|Title of Invention||
"LIPOSOMIC FORMULATION OF CLOBETASOL PROPIONATE"
|Abstract||A topical liposomal formulation of clobetasol propionate for use in the treatment of skin diseases such as atopic dermatitis and psoriasis. The formulation has good therapeutic effect and favorable distribution in the stratum corneum. The liposomes include clobetasol propionate as active substance in low dose.|
|Full Text||FIELD OF THE INVENTION
The present invention relates to a dermatological composition comprising liposomes with Clobetasol Propionate and is within the field of liposomal products used in human medicine.
BACKGROUND OF THE INVENTION
As is already known, Clobetasol Propionate is a powerful synthetic corticosteroid with anti-allergic, anti-exudative and anti-proliferative properties, which inhibit the inflammatory response produced by mechanical, chemical or immunological agents. It is prescribed in cream-form (CLOVATE*) or as an ointment (DECLOBAN®) for the short term treatment of dermatoses such as psoriasis, recidivist eczemas, flat lichen discoid lupus erythematosus, which are resistant to the commonly used external corticosteroids. Nevertheless, the use of this medicine has been restricted as maximum up to 14 days due to the side effects produced, particularly the hypophysis-adrenal inhibition, risk that increases with the preparation potency, as well as with the application surface and with the duration of the therapy.
The patent applications WO 93/15740, EP 0391033, WO 98/36753, WO 96/27376, JP11130679, as well as the patent US 5998395 relate to formulations comprising steroid anti-inflammatory drugs including Valerate and Clobetasol Propionate. In these compositions the Clobetasol Propionate is dispersed and although they head to increase the therapeutic activity of the preparation, they are not able to diminish the adverse effects associated to the use of the medication.
On the other hand, the patents US 5192528, US 4427649, WO 97/13500, US 4693999, WO 96/22764 and US 5190936 refer to liposomal formulations of corticosteroids but none comprise the encapsulation of the Clobetasol Propionate or any other derivative of the aforementioned corticosteroid in liposomes.
STATEMENT OF INVENTION
The present invention provides a topical liposomal formulation comprising a) Clobetasol Propionate encapsulated in liposomes; b) 0.5 to 4.0% of phospholipid such as herein described; c) 0.1 to 0.6% lipophilic additive; d) 0.01 % to 0.2 % of anti-oxidant agents; e) 8 to 15% organic solvent; f) 0.02% to 0.12% buffer agent to maintain the pH level of the water component in the interval of 5 to 7.5; g) 0.02% to 0.2% preservative substances; h) 0.85% gelling agents; and i) water.
'SUMMARY OF THE INVENTION
The present invention relates to a topical liposomal formulation having clobetsol propionate as an active agent. It uses low concentration of active ingredient and rende*$high concentrations of product to the skin, being attenuated more this effect in the stratum corneum. The formulation reduces the diffusion of active ingredient to systemic circulation and minimizes the side effects.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is related with a liposomal formulation of Clobetasol Propionate for topical use, which is characterized because it uses low concentrations of the active ingredient (between 0,01 and 0,03) and it renders high concentrations of the product in the skin, being accentuated more this effect in the stratum corneum, from where the product can be control released and it diminishes the diffusion of the active ingredient toward the systemic circulation minimizing this way the toxicity of the same, and therefore its adverse effects.
Tfre efficiency of the composition of the invention is similar to the classical cream and ointment formulations where the Clobetasol Propionate is diffused, although surprisingly it was observed that encapsulating said active ingredient in liposomas, its concentration can be reduced a half of the amount used in classic formulations of this active ingredient maintaining its powerful anti-inflammatory effect, due to the high affinity of the compounds belonging to the lipid double-layer of liposomes with the biologica! membranes, achieving a maxim local action.
The current invention provides a liposomal formulation for externai use, preferably in gel form, comprising Clobetasol Propionate to low concentrations, phospholipids, a lipophilic additive, antioxidant agents and a solvent for Clobetasol Propionate, like phospholipids, lipophilic additives and antioxidant agents, and also aqueous solution forming part of the liposomal suspension, as well as convenţional auxiliary agents in geles or the appropriate externai formulations: creams, ointments and lotions. The present composition preferably includes a liposomal suspension, of multi-lamellar vesicles with sizes varying between 2000 and 5000 nm, preferably between 3500 and 4500 nm.
