JPH0380773B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to pharmaceutical compositions and in particular to the formulation of substances for inhalation.

Sodium cromoglycate has been known for many years for the treatment of allergic conditions such as asthma, hay fever and vernal keratoconjunctivitis. However, this has the disadvantage that its duration of action is relatively short.

According to the present invention, a pharmaceutical composition comprising a liposome and sodium cromoglycate is provided.

By administering the liposomes of the invention directly to the site of the allergic condition, such as the lungs, it is possible to increase the level of sodium cromoglycate retention at that site, thereby achieving an increased duration of action.

The initial steps of liposome preparation according to the invention conveniently follow procedures described in the art. That is, the lipid starting material is dissolved in a solvent such as ethanol or chloroform, which is then evaporated.
The resulting lipid layer is then dispersed in a selected aqueous medium containing an appropriate concentration of sodium cromoglycate. However, contrary to common practice, it is preferred not to sonicate the liposomes thus prepared. This is because such treatment reduces the size of the liposomes.
Liposomes prepared by the method of the invention will typically have a range of sizes. The liposomes of the present invention preferably have a diameter of 100 nm to 10 μm, more preferably 1 μm to 7 μm. for example,
It is known that liposomes with diameters up to 5000 nm can be easily phagocytosed. Preferably, the liposomes are fractionated to remove substantially all those having a diameter of 100 nm or less, preferably 1 μm or less. Fractionation can be conveniently accomplished by column gel chromatography using, for example, cross-linked dextran or agarose, the size of the gel being chosen according to the desired liposome size. Alternatively, liposomes can be fractionated using ultracentrifugation or by dialysis using, for example, polycarbonate membrane filtration.

A wide variety of lipid materials can be used to form liposomes, including natural lecithins, such as those derived from eggs and soybeans, and synthetic lecithins. Lipids that are non-immunogenic and can be degraded by the organism are preferred. The properties of the lipid, such as its phase transition temperature, can have a significant influence on the retention and uptake of liposomes into the target organ, and for that reason well-defined synthetic lecithins are preferred over natural lecithins. . Synthetic lecithins that can be used, along with their respective phase transition temperatures, include di(tetradecanoyl)phosphatidylcholine (hereinafter abbreviated as "DTPC") (23°C);
Di(hexadecanoyl)phosphatidylcholine (hereinafter abbreviated as "DHPC") (41°C) and di(octadecanoyl)phosphatidylcholine (hereinafter abbreviated as "DOPC") (55°C). Preference is given to using di(hexadecanoyl)phosphatidylcholine as the sole lecithin or as the main amount of lecithin, optionally together with minor amounts of di(octadecanoyl) or di(tetradecanoyl) compounds. Other synthetic lecithins that may be used are unsaturated synthetic lecithins, such as di(oleyl).
phosphatidylcholine and di(linoleyl)phosphatidylcholine. Preferably, the synthetic lecithin or lipid mixture has a phase transition temperature in the range of 35-45°C. In addition to the predominant liposome-forming lipid or lipids, which is usually a phospholipid, to modify the structure of the liposome membrane to make it more fluid or more rigid depending on the nature of the predominant liposome-forming lipid or lipids. Other lipids such as cholesterol or cholesteryl stearate may be included (eg in proportions of 5-40% w/w of total lipids). A third component which may optionally be included is a substance imparting a negative charge, such as phosphatide acid, dicetyl phosphate or bovine ganglioside;
or a substance imparting a positive charge, such as stearylamine acetate or cetylpyridium chloride. The charged component may be included in a proportion of 1-20% w/w of total lipids.

A wide range of ratios of sodium cromoglycate to lipid can be used during formation, depending on the lipid used and the circumstances. However, generally 1 part by weight of sodium cromoglycate to 0.01 part of lipid
A range of from 100 parts by weight, preferably from 0.05 to 20 parts by weight, most preferably from 0.1 to 10 parts by weight, has been found to be suitable. It is preferred to use as high a proportion of sodium cromoglycate as possible.

The sodium cromoglycate concentration in the aqueous phase during liposome formation is preferably between 0.01 and 50.
mg/ml, and preferably 0.1 to 20 mg/ml,
For example 10 or 20 mg/ml.

The aqueous phase contains metal ions of groups a, b, b and b of the periodic table and reduction metals, especially Pb ⁺⁺ ,
Ca ⁺⁺ , Mg ⁺⁺ , Fe ⁺⁺ , Fe ⁺⁺⁺ and Zn ⁺⁺ ions
It is preferably contained in an amount of 20 ppm or less.

