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Description  |
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This invention relates to pharmaceutical compositions and more particularly
it relates to the formulation of substances for inhalation.
Sodium cromoglycate has been known for a number of years for the treatment
of allergic conditions, for example asthma, hay fever and vernal kerato
conjunctivitis; however it suffers from the disadvantage that it is of
relatively short duration of action.
According to the invention there is provided a pharmaceutical composition
comprising liposomes and sodium cromoglycate.
By administering the liposomes of this invention directly into the site of
the allergic condition, e.g. the lung, it is possible to effect an
increased level of retention of sodium cromoglycate at the site, thereby
obtaining increased duration of action.
The initial stages of the preparation of liposomes according to the present
invention may conveniently follow procedures described in the art, i.e.
the lipid starting materials being dissolved in a solvent, e.g. ethanol or
chloroform, which is then evaporated. The resulting lipid layer is then
dispersed in the selected aqueous medium containing an appropriate
concentration of sodium cromoglycate. In contradistinction to the usual
practice. however, it is preferred not to sonicate the liposomes thus
produced, since this reduces their size. The liposomes produced by our
procedure will usually be of a range of sizes. The liposomes of this
invention preferably have a diameter of between 100nm. and 10 .mu.m, more
preferably they have a diameter of between 1 .mu.m and 7 .mu.m. It is
known, for example, that liposomes having a diameter of up to 5000nm. may
be readily phagocytosed. It is preferred that the liposomes are
fractionated to remove substantially all those having a diameter less than
100nm., and preferably also those having a diameter less than 1 .mu.m.
Fractionation may conveniently be effected by column gel chromatography,
for example using cross linked dextran or agarose, the size of the gel
being selected according to the desired liposome size. Alternatively, the
liposomes may be fractionated using ultracentrifugation, or by dialysis,
e.g. using polycarbonate membrane filtration.
A wide variety of lipid materials may be used to form the liposomes
including natural lecithins, e.g. those derived from egg and soya bean,
and synthetic lecithins. Lipids which are non-immunogenic and
bio-degradable are preferred. The properties of the lipid, for example its
phase transition temperature, can have a marked effect on the retention
and uptake of the liposomes in the target organ and for this reason the
well defined synthetic lecithins are preferred to the natural lecithins.
Examples of synthetic lecithins which may be used, together with their
respective phase transition temperatures, are
di-(tetradecanoyl)phosphatidylcholine (DTPC) (23.degree. C.),
di-(hexadecanoyl)phosphatidylcholine (DHPC) (41.degree. C.) and
di-(octandecanoyl)phosphatidylcholine (DOPC) (55.degree. C.). We prefer to
use di-(hexadecanoyl) phosphatidylcholine as the sole or major lecithin,
optionally together with a minor proportion of the di-(octadecanoyl) or
the di-(tetradecanoyl) compound. Other synthetic lecithins which may be
used are unsaturated synthetic lecithins, for example
di-(oleyl)phosphatidylcholine and di-(linoleyl)phosphatidylcholine. We
prefer the synthetic lecithin, or the mixture of lipids, to have a phase
transition temperature in the range 35.degree.-45.degree. C. In addition
to the main liposome-forming lipid or lipids, which are usually
phospholipids, other lipids (e.g. in a proportion of 5-40% w/w of the
total lipids) may be included, for example cholesterol or cholesterol
stearate, to modify the structure of the liposome membrane, rendering it
more fluid or more rigid depending on the nature of the main
liposome-forming lipid or lipids. An optional third component is a
material which provides a negative charge, for example phosphatidic acid,
dicetyl phosphate or beef brain ganglioside, or one which provides a
positive charge for example stearylamine acetate or cetylpyridinium
chloride. The charged component may be included in a proportion of 1-20%
w/w of the total lipids.
A wide range of proportions of sodium cromoglycate to lipid during
formation may be used depending on the lipid and the conditions used.
