aerosolized cyclosporin A (soluble in ethanol or propylene glycol), clinical improvement (rejection of transplantation) when 20 mg of cyclosporin A was delivered to the lungs Phenomenon is different). In order to use the available cyclosporin A-DLPC liposome system, this amount should be inhaled by the aerosol for approximately 2 hours. This prolonged inhalation every day is annoying for the patient and requires eight refills of the nebulizer reservoir. Therefore, it was necessary to increase the cyclosporin A-DLPC reservoir concentration. However, it has been widely recognized in the prior art that it is not possible to spray liposomes above 50 mg / ml, because spraying larger concentrations will result in poor operation of the nebulizer jet.

The present invention was successful in achieving 20 to 30 mg / ml cyclosporin A: 150 ml to DLPC 150 ml / departure reservoir concentration. This change resulted in a minimum increase in particle size, with the GSD upregulated from 1.6 μm to 2.0 μm as demonstrated by cyclosporin A-DLPC (5 mg / 37.5 mg), which was not changed at all (FIG. One). Aerosol emissions of “high dose” 20-30 mg cyclosporine A-DLPC are significantly higher than 5 mg cyclosporine A-DLPC.

As shown in FIG. 2, the time required to deliver the estimated therapeutic dose in pulmonary allograft patients in the simulated human lung model (15 minutes for high dose cyclosporine A-DLPC) will be approximately 45 minutes or less. . Clearly, this time is based on dose results by other researchers using other cyclosporine A aerosols. Cyclosporin A-liposomes appear theoretically much more effective at lower doses and are less toxic than cyclosporin A in ethanol or propylene glycol, resulting in much shorter inhalation times. Increasing cyclosporin A-DLPC to above about 30 mg cyclosporin A-225 mg DLPC has proven inefficient.

The present invention demonstrates the utility of high dose cyclosporin A-DLPC liposome aerosols in the range of 20-25 mg cyclosporin A / 150-200 mg DLPC / ml; Amounts up to 30 mg / ml also fall into the high dose category. Other phospholipids may replace DLPC in such high dose cyclosporin A-liposomal formulations. Representative examples of suitable phospholipids are egg yolk phosphatidylcholide, hydrogenated soybean phosphatidylcholide, dimyristoylphosphatidylcholide, dioleoliyl-dipalmitoyloleylphosphatidylcolide and dipalmitoylphosphatidylcolide.

High dose cyclosporine A-liposomal aerosols have been proven useful for a variety of immunologically mediated lung diseases such as allograft rejection, obstructive bronchiolation, allergies, hypersensitivity, and asthma, and are useful in pediatric, adult, and elderly patients using different nebulizer systems. Proved. Different inhalation times are needed to treat various diseases.

It is useful to produce high dose cyclosporin A-DLPC liposomes because high dose cyclosporin A aerosol is used to treat lung transplant rejection episodes. In these studies, patients are treated with nebulized cyclosporin A-cremophore (50 mg cyclosporin A / ml). As shown in FIG. 5, the aerosol emissions (5 mg / ml and 20 mg / ml) of cyclosporin A-liposomes are significantly higher. Although cyclosporin A-cremopores are very irritating, these aerosols provide some clinical benefit. Thus, cyclosporin A-liposomes will be superior to those previously tested in similar patients. Cyclosporin A-DLPC liposome aerosol is also effective for treating asthma as described for cyclosporin A for oral administration.

Studies according to the invention have demonstrated that glucocorticoid budesonide produces stable liposomes that can be effectively sprayed and produces aerosols in the range of 1 to 3 μm MMAD. At this reservoir concentration, the typical inhalation time for delivering a daily dose for asthma treatment from an ATII nebulizer would be approximately 15 minutes. This may be clinically viable and practically performed.

Boehringer-Ingelheim tested glucocorticoid liposomes in nebulizer apparatus. The design of these devices is required to deliver between 100 and 200 μg of glucocorticoids per 20 μl of performance. In simple mathematical terms, it was found that 5,000 to 10,000 μg / ml in the device reservoir was required. In experiments with the device, budesonide in ethanol vehicle was tested.