The aqueous phase can be made isotonic using sodium chloride. Additionally, the aqueous phase may also contain potassium chloride.

The aqueous phase can be adjusted to a pH of 6 to 50% by addition of a suitable acid or base or by addition of a suitable buffer such as tris(hydroxymethyl)methanamine (Tris).
8, preferably PH6.5-7.5.

The concentration of lipids dispersed in the aqueous phase is preferably
0.1-150mg/ml, more preferably 0.5-50mg/ml
And most preferably 1 to 30 mg/ml.

It is preferred that the liposome formulation has a half-life (efflux rate) of about 12-48 hours, preferably 12-24 hours at 37°C. Half-life can be measured by conventional methods, such as the dilution method. The half-life of the formulation can be varied by varying the proportions of various lipids used to make the liposomes.

The compositions of the invention can be used to treat asthma by instilling an atomized aqueous suspension of sodium cromoglycate-liposomes into the lungs. The compositions of the invention may be used as eye drops in the treatment of allergic eye conditions such as vernal keratoconjunctivitis, ocular symptoms of hay fever and/or peripheral infiltrates.

The composition may also be used by esophageal administration in the treatment of gastrointestinal diseases such as ulcerative colitis and food allergies. Enemas incorporating the compositions of the invention can be used in particular for the treatment of intestinal diseases of allergic origin. The compositions of the invention may also be used, for example, in the treatment of hay fever, by administration to the nose as a nasal spray, and in the treatment of skin conditions, such as chronic skin diseases in mammals, particularly humans. Skin diseases that can be treated include those involving skin breast cell and/or antibody-antigen reactions, and eczema, drug rash, psoriasis, dermatitis, pemphigus herpetiformis and chronic skin ulcers.

The composition prepared as described above is an aqueous suspension of liposomes in which sodium cromoglycate is partitioned between a free aqueous phase and a liposome phase.

These aqueous formulations have useful and unexpected properties in that the aqueous phase can provide an initial "priming" amount of sodium cromoglycate and the liposomal phase can provide a maintenance amount of sodium cromoglycate. I found out. This has the effect of increasing the duration of action of sodium cromoglycate.

According to the invention, therefore, an aqueous suspension is provided consisting of sodium cromoglycate distributed between a free aqueous phase and a liposomal phase.

The total concentration of sodium cromoglycate in the aqueous suspension is between 0.01 and 50 mg/ml, preferably between 0.1 and 20
Preferably it is mg/ml.

The percentage of sodium chromoglycate associated with the liposomes is 2-35% w/w, e.g. 4-20
% is preferable. The percentage of sodium chromoglycate associated with liposomes can be determined by conventional methods, such as centrifugation.

Alternatively, an aqueous suspension of sodium cromoglycate partitioned between the aqueous phase and the liposome phase can be concentrated to form a liposome gel, for example by centrifugation, ultrafiltration or dialysis. This gel can be used in several ways. For example, it can be incorporated into an ointment base and resuspended in water or an isotonic buffered saline solution, which may optionally contain sodium cromoglycate. Such formulations can be prepared immediately before use from the liposomal gel and suitable excipients.

The dosage given will vary depending on the particular composition used, the condition being treated and its severity. It is preferred to use an effective amount of sodium cromoglycate liposomes in the treatment of these conditions (e.g. 0.1-20
mg).

The invention is illustrated by the following examples, but is not limited thereto.

General Preparation of Liposome-Containing Sodium Chromoglycates: Add the desired amount (e.g. 20 mg) of a suitable phospholipid or phospholipid mixture (e.g. lecithin, DTPC, DHPC or DOPC), if desired, to any other lipid-soluble component (e.g. cholesterol). , cholesteryl stearate) into a round bottom flask. The lipid component is dissolved in a small amount (approximately 5 ml) of a suitable solvent (eg ethanol) and evaporated to dryness under reduced pressure using a rotating membrane evaporator to obtain a thin film of phospholipid on the internal surface of the flask.