However we have in general found that a range of one part by weight of
sodium cromoglycate to from 0.01 to 100, preferably 0.05 to 20, most
preferably 0.1 to 10 parts by weight of lipid is appropriate. We prefer to
use as high a proportion of sodium cromoglycate as is practicable.
The concentration of sodium cromoglycate in the aqueous phase during
liposome formation is preferably in the range 0.01 to 50mg/ml, and more
preferably 0.1 to 20mg/ml, e.g. 10 or 20mg/ml.
We prefer the aqueous phase to contain less than 20 ppm of metal ions in
group IIa, IB, IIB and IVb of the periodic table, and of the transition
metals, in particular Pb.sup.++, Ca.sup.++, Mg.sup.++, Fe.sup.++
Fe.sup.+++ and Zn.sup.++ ions.
The aqueous phase may be made isotonic, using sodium chloride. In addition
the aqueous phase may contain potassium chloride.
The aqueous phase may be adjusted to a pH of between 6 and 8, and
preferably 6.5 to 7.5 by the addition of acid or base as appropriate, or
by the addition of a suitable buffering agent, e.g.
tris(hydroxymethyl)methanamine (Tris).
The concentration of lipid dispersed in the aqueous phase is preferably in
the range 0.1 to 150mg/ml, more preferably 0.5 to 50mg/ml and most
preferably 1 to 30mg/ml.
We prefer the liposome formulation to have a half life (efflux rate) at
37.degree. C. of from about 12 to 48 and preferably 12 to 24 hours. Half
lives may be measured using conventional techniques, e.g. by dilution
methods. The half life of the formulation may be varied by varying the
proportion of the various lipids used to make the liposome.
The compositions of the invention may be used for the treatment of asthma,
by instilling a nebulised aqueous suspension of the sodium cromoglycate
liposomes into the lungs. The compositions of the invention may be used as
eye drops in the treatment of allergic eye conditions, e.g. vernal kerator
conjunctivities, the occular symptoms of hay fever and/or marginal
infiltration.
The compositions may also be used in the treatment of diseases of
gastro-intestinal tract, e.g. ulcerative colitis, and food allergies, by
oesophageal administration. Enemas incorporating the compositions may be
used in the treatment of bowel diseases, particularly of allergic orgin.
The compositions may also be used in the treatment of hay fever, by
administration to the nose, e.g. as a nasal spray, and in the treatment of
skin conditions, e.g. chronic dermatoses in mammals, notably man.
Dermatoses which may be treated include those involving skin mast cells
and/or antibody anitgen reactions and include eczemas, drug eruptions,
psoriasis, dermatitis, herpetiformis pemphigus and chronic skin ulcers.
The compositions produced as described above are aqueous suspensions of
liposomes in which sodium cromoglycate is partitioned between the free
aqueous phase and the liposome phase.
We find that these aqueous formulations have useful and unexpected
properties, in that the aqueous phase can provide an initial `priming`
dose of sodium cromoglycate, and the liposome phase can provide a
maintenance dose of sodium cromoglycate. This has the effect of increasing
the duration of action of sodium cromoglycate.
According to the invention, we therefore provide an aqueous suspension
comprising sodium cromoglycate partitioned between a free aqueous phase
and a liposome phase.
We prefer the total concentration of sodium cromoglycate in the aqueous
suspension to be from 0.01 to 50mg/ml, and preferably 0.1 to 20mg/ml.
We prefer the percentage of sodium cromoglycte associated with the
liposomes to be from 2 to 35% w/w, e.g from 4 to 20%. The percentage of
sodium cromoglycate associated with the liposomes can be determined by
conventional methods, e.g. centrifugation.
Alternatively, the aqueous suspension of sodium cromoglycate partitioned
between an aqueous phase and a liposome phase, may be concentrated, e.g.
by centrifugation, ultrafiltration or dialysis, to give a liposome gel.