Based on prior experience, liposome preparations may be used to produce concentrated, viscous suspending agents. In the previous experiment with budesonide, a ratio of 1:25 (budenoside to DLPC weight ratio) was used. Based on the high content of DLPC required, the various budesonide-DLPC ratios were examined to confirm that a ratio of 1:15 was suitable. The formulation ratio is then gradually increased, first to 5 mg budesonide: 75 mg DLPC / ml and finally to 10 mg budesonide: 150 mg DLPC / ml. Increasing the ratio with other glucocorticoids (beclomethasone dipropionate or flunisoleide) is even more difficult due to unstable formulations. The 10 mg budesonide-150 mg DLPC formulation is stable and can be efficiently sprayed using an ATII nebulizer.

3 shows that increasing the concentration results in larger aerosol particles as the MMAD increases from 1.2 μm to 2.0 μm for high dose budesonide-DLPC. FIG. 4 shows that after 15 minutes of inhalation of this high dose budesonide-formulation, approximately 6 mg of budesonide can be inhaled six times more than the clinically most daily dose. The relationship between the aerosol emissions of low dose budesonide-DLPC and the aerosol emissions of high dose budesonide-DLPC is not proportional.

An exemplary “high dose” budesonide-DLPC liposome aerosol is in the range of about 12.5 mg budesonide / 225 mg DLPC / ml. Other phospholipids may replace DLPC in the high dose budesonide-liposomal formulations of the invention. These high dose budesonide-DLPC liposome aerosol formulations are clinically useful for the treatment of certain inflammatory lung diseases such as asthma and interstitial fibrosis, as well as immunologically mediated pulmonary allograft rejection, obstructive bronchiolitis, allergies and hypersensitivity. . This has proven useful in pediatric, adult and elderly patients using different nebulizer systems.

The following examples are intended to illustrate various aspects of the invention and do not limit the invention in any way.

Example 1

Liposomal Formulations: Preparation of High Dose Drugs / Liposomes

A freeze drying process was developed in the present invention to optimally formulate various drugs / liposomes. The optimal cyclosporin A to DLPC ratio was found to be 1: 7.5 weight ratio. To determine the maximum concentration to match the spray, an aerosol spray formulation was prepared with 10-30 mg of cyclosporin A and 75-225 mg of DLPC at an optimal CsA to DLPC ratio (1: 7.5 weight ratio). Judging by aerosol emissions and particle size for inhalation, a formulation containing 21.3 mg cyclosporine A: 160 mg DLPC was determined to be optimal. For optimized high dose cyclosporin A-liposomes, 100 mg of cyclosporin A (Sandoz Pharmaceuticals or Chemwerth Chemical Company) was synthesized with the synthetic alpha-lecithin 1,2-dilauroyl-sn-glycero-3-phosphocholine (Avanti Polar And 750 mg of DLPC from Lipids. While operating in a 37 ° C. greenhouse, the drug / DLPC is mixed in 20 ml tertiary butanol with stirring as described in Waldrep et al., Int'l J. of Pharmaceutics 97: 205-12 (1993). After mixing, the drug / lipid mixture is pipetted into glass vials, quickly frozen, and then lyophilized overnight to remove t-butanol, leaving a powder mixture. Multilayer liposomes are prepared by adding 10 ml of ultrapure water above phase transition temperature (Tc) at 25 ° C. to deliver a final standard drug concentration of 1 to 30 mg cyclosporin A: 7.5 to 225 mg DLPC / ml. The mixture is incubated at room temperature for 30 minutes with intermittent mixing to prepare multilayer vesicular liposomes. As another method, such liposome preparations can be prepared by rotary evaporation. An aliquot is taken to determine drug concentration by HPLC. This simple liposome preparation method is chosen because it can be easily increased in large batch production.