The compositions of the invention, comprise, in weight of the total weight of the composition the following essential components:
- Phospholipids suitable for use in the formulation are, for example, phosphatidyl
cholines, phosphatidylethanolamines and phosphatidylserines, and mixtures thereof,
in particular those soluble along with Clobetasol Propionate in a non-toxic, organic
and pharmaceutically acceptable solvent. Among them are preferred the
phosphatidylcholine or lecithin, soyabean-lecithin for example. The amount of
phospholipid in the present formulation may vary between 1.0 and 1.5 %, and is
preferably 1.2 %.
- Lipophilic additives to selectively modify the characteristics of the liposomes. These
may include cholesterol, phosphatidic acid, estearylamine, cholesterol being the
ideal additive. The amount of lipophilic additive may vary between 0.1 and 0.6 %,
preferably between 0.2 and 0.4 % and is preferably 0.3 %. In general the ratio of
lipophilic additive to phospholipid varies between 1:1 and 1:3, and in this case was
anti-oxidant agents for liposome preparation, among them a-tocopherol and
butylated hydroxytoluene (BHT) may be used as anti-oxidant agents. BHT is the
For the formulation preparation, the above mentioned phospholipids, additives, anti-oxidants and active ingredient (Clobetasol Propionate) are dissolved in a water-soluble, non-toxic, and pharmaceutically acceptable organic solvent.
- The organic solvent used in the liposomal suspensions may be a glycol, particularly
propylene glycol, in quantities ranging between 8 and 15 %, particularly between
10.0 and 12.5 % and preferably 11.8 %.
The term "organic component" used here refers to mixtures composed of the phospholipid, lipophilic additive, anti-oxidant and the organic solvent.
- The active ingredient, Clobetasol Propionate is dissolved in the organic component.
The quantity of the active ingredient in the final composition varies between 0.01 -
0.06 %, preferably between 0.02 - 0.055 % and preferably between 0.02 - 0.05 %.
- The liquid component of the present formulation is formed principally of water and
may contain various additives such as electrolytes or preferably a buffer system,
preferably the Tris HCI buffer. Such a buffer system will maintain the pH of the liquid
component at an interval of between 5 and 7.5, preferably between 6 and 7.2 and
preferably between 6.5 and 7.
The present invention includes the finished gel product for externai use containing preservatives, anti-oxidants and the normal ingredients used in gel manufacture.
- Among the preservatives that may be used to avoid degradation by microorganisms
are benzoic acid, methylparaben and propylparaben. The two latter preservatives are
preferably used in quantities of 0.2 % and 0.02 % respectively.
- Among the anti-oxidants that rnay be used for preserving the composition are
tetracetic ethylenediamine acid (EDTA), disodic edetate and sodium methabisulphite.
The optimum being either EDTA or sodium methabisulphite at 0.01 % and 0.2 %
- The composition of the invention also includes suitable gelling agents for obtained
the end form for externai use, these agents may for example
hydroxypropylmethylcellulose, hydroxyropylcellulose, their mixtures or carbopol 940,
being preferred carbopol 940, together with triethanolamine. The amount of these
gelling agents will depend of the remainder ingredients and in general is
apţroximately from 0.5 to 1.0%, and in particular is about 0.85% for carbopol 940
and 0.65% for triethalonamine.
For preventing degradation of the materials used during the preparation of the
liposomal formulation, it is advantageous to purge all solutions with an inert gas like
argon or nitrogen, and to carry out all the steps under inert atmosphere.
The ideal proportions in the fmished product, based on the total weight of the
(a) 0.01 to 0.03 % of Clobetasol Propionate;
(b) 0.5 to 4.0 % of phospholipid;
(c) 0.1 to 0.6 % of cholesterol;
(d) 8 to 15 % of propylene glycol;
(e) 0.003 to 0.007 % of BHT;
(f) buffer agent to maintain the pH of the liquid component at an interval of 5
(g) sufficient dermatologically acceptable preservatives and anti-oxidants to
(h) a basic agent to regulate the pH of the gel;
(i) 0.5 to 1.0 % of gelling agents;
The preferred compositions would be those in which: the quantity of Clobetasol Propionate is 0.02 to 0.025 % the quantity of phospholipid is 1.0 to 1.5 % the quantity of cholesterol is 0.2 to 0.4 % the quantity of propylene glycol is 10 to 12.5 % the quantity of gelling agent is 0.6 to 0.9 %
The optimum composition includes approximately (where weights are based on the total weight of the composition):
(a) 0.025 % of Clobetasol Propionate
(b) 1.2 % of phospholipid
(c) 0.3 % of cholesterol
(d) 11.8 % of propylene glycol
(e) 0.005 % de BHT
(f) 0.1 % of Tris (hydroxymethyl) - aminomethane and 0.02 % of
(g) 0.2% of methylparaben and 0.02 % of propylparaben
(h) 0.01 % of EDTA and 0.2 % of sodium methabisulphite
(i) 0.65 % of triethanolamine
(j) 0.85 % of Carbopol 940
The liposomal formulation of Clobetasol Propionate in the present invention is preferably prepared in the following way:
(a) heating the lipophilic additive and a portion of the organic solvent to between
100 and 140°C, preferably between 120 and 135°C in an iniţial beaker leaving the
mixture to cool to between 70 and 90°C, dissolving in this the phospholipid, the
active ingredient and the anti-oxidant and stirring until complete dissolving has
(b) heating in a second beaker the buffer solution, preferably at between 70 and
90°C, and adding part of this to the first beaker whose temperature should be
maintained at between the 70 to 90°C mark until a paste is obtained;
(c) transferring the remainder of the second glass, still at between 70 and 90°C, to
the first glass and mixing the combination in a high efficiency mixer;
(d) dissolving the preservatives in the organic solvent in a third beaker, preferably
in the mixer, until complete dissolution;
(e) adding to a portion of the water the contents of the third beaker, preferably in a
mixer, dissolving the anti-oxidants and the gelling agent, adding to the rest of the
water dissolved triethanolamine and the contents of the first beaker, preferably
maintaining the mixing action to homogenise.