This gel may be used in several ways, e.g. it may be incorporated in an
ointment base, resuspended in water or an isotomic, buffered saline
solution, which may optionally contain sodium cromoglycate. Such
formulations may be made up from the liposome gel, and suitable excipients
immediately prior to use.
The dosage given will vary with the particular compositions used, the
condition to be treated and its severity. We prefer to use an effective
amount of sodium cromoglycate liposomes (e.g. for inhalation treatment of
asthma, from 0.1 to 20mg) in the treatment of these conditions.
The invention is illustrated, but in no way limited by the following
Examples.
General procedure for preparing sodium cromoglycate containing liposomes
The desired quantity (e.g. 20mg) of the appropriate phospholipid or mixture
of phospholipids (e.g. egg lecithin, DTPC, DHPC or DOPC), together if
desired with any other lipid soluble components (e.g. cholesterol,
cholesterol stearate) is weighted into a round bottom flask. The lipid
component is dissolved in a small quantity (ca 5ml) of a suitable solvent
(e.g. ethanol), and evaporated to dryness under reduced pressure using a
rotary film evaporator, to leave a thin film of phospholipid on the inner
surface of the flask.
An aqueous solution of sodium cromoglycate of appropriate concentration
(e.g. 1mg/ml) is prepared by dissolving a weighed amount of sodium
cromoglycate in 20ml of an aqueous medium (e.g. 0.9% w/v saline solition,
buffer solution, etc) and if desired the pH of the resulting solution can
be adjusted to a selected value by the addition of acid or alkali. The
aqueous solution of the sodium cromoglycate is warmed to a temperature
20.degree. C. above the phase transition temperature of the lipid(s),
added to the lipid film in the flask, and the flask gently shaken until
all the lipid film is dispersed. The resulting suspension contains
liposomes ranging from 200nm to 10um in size.
The suspension was allowed to equilibrate for 48 hours, at 37.degree. C.
These suspensions contain sodium cromoglycate partitioning between the free
aqueous phase and the liposome phase.
After 24 hours the suspension in most cases separates out to form a
colloidal white precipitate, which is readily redispersed on shaking.
The following sodium cromoglycate liposomes compositions were prepared
using the above general procedure:
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1. Egg lecithin 20 mg
Sodium cromoglycate 200 mg
Demineralised water 20 ml
2. Egg lecithin 20 mg
Sodium cromoglycate 20 mg
Demineralised water 20 ml
3. DTPC 20 mg
Sodium cromoglycate 2 mg
0.9% w/v saline solution
20 ml
4. DTPC 20 mg
Sodium cromoglycate 20 mg
0.9% w/v saline solution
20 ml
5. DTPC 20 mg
Sodium cromoglycate 200 mg
0.9% w/v saline solution
20 ml
6. DTPC 200 mg
Sodium cromoglycate 200 mg
0.9% w/v saline solution
20 ml
7. DTPC 400 mg
Sodium cromoglycate 200 mg
0.9% w/v saline solution
20 ml
8. DHPC 200 mg
Sodium cromoglycate 200 mg
0.9% w/v saline solution
20 ml
9. DOPC 200 mg
Sodium cromoglycate 200 mg
Demineralised water 20 ml
10. DTPC 133 mg
Cholesteryl stearate 67 mg
Sodium cromoglycate 200 mg
Demineralised water 20 ml
11. DHPC 133 mg
Cholesterol stearate 67 mg
Sodium cromoglycate 200 mg
Demineralised water 20 ml
12. DHPC 133 mg
Cholesterol 67 mg
Sodium cromoglycate 200 mg
Demineralised water 20 ml
13. DHPC 20 mg
Sodium cromoglycate 200 mg
0.9% w/v saline solution
20 ml
14. DHPC 75 mg
Sodium cromoglycate 102.5 mg
150 mM potassium chloride
10 mM Tris buffer, pH 7.4
10 ml
in water
15. DHPC 70 mg
DTPC 30 mg
Sodium cromoglycate 100 mg
0.9 w/v saline solution 10 ml
16. DHPC 180 mg
Sodium cromoglycate 200 mg
Cetylpyridinium chloride
20 mg
0.9 w/v saline solution 200 ml
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Determination of percentage sodium cromoglycate associated with liposomes
The equilibrated, sodium cromoglycate liposome dispersion is centrifuged at
70,000G for one hour. Aliquots of the supernatant are assayed in an
ultraviolet spectrophotometer, at 326nm, to determine concentration of
free sodium cromoglycate.