After swelling, an amount of liposome preparation is subjected to a microscope to check for size and presence of drug crystals before and after spraying using eyepieces. Drug-lipid binding (encapsulation efficacy) is determined using Percol gradient analysis as described in O'Riordan, et al., J. of Aerosol Med., In press (1996). It is not necessary to reduce the size of the multilayer vesicular high dose cyclosporin A-DLPC drug-liposomal formulation prior to spraying, which is the size of such drug-liposomes (heterogeneous starting mixture of 2.2 to 11.6 μm after swelling) that will reduce the jet diameter of the nebulizer. This is because it is further reduced during spraying (and continuous reflux) by the shear force generated by the extrusion through. These liposomes in the aerosol droplets range in size from 271 to 555 nm. The aqueous particles in the aerosol contain one to several liposomes. The diameters of these liposomes are smaller than the aqueous aerosol particles with which they are involved (Waldrep et al., Int'l J. of Pharmaceutics 97: 205-12 (1993)). After swelling, the formulation is used for spraying within a few hours. Sterile formulations are kept for several months at room temperature or in the refrigerator.

For high dose budesonide-DLPC liposome preparations, the optimal drug to lipid ratio is determined by testing different preparations at a budesonide-DLPC ratio of 1: 1 to 1:20. The 1:15 weight ratio was chosen as the optimal ratio for the high dose budesonide-DLPC formulation. Such high dose formulations are prepared by mixing 10-150 mg of budesonide with 150-225 mg of DLPC (as described above for cyclosporin A-DLPC). While operating in a 37 ° C. greenhouse, the drug / DLPC is mixed in 20 ml tertiary butanol with stirring. After mixing, the drug / lipid mixture is pipetted into glass vials, quickly frozen, and then lyophilized overnight to remove t-butanol, leaving a powder mixture. Multi-lamellar liposomes are prepared by adding 10 ml of ultrapure water above phase transition temperature (Tc) at 25 ° C. to deliver a final standard drug concentration of 1-15 mg budesonide: 15-225 mg DLPC / ml of solution. The mixture is incubated at room temperature for 30 minutes with intermittent mixing to prepare multilamellar vesicular liposomes. An aliquot is taken and subjected to HPLC analysis to determine drug concentration. As another method, such liposome preparations can be prepared by rotary evaporation. 8 depicts percol gradient analysis of Bud-DLPC liposomes (O'Riordan, et al., J. of Aerosol Med., In press (1996)). Once swelled, the multilayer vesicular budesonide-DLPC liposomes are stable for several weeks at room temperature. Sterile formulations are stable for several months. Benzalkonium chloride (10 mg / L) may be added as preservative.

Example 2

Liposomal Aerosols: Aerosol Drug-Liposome Treatment

In order to produce drug-liposomal aerosols, Aerotech II nebulizers (CIS-USA, Bedford USA) are used, but other commercial nebulizers may be used. ATII is a high-dose, efficient nebulizer that has been demonstrated to produce liposome aerosols in the size range 1 to 3 μm MMAD, which is optimal for peripheral lung transport. Vidgren, et al., Int'l J. of Pharmaceutics 115: 209 -16 (1994)]. The dry dry source is delivered to the nebulizer and its internal dry dry intake is measured via a regulated flow meter of 10 l / min. An initial reservoir volume of 5 ml is sufficient for aerosols for 15-20 minutes. If the processing period becomes longer, it is necessary to recharge the reservoir.

Example 3

Drug-Liposome Aerosol Particle Size Distribution

As Andersen's simulator of human lungs, using Andersen 1 ACFM non-volatile ambient particle size sampler (Graseby Andersen Instruments Inc., Atlanta, GA), see Waldrep et al., J. of Aerosol Med. 7: 1994 (1994)] to determine the aerodynamic particle size classification of drug-liposomal aerosols. Drug-liposomal aerosols generated from ATII nebulizers are collected using a vacuum pump (1 ACFM) by impacting on 8 aluminum stages at a standard sampling time of 0.5 minutes per experiment. At each stage drug concentrations in aerosol droplets of size 0-10 μm were collected (0 = 9.0-10.0 μm; 1 = 5.8-9.0 μm; 2 = 4.7-5.8 μm; 3 = 3.3-4.7 μm; 4 = 2.1- 3.3 μm; 5 = 1.1-2.1 μm; 6 = 0.65-1.1 μm; 7 = 0.43-0.65 μm) Determined after eluting with 10 ml of ethanol or methanol and HPLC analysis. USP artificial throat attached to the inlet of the impactor is used to remove a few aerosol particles larger than 10 μm. The final stage used a filter for glass fiber collection. After determining the drug concentration for each stage by HPLC, the mass-centered aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the drug-liposomes represent the cut-off diameters effective as vertical lines and below the particle range as horizontal lines. Calculation is based on a logarithmic probability scale indicating accumulation rate (by concentration) (KaleidaGraph 3.0). MMAD and GSD are determined by liposome drug content distributed in aerosol-comprising droplet arrangements (Waldrep et al., Int'l J. of Pharmaceutics 97: 205-12 (1993)). MMAD and GSD were determined by droplet arrangement rather than liposome size. The usefulness of these MMAD and GSD calculation methods was independently verified using a model 3300 TSI laser aerosol particle size classifier.