An aqueous solution of sodium cromoglycate at an appropriate concentration (e.g. 1 mg/1 ml) is added to an aqueous medium (e.g. 0.9
% w/v saline solution, buffer solution, etc.) and dissolve a weighed amount of sodium chromoglycate in 20 ml and, if desired, adjust the pH of the resulting solution to a range of 6 to 8 by addition of acid or alkali. by adjusting to a selected value within. An aqueous solution of sodium chromoglycate is warmed to 20° C. above the phase transition temperature of the lipid(s), added to the lipid film in the flask, and the flask is gently shaken until all the lipid film is dispersed. The resulting suspension contains liposomes with a size of 200 nm to 10 μm.

This suspension was equilibrated at 37°C for 48 hours.

These suspensions contain sodium cromoglycate distributed between a free aqueous phase and a liposomal phase.

After 24 hours, the suspension often separates to form a colloidal white precipitate, which is easily redispersed on shaking.

The sodium cromoglycate liposome compositions exemplified below were prepared using the general procedure described above.

1 Egg lecithin 20mg Sodium cromoglycate 200mg Demineralized water 20ml 2 Egg lecithin 20mg Sodium cromoglycate 20mg Demineralized water 20ml 3 DTPC 20mg Sodium cromoglycate 2mg 0.9% w/v salt solution 20ml 4 DTPC 20mg Sodium cromoglycate 2mg 0.9% w/v saline solution 20ml 5 DTPC 20mg Sodium cromoglycate 200mg 0.9% w/v saline solution 20ml 6 DTPC 200mg Sodium cromoglycate 200mg 0.9% w/v saline solution 20ml 7 DTPC 400mg Sodium cromoglycate 200mg 0.9% w/v saline solution 20ml 8 DHPC 200mg Sodium cromoglycate 200mg 0.9% w/v saline solution 20ml 9 DOPC 200mg Sodium cromoglycate 200mg Demineralized water 20ml 10 DTPC 133mg Cholesterol stearate 67mg Sodium cromoglycate 200mg Demineralized water 20ml 11 DHPC 133mg Cholesteryl stearate 67mg Sodium cromoglycate 200mg Demineralized water 20ml 12 DHPC 133mg Cholesterol 67mg Sodium cromoglycate 200mg Demineralized water 20ml 13 DHPC 20mg Sodium cromoglycate 200mg 0.9% w/v saline solution 20ml 14 DHPC 7 5mg sodium Cromoglycate 102.5mg subsalt potassium in water (150mM) and PH7.4
Tris buffer (10mM)) 10ml 15 DHPC 70mg DTPC 30mg Sodium cromoglycate 200mg 0.9% w/v saline solution 10ml 16 DHPC 180mg Sodium cromoglycate 200mg Cetylpyridinium chloride 20mg 0.9% w/v saline solution 200ml Associated with liposomes Measurement of % Sodium Chromoglycate The equilibrated sodium chromoglycate liposome dispersion is centrifuged at 70000G for 1 hour.
A portion of the supernatant is analyzed in an ultraviolet spectrophotometer at 326 nm to determine the free sodium chromoglycate concentration.

The percentage of sodium chromoglycate associated with liposomes is determined from the following relationship.

% of sodium cromoglycate associated with liposomes = [total cromoglycate] - [cromoglycate in supernatant] / [total cromoglycate] × 10
0 The following association % was measured.

Example 5 4.5% w/v Example 13 8.23% w/v Example 14 14.00% w/v Sodium cromoglycate release rate from liposomes and liposome half-life Sodium cromoglycate release rate from liposomes It can be determined by centrifuging at 70000G as described above, discarding the supernatant and resuspending in isotonic saline buffered to pH 7.4. 37℃
A portion of the stirred resuspended liposomes was centrifuged at intervals, and the sodium chromoglycate concentration in the supernatant was measured by ultraviolet spectrophotometry. The release constant k of liposomes is determined by plotting ln [chromoglycate released] versus time.

The half-life of liposomes, t1/2, is expressed by the relationship t1/2=ln2/k is given by

The half-life of liposomes is also determined by M. Ahmed et al., “Biochemical Pharmacology,” Vol. 29, No. 2361.
It can also be measured using the dilution method described on page 2365 (1980).

Measurement of Flow Rate and Permeability Coefficients [Membrane Preparation] White, colorless mice of either sex, 10-12 weeks old, were sacrificed by twisting the neck and the skin on the back was removed with minimal manipulation. Subcutaneous fat, visible as individual balls, was removed. The skin samples were inspected for any signs of damage prior to use. One skin sample was used for the diffusion cell and placed face side up over the opening in the upper part of the diffusion cell and secured with a "0" ring. Excess skin was scraped off before assembling the cells.