At stage (c) any type of mixing machine or homogenizer which employs an intense cutting action on the mixture may be used. Generally, a mixer capable of reaching speeds of 2000 to 16000 rpm, particularly between 2500 and 6000 r.p.m. is ideal. In the following stages any mixing method may be used, particularly those which use a propeller-type mixing rod.
The liposomes prepared in the described manner, generally contain the highest amount of encapsulated active ingredient and the separation of the non-encapsulated liposomes is not necessary.
Example 1. Preparation of the formulation of encapsulated Clobetasol Propionate.
The following proportions of components in the formulation were used (Table 1).
1. The cholesterol is heated in approximately a 6th part of the propylene glycol to
120 - 135°C. The mixture is left to cool to approximately 70 - 90°C. The
Clobetasol Propionate, soyabean-lecithin and the butylated hydroxytoluene are
dissolved in this mixture at 70 - 90°C. Complete dissolving is achieved through
2. 0.1 g of Tris (hydroxymethyl)-aminomethane and 0.02 g of hydrochlorhydric acid
are dissolved in approximately a quarter part of the water in a separate beaker
which is kept at 70 - 90°C. Approximately 5 ml of this solution is extracted and
added to the mixture obtained in step 1. This is mixed until a paste is formed.
3. The remainder of step two's mixture is added to the paste at between 70 and
90°C. This is homogenized for five minutes at 3000 r.p.m. in a high efficiency
4r The parabens are dissolved in the remaining propylene glycol in a separate beaker and complete dissolution is obtained through constant stirring.
5. Approximately half of the water is added to the result of stage 4 and stirred constantly. The EDTA and sodium methabisulphite are added and constantly stirred until completely dissolved. The carbopol 940 is gradually added and stirred vigorously and constantly for 30 minutes. The dissolved triethanolamine is added to the rest of the water and the result of stage 3 is immediately incorporated.
The homogenizer used was the POLYTRON® disperser with its efficient cutting
action and the mixer used in stages 4 and 5 was a mechanical stirrer IKA which has
a propeller rod.
The liposomal gel obtained from stage 5 can be apportioned into appropriate
containers such as aluminium tubes coated in PVC.
Example 2. Trial formulation in edema in ear of mice induced by croton oii. Groups of ten animals were made up (male mice weighing from 28 to 32 g) and the edema was induced in the right ear through the application of 50 ui of croton oii (5 %) and ethanol (95 %) (v/v). The same volume of ethanol was applied in the left ear. Topica! administration of the trial formulations was applied 30 minutes before the induction of the edema. In every case, 20 mg of the sample was applied to each ear. The negative control group only received the croton oii. The following experimental groups were formed:
1 ,2,3,4,5 and 6 hours after the induction of the edema all the groups of animals were killed by cervical dislocation. Both ears were removed to obtain and weigh discs of 5 mm in diameter.
In Table 2 the average values of weight difference of the ear according to treatment groups are shown. It may be seen that all the experimental groups are statistically different (p TABLE 2
The statistica! analysis demonstrated that there were no significant differences at any of the studied times between the ointment, the cream and the liposomal gel at a dose of 5 (0.025 %) ug of Clobetasol Propionate per ear. A greater inhibition of the edema was found in the group treated with the ointment at the interval of 4 and 5 hours respectively. The inhibitory effect on the edema induced by the croton oii is shown in Figure 1.