Example 4

Assessment of inhaled dose

To assess the inhaled dose of Bec-DLPC liposomes, nebulized samples were collected in a simulated human lung system as described in Smaldone et al., Am Rev Respir Dis 143: 727-37 (1991). do. Using a Harvard respirator, aerosol samples are collected from a ATII nebulizer (flow rate 10 l / min) onto Whatman GF / F filters using 15 breaths per minute at a tidal volume of 500 ml. This basic tidal volume 500 (450 for women) is determined for men from monograms adjusted for number of breaths, weight and gender. Aerosol samples are collected over a 15 minute spray time. The amount of cyclosporin A or budesonide accumulated on the filter is determined after extraction by HPLC analysis.

Example 5

Analysis of Pulmonary Artery Cyclosporin A: Solid Phase Extraction

Perform the following steps:

1. After inhaling cyclosporin A-DLPC liposome aerosol, mouse lung tissue is obtained. Add 10 μg of internal CSD standard (10 μl of 1 mg / ml stock). The tissue is homogenized in a blender or Wig-L-Bug tube (using 4-5 beads per tube).

2. The homogenized tissue as above is extracted in 1 ml of ultrapure water for 1-2 minutes. These volumes are for one tissue sample and if more than one tissue sample is collected it is diluted.

3. Add 2 ml of 98% acetonitrile / 2% methanol and shake the sample.

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

5. Collect the supernatant and add 5 ml ultrapure water to every 1 ml used for tissue extraction.

6. Prepare a Sep-Pak C18 column (Waters Sep-Pak Lite for single mouse tissue) and wash with 5 ml of 95% ethanol and 5 ml of ultrapure water. The sample is added slowly and washed with 5 ml of ultrapure water followed by 5 ml of 50% acetonitrile.

7. Transfer this eluate to a collection tube, elute with 1 ml methanol and elute with 0.5 ml water.

8. Wash the eluate twice with 1.5 ml hexane and remove contaminants by flushing the top layer.

9. The extracted eluate is evaporated to dryness using the reaction-vapor temperature fixed at the bottom with minimal air flow.

10. Reconstitute on 0.3 ml CSA mobile and sample analyze on HPLC.

Example 6

Drug Analysis by High Pressure Liquid Chromatography (HPLC): Budesonide Analysis

HPLC analysis is utilized for various purposes to determine the budesonide content of the liposome formulation, the encapsulation efficacy, and the budesonide content of the aerosol sample obtained using the lung simulator. The budesonide concentration is determined by HPLC analysis using a Waters WISP 717 Autosampler and Waters Nova-Pack C18 (3.9 x 150 mm) at room temperature. Peak detection is performed at 238 nm using a variable UV / Vis wavelength detector as quantified by Waters Millennium 2010 Chromatography Manager Version 2.15. The mobile phase utilized in these studies is 50:50 ethanol / water at a flow rate of 0.6 ml / min (Andersen & Ryrfeldt, J Pharm Pharmacol 36: 763-65 (1984)). Analytical samples are directly dissolved in ethanol (to dissolve the liposomes). Drug standards are prepared from ethanol stock maintained at -80 ° C.