The percentage of sodium cromoglycate associated with the liposomes is
determined from the relationship:
##EQU1##
The following percentage associations were determined:
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Example
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5 4.5% w/w
13 8.23% w/w
14 14.00% w/w
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Rate of sodium cromoglycate release from liposomes, and liposome half-life
The rate of sodium cromoglycate release from the liposomes may be
determined by centrifuging the sodium cromoglycate liposomes at 70,000G as
above, discarding the supernatant and resuspending in isotomic saline,
buffered at pH7.4. Aliquots of the resuspended liposomes, agitated at
37.degree.C., were centrifuged at intervals, and the concentration of
sodium cromoglycate in the supernatant determined by u.v.
spectrophotometry. The release constant, k, of the liposome is determined
by plotting 1n [cromoglycate released]v time. The half-life of the
liposome, t.sub.1/2, is given by the relationship t.sub.1/2 =(1n2)/k.
Liposome half lives may also be determined using the dilution method
described by M Ahmed et al, Biochemical Pharmacology, 29, 2361-2365,
(1980).
Determination of flux and permeability coefficient
Preparation of Membranes
Albino hairless mice of either sex and aged 10 to 12 weeks were sacrificed
by cervical dislocation and the dorsal skin removed with the minimum of
handling. Any subcutaneous fat, visible as discrete globules, was removed.
The skin samples were examined for any signs of damage before use. One
skin sample was used per diffusion cell and was mounted, epidermal side
up, over the opening in the upper section of the diffusion cell and was
then secured with an `O` ring. Excess skin was trimmed away before
assembly of the cell.
Diffusion Cell Assembly
A small amount of silicone grease was applied to the `O` ring of the upper
section after securing the membrane. The upper section was then pushed
firmly into the lower chamber until correctly positioned. The chamber was
then filled with saline pre-equilibrated to 37.degree.. The volume of each
cell was adjusted individually to ensure that the skin membrane remained
level. The fill volume was then marked on the sidearm.
Experimental Procedure
The set of eight diffusion cells were mounted on a carrier plate held in a
thermostatically controlled water bath set at 37.degree. C. and were
allowed to equilibrate. Each cell was positioned over an underwater
magnetic stirrer motor and the water level was adjusted to be
approximately the same as the skin surfaces. This ensured that the
temperature of the skin surface remained at 30.degree..
The vehicle to be studied was applied, either by delivery from a
micropipette. The preparation was then evenly distributed over the exposed
skin surface using a small glass rod. The weight of each aliquot applied
was determined by accurately weighing at least 10 samples delivered by the
micropipette or syringe.
Following application of the vehicle the magnetic stirrers were switched on
and at appropriate time intervals 1.0ml samples of the receptor fluid were
removed via the side arms and immediately replaced with fresh saline
pre-equilibrated to 37.degree.. The samples were then deep frozen until
analysed for the drug by High Performance Liquid Chromatography (HPLC).
At least three replicate diffusion cells were used for each formulation
studied.
Data Handling
Assuming that only passive diffusion occurs during the transport of the
drug across the skin, the rate of penetration can be given by Fick's law.
J=P.DELTA.C
Where
J is the flux, the amount of drug diffusing per unit area per unit time,
P is the permeability coefficient
.DELTA.C is the concentration difference across the stratum corneum.
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Description |
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