TTO inhibitory response was similar for the groups treated with clobetasol propionate
but it was higher for the groups treated with the cream and liposomal gel at a dose of
Comparing the results obtained for different groups treated with liposomal gel, at
maximum inflammation time (5 hours) the highest inhibition of the edema was
Example 3. Trial formulation in granuloma induced by cotton pellet.
Male Wistar rats with a body weight of between 250 and 300 g were used. These
were kept in a temperature-controlled room of 2°C ± 20°C and a 12-hour
The animals were anaesthetized intraperitoneally (IP) with 30 mg/kg of sodic
phentobarbital. An incision was made in the dorsal area and two cotton pellets of 35
± 2 mg were inserted, settling 2 cm from the wound. This was sewn and an antiseptic
solution was applied to avoid sepsis. The animals were topical treated (0.3 g
sample/cotton pellet) for 7 days and on the eighth they were killed in an ether
atmosphere and the granulomas were extracted and weighed. The granulomas were
dried in an oven at a constant 65°C over approximately 24 hours to determine the
water content of the granuloma (wet weight - dry weight) and the fibre granularity (dry
weight - cotton pellet weight). The following experimental groups were formed:
An increased dry weight of the cotton pellet was observed in all treated groups during
The subcutaneous insertion of the cotton pellets provoked a vascular fibrous capsule
with the presence of mononuclear cells; it resulted in a granuloma at the site of
The water and fibre granularity content obtained after the application of the trial
substances for seven days appear in figures 2 and 3.
Plgure 2 shows the results obtained for different formulations containing Clobetasol
Propionate. It was observed in all groups an inhibitory effect with the following order:
Cream>liposomal gel of table 1 > Ointment. It may be seen that all the experimental
groups are statistically different (p control group. From the results it can be concluded that the Ointment, the cream and
the gel exert an inhibitory effect at the assayed doses.
Figure 3 shows the results obtained for different formulations containing Clobetasol
Propionate. It was observed in all groups an inhibitory effect with the following order:
Cream>liposomal gel of table 1> Ointment. It may be seen that all the experimental
groups are statistically different (p control group. From the results it can be concluded that the Ointment, the cream and
the liposomal gel exert an inhibitory effect at the assayed dose.
The encapsulation of Clobetasol Propionate into liposomes is an advantage because
the dose may be reduced to a half of the convenţional dose used for other
formulations and even the same therapeutic effect is obtained.
The results obtained in the pre-clinical study indicate that the liposomal gel of
Clobetasol Propionate 0.025% have a topical anti-inflammatory effect in the
experimental model evaluated, similar and in some cases superior to the effect
observed in the convenţional cream and ointment.
Example 4. Study of "in vitro" percutaneous absorption.
Formerly the radiolabelling of the Clobetasol Propionate with tritium was carried out
through an isotopic, catalytic, heterogeneous exchange in solid phase. A specific
activity of 150 mCi/mmol at 95 % radiochemical purity was obtained. The labelled
Clobetasol Propionate was mixed with the different subject formulations, these being:
Liposomal gel of Clobetasol Propionate at 0.025 %
Clobetasol Propionate gel at 0.05 %
Clobetasol Propionate cream at 0.05 %
Clobetasol Propionate ointment at 0.05 %
The human skin used in the "in vitro" percutaneous absorption study was obtained
f rom aesthetic breast surgery.
Samples were taken from the receptor compartment 24 hours after the formulations
were applied on the skin. Subsequently, the "stripping" process was carried out and
the stratum corneum, epidermis and dermis layers were separated. Measurement of
the radioactivity in each of the samples taken was through liquid scintillation. The
stripping in this manner fundamentally represents the residue of the application,
although the most superficial layers of the stratum corneum are also present.
The percentages of radioactivity accumulated in the samples, measured in relation to
the activity applied in each case, are shown in Figure 4. It is demonstrated that the
liposomal formulation deposit larger concentrations of the Clobetasol Propionate on
the skin (approximately 50% of the applied dose), this being more pronounced on the
stratum corneum with lower diffusion towards the receiving compartment as
compared to the convenţional formulations, even when the Clobetasol Propionate
dose was reduced to a half.
On the other hand, it was observed that in the non-liposomal formulations the
greatest quantity of Clobetasol Propionate was found in the receiving compartment,
particularly with the ointment at 70 %. From these results one may expect a lower
diffusion of the Clobetasol Propionate incorporated in liposomes towards the
systemic circulation after "in vivo" application.
Brief Description of the Figures
Figure 1: Shows the inhibitory effect of the formulation against the edema induced by
the croton oii during the study period, through comparison of differences in ear
weight (milligrams) against time in hours.