Example 7

Drug Analysis by High Pressure Liquid Chromatography (HPLC): Cyclosporin A Assay

Cyclosporin A in liposome formulations and aerosol samples (to determine cyclosporin A content and encapsulation efficacy) are measured by HPLC. A Waters (Milford, MA) WISP automated sample injector and a Supelco LC-1 column heated to 75 ° C are used for this analysis. The mobile phase is 50% acetonitrile, 20% methanol and 30% water. Charles et al., Ther. Drug Monitor, 10: 97-100 (1988). Peaks at 214 nm are detected using a variable wavelength detector and quantified using Waters Millennium 2010 Chromatography Manager Version 2.15. Analytical samples are directly dissolved in ethanol (to dissolve the liposomes). Drug standards are prepared from ethanol stock maintained at -80 ° C.

Example 8

Drug Analysis by High Pressure Liquid Chromatography (HPLC): DLPC Analysis

See, Grit and Commelin, Chem. & Phys. of Lipids 62: 113-22 (1992). Waters 717 WISP Autosampler and Sperisorb S5 amino column (0.25 cm x 4.6 mm, 5 μm) were prepared with acetonitrile, methanol and 10 mM ammonium / trifluoroacetic acid, pH 4.8 (64: 28: 8 v: v v) Use with mobile phase. Peaks are detected with a mass evaporative detector (SEDEX 55, Sedre, France) and quantified by Waters Millennium 2010 Chromatography Manager Version 2.15. Analytical samples are dissolved directly in ethanol or methanol (to dissolve the liposomes).

Example 9

Lung Model for Drug Testing in Mice: Acute Inflammation Assay: (LPS) Bronchial Intestinal Technique

Gram negative cell wall lipopolysaccharide (LPS) is used to induce acute pulmonary artery inflammation at regenerative levels in mice. E. E. generated from PBsj 1600 nebulizer (100 μg / ml reservoir concentration; 60 ng delivery capacity). 10 minutes exposure to E. coli 055: B5 LPS (Sigma) aerosol induced a strong inflammatory response as measured by accumulation of PMN in the alveoli in response to the synthesis of chemotactic cytokines (detectable at 3 hours; irritating; Peak reaction after 6 hours). Several hours after exposure to LPS aerosol, mice are sacrificed by methoxyflurane anesthesia and bleeding through the abdominal aorta. The trachea is surgically exposed and cannulated using PE50 tubing (outer diameter 0.965 mm, Clay Adams). The lungs are washed five times with about 0.1 ml volume using a total volume of 2.0 ml Hanks balanced salt solution (HBSS; Ca / Mg free of EDTA). The yield is typically about 85% elongated fluid recovery. The leukocytes thus generated are counted with a hemocytometer, centrifuged and stained. From different counts, drug efficacy is recognized by a decrease in leukocyte counts and by a reduced number of PMN and / or myeloperoxidase positive cells for residual macrophages and / or myeloperoxidase negative cells. Using this assay as a standard, drug-liposomal aerosol regimens for biological activity are tested through reduction of acute inflammatory intracellular influx of the airways. FIG. 7 depicts the anti-inflammatory effect of high doses of Bud-DLPC on pulmonary bronchoalveolar lavage (BAL) leukocytes in response to LPS (endotoxin) challenge.

Example 10

Cytology: Lung Intestine

Cell preparations (egen, thymic cells, lymph nodes or splenocytes) are counted in a hemocytometer, centrifuged onto slides (using Miles Cyto-Tek) and stained with Wright-GMSA. May-Grunwald-Giemsa or leukocyte peroxidase is stained according to this formulation. The coefficient of discrimination is measured by microscopic observation under oil immersion. The biological effect of the drug-liposomal aerosol regimen is recognized by a decrease in the total white blood cell count and by a reduced number of PMN or myeloid peroxidase positive cells for residual macrophages.

Example 11

Inhibition of antigen / mitogen-induced lymphocyte embryonic development in vitro by CsA isolated from mouse lung tissue following aerosol delivery of CsA-DLPC liposomes

Biological Activity of CsA Transported to Lungs by Liposomal Aerosols

For testing, a primary immune response is generated in pulmonary-bound lymph nodes within the bronchial sheath-bound lymphoid tissue and mediastinum. Balb / c mice were immunized locally with intranasal precipitated ovalbumin (AP-OVA (80 μg) supplemented with Bordetella pertussis vaccine), and then mice were sacrificed after 7 days and mediastinal After removal, lymphocytes are isolated for in vitro analysis. Proliferation assays were activated with sensitized antigen ovalbumin or non-specific T-cell mitogens to modify stimulation of lymphocytes, Con A + coculture with CsA isolated from mouse lung tissue by solid phase extraction and HPLC Quantify by The uptake of ³ [H] -TdR into DNA is measured between 48 and 72 hours. Inhibition of specific or non-specific lymphocyte activation is demonstrated by eliminating or reducing antigen-specific stimulation or mitogen reactivity inhibition.