[Assembly of diffusion cells]

After fixing the membrane, a small amount of silicone grease was applied to the "0" ring on the upper part. The upper portion was then pressed firmly until it was properly seated within the lower chamber. The chamber was then filled with saline that had been equilibrated to 37°C. The volume of each cell was adjusted individually so that the skin membrane remained level. The filling volume was marked on the side arm.

[Experimental operation]

A set of eight diffusion cells was placed on a carrier plate held in a thermostatically controlled 37°C water bath set and equilibrated. Each cell was placed on a magnetic stirrer motor under water and the water level was adjusted to approximately the same level as the skin surface. This ensured that the skin surface temperature remained at 30°C.

The vehicle to be studied was applied by adding from a micropipette. The formulation was then distributed evenly over the exposed skin surface using a small glass rod. The weight of each species applied was determined by accurately weighing at least 10 samples added with a micropipette or syringe.

Following vehicle application, the magnetic stirrer is turned on and at appropriate time intervals 1.0 ml samples of the receiver fluid are removed from the side arms and immediately replaced with fresh saline solution previously equilibrated to 37°C. Replaced with. These samples were then frozen until analyzed for drug by high performance liquid chromatography (HPLC).

Diffusion cells were used with at least three replicates for each formulation studied.

〔Data processing〕

Assuming that only passive diffusion occurs while the drug is transported through the skin, the permeation rate can be given by Fick's Law.

Here, J is the flow rate, that is, the amount of drug diffusing per unit area per unit time, P is the permeability coefficient, and ΔC is the concentration difference in the stratum corneum.

Test Examples Methods Liposome Preparation 266 mg of di(hexadecanoyl)phosphazylcholine (DHPC) was placed in a long neck round bottom flask and dissolved in diethyl ether/chloroform (1:1). Ten milliliters of a sterile 3.2% solution of sodium chromoglycate (SCG) in 0.9% NaCl was added to the flask and sonicated for 6 minutes at 50°C to promote emulsification. The organic solvent was slowly removed at 45°C,
The suspension was passed through a polycarbonate membrane filter (pore size =
1.0 μm), kept at 45° C. for 1 hour, and then placed in a dialysis tank to remove free chromoglycate. Add this liposome preparation to 0.9% NaCl100
The solution was dialyzed for 120 hours at 4° C. with 4 changes over 24 hours. Immediately prior to administration to volunteers, liposomes were removed from the tank and analyzed for free and total sodium cromoglycate.

Human studies Five healthy non-smoking males, aged 18-40 years, with no known hypersensitivity to sodium cromoglycate, and with normal lung function (respiratory capacity, FEV, FEV ₁ ) participated in this study. did. Each person received an air jet nebulizer (Hudson; Henleys Medical Supplies Ltd.,
20 mg of sodium cromoglycate delivered by UK) was inhaled as a 0.9% aqueous solution in NaCl b DHPC/Col (1:1) liposomal formulation.

The nebulized product was generated by compressed air at 172 kPa (25 psi) and inhaled through the mouthpiece. Volunteers maintained a deep, slow inhalation with a breath-holding period before exhalation, with a breathing rate of 6 to 8 cycles per minute. Nebulization lasted approximately 8 minutes and 15 minutes for free and liposomal sodium cromoglycate, respectively, times calculated for delivery of a 20 mg dose.

0, 2.5, 5, 7.5, 10, 15 after starting inhalation of medicine
20, 30, 40, 50 and 60 minutes, and 2, 3, 4,
At 6, 8 and 10 hours, 5 ml blood samples were taken through an indwelling catheter inserted into a forearm vein. After 10 hours of sample, remove the catheter and remove the 24
Two additional blood samples were taken by venipuncture at 50 and 25 hours. Blood sample at 3000 rpm for 10 minutes
The plasma was transferred to lithium heparin tubes and stored at -20°C.

Plasma concentrations of sodium cromoglycate after inhalation of therapeutic doses of the drug were determined by Brown et al., J. Clin.
Pharmacol., 1981, 11, 425 and Ann.Clin.
It was determined using a radioimmunoassay developed by Biochem. 1983, 20, 31.

Results and Discussion The mean plasma concentration time profiles for free drug and liposomal drug are shown in the figure.