Figure 2: Shows the water content values obtained after 7 days of treatment with the
various trial substances.
Figure 3: Shows the fibro-granularity content after 7 days of treatment with the
various trial substances. In the figure, equal letters show no significant differences
according to Duncan's test.
Figure 4: Shows the radioactivity count accumulated in the analyzed samples and for
each of the evaluated formulations in relation to the radioactivity applied.
1. A topical liposomal formulation comprising;
(a) Clobetasol Propionate encapsulated in liposomes;
(b) 0.5 to 4.0% of phospholipid such as herein described;
(c) 0.1 to 0.6% lipophilic additive;
(d) 0.01 % to 0.2 % of anti-oxidant agents;
(e) 8 to 15% organic solvent;
(f) 0.02% to 0.12% buffer agent to maintain the pH level of the water
component in the interval of 5 to 7.5;
(g) 0.02% to 0.2% preservative substances;
(h) 0.85% gelling agents; and
2. A formulation as claimed in claim 1, wherein the liposomes are multi-lamellar
3. A formulation as claimed in claim 2, wherein the liposome size is in the range of
2000 and 5000 nm, preferably between 3500 and 4500 nm.
4. A formulation as claimed in claim 1, wherein the phospholipid is selected from
the group consisting in phosphatidylcholine, phosphatidylethanolamine and
phosphatidylserine, lysophosphatidylcholine and mixtures thereof.
5. A formulation as claimed in claim 4, wherein the phospholipid is lecithin,
preferably soyabean- lecithin.
6. A formulation as claimed in claim 5, wherein the lecithin is between 0.5 and 4.0%
(w/w), preferably between 1.0% and 1.5%, and in particular it is about 1.2%.
7. A formulation as claimed in claim 1, wherein the lipophilic additive is selected
from the group consisting in cholesterol, phosphatidic acid and estearylamine,
being cholesterol the preferred additive.
8. A formulation as claimed in claim 7, wherein the amount of lipophilic additive is
between 0.1 and 0.6% (w/w), preferably between 0.2 and 0.4% and in particular it
is approximately 0.3%.
9. A formulation as claimed in claim 1 wherein the relationship between quantity of
preservative lipophilic and the quantity of phospholipid varies between 1:1 and
1:3, and in particular it is approximately 1:2.5.
10. A formulation as claimed in claim 1 wherein the anti-oxidant agent used in the
preparation of the liposomes is a-tocopherol or butylated hydroxytoluene (BTH),
being preferably BHT which is between 0.003 to 0.007% (weight/weight).
11. A formulation as claimed in claim 1 wherein the anti-oxidant agent used for the
preservation in the in the end form are ethylene diamine tetracetic acid (EDTA),
disodic edetate and sodium methabisulphite being preferably either EDTA or
sodium methabisulphite at 0.01 % and 0.2 % respectively.
12. A formulation as claimed in claim 11 wherein the organic solvent is a glycol, and
preferably it is propylene glycol.
13. A formulation as claimed in claim 12 wherein the organic solvent is between 8
and 15% (w/w), in particular between 10.0 and 12.5% and preferably at 11.8%.
14. A formulation as claimed in claim 1 wherein the buffer agent for maintaining the
pH of the watery component between 5 at 7.5 is Tris (hydroxymethyl)-
aminomethane at 0.1 % (weight/weight) and hydrochlorhydric acid at 0.02%
15. A formulation as claimed in claim 1 wherein the preservative substances is
methylparaben to 0.2% (w/w) and propylparaben at 0.02% (w/w).
16. A formulation as claimed in claim 1 wherein the gelling agent is selected of the
group consisting in hydroxypropylmethylcellulose, hydroxyropylcellulose, their
mixtures or carbopol 940, being preferred carbopol 940 at 0.85%.
17. A formulation as claimed in any of claims from 1 to 16 in the form of gel.
18. A liposomal formulation for topical use substantially as herein described with
reference to foregoing examples.
|Indian Patent Application Number||IN/PCT/2003/00001/DEL|
|PG Journal Number||26/2010|
|Date of Filing||01-Jan-2003|
|Name of Patentee||CENTRO DE INVESTIGACION Y DESARROLLO DE MEDICAMENTOS(CIDEM)|
|Applicant Address||AVE 26, NO. 1605 ENTRE BOYEROS Y PUENTES GRANDES, PLAZA DE LA REVOLUCION, CIUDAD HABANA 10600 CUBA|
|PCT International Classification Number||A61K 9/00|
|PCT International Application Number||PCT/CU01/00005|
|PCT International Filing date||2001-07-17|