Example 12

Physical and chemical analysis:

Surface Tension & Viscosity : Surface tension (dynes / cm) is measured using a Tensiomat (Model 21, Fisher Scientific, Indiana, PA). Platinum iridium rings of known dimensions are cleaned from the surface of the liquid to be tested under precisely controlled conditions. The "visible" value read from the instrument is multiplied by the correction factor F (inserting the ring measurement dimensions, the density of the liquid and other variables) (as directed by the manufacturer). Viscosity measurements are performed using a Gilmont Falling Ball Viscometer (Gilmont Instruments, Barrington, IL). Viscosity (centipoise) is measured at ambient room temperature.

Determination of the size of drug-liposomes : Determination of the particle size of drug liposome solutions by quasi-elastic light scattering using a Nicomp® Model 370, submicron particle size classifier (Program Version 5.0, Nicomp Particle Size Classification System, Santa Barbara, CA) do. Drug-liposomal samples dispersed in water are analyzed according to the manufacturer's instructions and the data is shown as intensity weighed vesicle sizes. Drug-liposomal mean particle diameters were initially determined from reservoir samples, 10 minutes after spraying, as described in Waldrep et al., Int'l J. of Pharmaceutics 97: 205-12 (1993), and Measured on recovered aerosol sample using AGI-4 impinger.

The results (plotted by DLPC concentration) in FIG. 9 indicate that the aerosol emissions of DLPC liposomes were increased up to a concentration of 170 mg / ml, and the emissions were reduced at higher concentrations. Expanding these data to CsA-DLPC liposomes showed similar results, with maximum liposome aerosol emissions at 21.3 mg CsA: 160 mg DLPC / ml (FIG. 9). For Bud-DLPC liposomes, a formulation consisting of 12.5 mg Bud: 187.5 mg DLPC / ml was used to demonstrate maximum aerosol DLPC emissions. Analysis of the nebulizer liquid vesicle release amount shown in FIG. 10 (plotted by DLPC) shows a concentration dependent reduced emissions as measured by mass aerosolization per minute.

Increasing the liposome concentration similarly increases aerosol emissions to the critical point simultaneously (FIG. 11) (plot by drug concentration). Measurement results for aerosolized drug emissions of CsA and Bud by HPLC analysis showed the maximum concentration for spraying (FIG. 11). For CsA-DLPC liposomes, the maximum emission is 21.3 mg CsA: 160 mg / ml. For Bud-DLPC, the maximum emissions are 12.5 mg Bud: 187.5 mg DLPC / ml. Physicochemical analysis of these liposome formulations showed that the viscosity increased side by side (plot by DLPC concentration) (FIG. 12). The results for DLPC, Bud-DLPC and CsA-DLPC are similar. The viscosity of Bud-DLPC formulation is about 20% lower than DLPC alone. The viscosity of CsA-DLPC is consistently the lowest and does not vary from 16 mg CsA / 120 mg DLPC to 24 mg CsA / 180 mg DLPC / ml. These results show that for each formulation, there is a maximum viscosity consistent with the sprayed aerosol emissions; If these limits are exceeded, increasing drug-liposome concentrations will not increase emissions.

The results in FIG. 13 (plotted by DLPC concentration) indicate that adding CsA and Bud to DLPC liposomes reduces the surface tension of the formulation. The decrease in surface tension is concentration dependent until some time and stabilizes around about 100 mg DLPC / ml. There is no obvious correlation between the aerosol emissions of the liposome formulations and the surface tension. However, increasing the concentration of the liposome formulation results in inverse proportion to the surface tension and viscosity measurements.