Patel et al., J. Clin. Pharmacol., 1986, 21, 231
shows that sodium cromoglycate provides up to 66% protection against exertion-induced edge breathing when plasma concentrations are 4 ng/ml or higher, and approximately 50% protection occurs at plasma concentrations of 2 ng/ml. showed that. Conventional treatment with pressure pack aerosol (1 mg or 5 mg per drive) resulted in plasma concentrations of sodium cromoglycate of less than 5 ng/ml after 4 drives, with a short duration of action (approximately 4 hours). .

This study showed that plasma levels of sodium cromoglycate could be kept at detectable levels for more than 24 hours after inhalation of a single dose of a liposomal formulation, while the same dose was administered as a solution. It was demonstrated that the drug was undetectable in 24-hour plasma samples.

The clinical use of sodium cromoglycate is in the prophylactic treatment of asthma, so it is desirable to maintain constant therapeutic plasma concentrations of the drug. To achieve this, sodium cromoglycate is usually administered in a multiple dose format, 4 to 8 times at a time (BNF, 1986). Repeated drug administration results in drug accumulation in the body, which can be controlled to obtain a desired plasma concentration time profile. It is possible to calculate the maximum, minimum, and average steady state concentrations of sodium cromoglycate after repeated administration of the liposomal formulation prepared in this study. Multiple dosing with a formulation producing the plasma concentration-time profile shown in the figure resulted in maximum and minimum plasma sodium cromoglycate levels of 6.53 and 2.26 ng/ml, respectively, with a mean steady state of 3.17 ng/ml. concentration. Thus, plasma levels of sodium cromoglycate, which is known to have a protective effect during exertion-induced asthma (Patel et al., 1986), can be maintained by inhaling this formulation at 24-hour intervals. It will be possible.

CONCLUSIONS: Repeated administration of such liposomal formulations at 24-hour intervals will maintain plasma concentrations at levels known to be associated with therapeutic effects. In contrast, conventional treatment with nebulized sodium cromoglycate solutions requires dosing several times a day.

[Brief explanation of drawings]

The diagram shows sodium cromoglycate (SCG)
This is a graph showing the relationship between the elapsed time and the concentration of SCG in plasma when 20 mg is administered to humans in the form of an aqueous solution (●-●) or in the form of liposomes (○-○).

Claims (1)

[Scope of Claims] 1. An aqueous suspension comprising liposomes and sodium cromoglycate, wherein the sodium cromoglycate is distributed between a free aqueous phase and a liposome phase, the liposomes comprising one or more types of liposomes. A pharmaceutical composition comprising more than one natural or synthetic lecithin, wherein the weight ratio of sodium cromoglycate to lipid in said liposomes ranges from 0.01 to 100. 2. The pharmaceutical composition according to claim 1, wherein the liposomes have a diameter of 100 nm to 10 μm. 3. A pharmaceutical composition according to claim 1, wherein the lecithin or lecithin mixture has a phase transition temperature of 35-45°C. 4. The pharmaceutical composition according to claim 1, wherein the liposome contains cholesterol and/or cholesteryl stearate. 5. The liposome contains an additional component having a negative or positive charge selected from the group consisting of phosphatide acid, dicetyl phosphate, bovine brain ganglioside, stearylamine acetate and cetylpyridinium chloride. Pharmaceutical composition of. 6 The total concentration of sodium cromoglycate is 0.01
50 mg/ml. 7. The pharmaceutical composition of claim 1, wherein the percentage of sodium cromoglycate associated with the liposomes is between 2 and 35% w/w. 8 Lecithins such as di(tetradecanoyl)phosphatidylcholine, di(hexadecanoyl)phosphatidylcholine or di(octadecanoyl)
consisting of sodium cromoglycate with a total concentration of 0.1 to 20 mg/ml distributed between a liposome phase consisting of one or more phosphatidylcholines and an aqueous phase, with a lecithin concentration dispersed in the aqueous phase of 1
~30 mg/ml and the percentage of sodium cromoglycate associated with liposomes is 2-35%
A pharmaceutical composition according to claim 1, which is w/w. 9. An aqueous suspension comprising liposomes and sodium cromoglycate, wherein the sodium cromoglycate is partitioned between a free aqueous phase and a liposomal phase, the liposomes containing one or more natural or synthetic In preparing a pharmaceutical composition consisting of lecithin, the weight ratio of sodium chromoglycate to lipid in the liposome is in the range of 0.01 to 100, 0.01 to 50 mg/
A method characterized in that an amount of lipid is dispersed in ml of an aqueous sodium cromoglycate solution such that the concentration of the lipid is 0.1 to 150 mg/ml.