Analysis of the drug-liposomal formulations prior to spraying by quasi-elastic light scattering showed a heterogeneous starting size range of about 2.2 to 11.6 μm, which is at or near the precise upper limit of the Nicomp 370. After spraying, minimal differences were detected in any of the formulations. The size range of the liposomes in the nebulizer reservoir ranges from 294 to 502 nm and the aerosol samples collected by the AGI-4 impinger range from 271 to 555 nm.

A high dose formulation of drug-liposome consisting of 10 mg Bud: 150 mg DLPC and CsA 20 mg: DLPC 150 mg is selected for further aerosol studies. Analysis using the Andersen Cascade Impactor showed 2.0 μm MMDA / 1.5 GSD for Bud-DLPC and 2.0 μm / 1.8 for CsA-DLPC (Table 2). Analysis of these formulations in a simulated human model of 15 BPM and 500 ml tidal volume revealed that it took 3 minutes inhalation time to inhale 1,000 μg of Bud daily dose in liposomes and 12 minutes to inhale below 5,000 μg. Appeared (Table 2). CsA-DLPC inhalation results in the simulated lung model require 4 minutes to inhale 5,000 μg of nebulized CsA in liposomes for high dose CsA-DLPC; It indicates that 11.5 minutes is required to inhale 15,000 μg of CsA (Table 2). These results indicate the high ability of liposomes for aerosol drug delivery.

The present invention relates to high dose cyclosporin A liposome aerosol compositions comprising up to about 30 mg / ml cyclosporin A in about 225 mg or less of phospholipid per ml of starting reservoir concentration. Preferably, the liposome aerosol composition comprises up to about 21.3 mg / ml cyclosporin A in up to about 160 mg of phospholipid per ml of starting reservoir concentration. Generally, in the cyclosporin A liposome aerosol compositions of the present invention, the particle size as measured by the center of mass aerodynamic diameter is in the range of about 1.0 to about 3.0 μm. In addition, for cyclosporin A liposome aerosol compositions, the ratio of cyclosporin A to phospholipids is from about 1 to about 7.5. Preferably, such phospholipids are selected from the group consisting of egg yolk phosphatidylcholide, hydrogenated soybean phosphatidylcholide, dimyristoylphosphatidylcholide, dioleoliyl-dipalmitoyloleyl phosphatidylcholide and dipalmitoyl phosphatidylcolide . In general, the cyclosporin A liposome aerosol compositions of the invention can be used to treat immunologically mediated lung diseases. Preferably, such immunologically mediated lung disease is selected from the group consisting of allograft rejection, obstructive bronchiolitis, allergy, hypersensitivity and asthma.

The invention also relates to high dose budesonide-liposomal aerosol compositions comprising up to about 15 mg / ml of budesonide in up to about 225 mg of phospholipid per ml of starting reservoir concentration. Most preferably, the high dose of budesonide-liposomal aerosol composition comprises up to about 15 mg / ml of budesonide in up to about 225 mg of phospholipid per ml of starting reservoir concentration. For the budesonide-liposomal aerosol composition of the present invention, the particle size as measured by the center of mass aerodynamic diameter is in the range of about 1.0 to about 2.0 μm. Generally, for budesonide-liposomal aerosol compositions, the ratio of budesonide to phospholipids is from about 1 to about 15. Representative examples of phospholipids are as mentioned above. Typically, the budesonide-liposomal aerosol compositions of the present invention can be used to treat immunologically mediated lung diseases and inflammatory lung diseases. Representative examples of such immunologically mediated lung diseases and inflammatory lung diseases are as mentioned above. Cyclosporin A indirectly inhibits inflammation by blocking the immune response. Budesonide suppresses both immune response and inflammation. These lung diseases can have both components.

The invention further includes treating a patient with an immunologically mediated lung disease, comprising administering to the patient with an immunologically mediated lung disease a pharmacologically acceptable dose of a high dose cyclosporin A liposome aerosol composition. It is about how to. The invention also treats a patient with an immunologically mediated lung disease, comprising administering to the patient with an immunologically mediated lung disease a pharmacologically acceptable dose of a high dose budesonide liposome aerosol composition. It is about how to. Concentrations for the preparation and administration of suitable pharmaceutical compositions will be readily apparent to those skilled in the art from the teachings presented herein.

The invention also relates to high dose cyclosporin A liposome aerosol compositions comprising up to about 30 mg / ml cyclosporin A in about 225 mg of dilauroylphosphatidylcholine per ml of starting reservoir concentration. Additionally, there is provided a high dose of budesonide liposome aerosol composition comprising up to about 15 mg / ml of budesonide in about 225 mg of dilauroylphosphatidylcholine per ml of starting reservoir concentration.

The present invention generally relates to high dose pharmaceutical liposome aerosol compositions comprising about 12 to 30 mg / ml of pharmaceutical compound and about 130 to 375 mg of phospholipid per ml of starting reservoir concentration. For example, the present invention relates to anti-inflammatory glucocorticoids, immunosuppressive compounds, antifungal compounds, antibiotic compounds, anti-viral compounds and anticancer compounds carried through high dose liposome aerosol compositions in phospholipids.

All patents or publications mentioned in the specification are indicative of the level of skill in the art to which this invention pertains. These patents and publications are incorporated herein by reference to the same extent as if each individual publication was specifically and individually indicated.

Those skilled in the art will readily recognize that the present invention is well adapted to carry out the objects and to obtain the uses and advantages mentioned. Embodiments of the present invention, together with the described methods, processes, treatments, molecules and specific compounds, are merely representative of preferred embodiments and do not limit the scope of the invention. Changes in the methods and uses encompassed within the subject matter of the present invention as defined by the claims will be apparent to those skilled in the art.

Claims (12)

High dose cyclosporin A liposomes containing 5 mg / m1 to 30 mg / ml cyclosporin A and 37.5 mg to 225 mg of phospholipid per ml of starting reservoir concentration, with a particle size ranging from 1.0 to 3.0 μm as measured by center of mass aerodynamic diameter Aerosol composition. The cyclosporin A liposome aerosol composition of claim 1 comprising 5 mg / ml to 21.3 mg / m cyclosporin A and 37.5 mg to 160 mg of phospholipid per ml of starting reservoir concentration. The cyclosporin A liposome aerosol composition according to claim 1, wherein the ratio of cyclosporin A to phospholipid is _1 to _7.5. The phospholipid of claim 1, wherein the phospholipid is selected from the group consisting of egg yolk phosphatidylcholine, hydrogenated soybean phosphatidylcholine, dimyristoylphosphatidylcholine, dilauroylphosphatidylcholine, dioleoyl-dipalmitoyloleylphosphatidylcholine and dipalmitoyl phosphatidylcholine Cyclosporine A liposome aerosol composition. The cyclosporin A liposome aerosol composition of claim 1 for treating allograft rejection, obstructive bronchiolitis, allergy, hypersensitivity and asthma. A high dose of liposome with a particle size ranging from 1.0 to 3.0 μm, comprising 1 mg / m1 to 15 mg / ml of budesonide in phospholipid 15 mg to 225 mg per ml of starting reservoir concentration, and measured by mass center aerodynamic diameter Budesonide-liposomal aerosol composition. 7. The budesonide-liposomal aerosol composition of claim 6 comprising 1 mg / m1 to 12.5 mg / ml of budesonide in 15 mg to 187.5 mg of phospholipid per ml of starting reservoir concentration. 7. The budesonide-liposomal aerosol composition of claim 6, wherein the ratio of budesonide to phospholipids is from _1 to _15. 7. The phospholipid of claim 6, wherein the phospholipid is selected from the group consisting of egg yolk phosphatidylcholine, hydrogenated soybean phosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dioleoliyl-dipalmitoyloleylphosphatidylcholine and dipalmitoyl phosphatidylcholine Budesonide-liposomal aerosol composition. The budesonide-liposomal aerosol composition according to claim 6 for use in treating allograft rejection, obstructive bronchiolitis, allergies, hypersensitivity and asthma. The high dose cyclosporin A liposome aerosol composition according to claim 2, wherein the phospholipid is dilauroylphosphatidylcholine. 8. The high dose of budesonide-liposome aerosol composition according to claim 7, wherein the phospholipid is dilauroylphosphatidylcholine.