Imported: 10 Mar '17 | Published: 27 Nov '08
USPTO - Utility Patents
The present invention relates to a composition and a method for preparing a liposome, the liposome including a lipid bilayer and an aqueous core contains a hydrophobic or a hydrophilic drug and a componentVitamin E derivative (d- tocopheryl polyethylene glycol 1000 succinate; TPGS). TPGS is able to increase the encapsulation efficiency of drug in liposome as well as to enhance the stability of drug in liposomes. Such liposome is capable to increase the skin permeation of drugs. The preparation method comprises the following steps: (1) adding the drug to a Vitamin E derivative solution to form a mixture; and (2) adding at least one phosphatidyl choline to the mixture, after hydration from either sonication or homogenization.
This application is a continuation application of parent U.S. application Ser. No. 11/024,799, filed Dec. 30, 2004, (of which the entire disclosure of the parent application is hereby incorporated by reference).
1. Field of the Invention
The present invention relates to a composition and a preparation method of a liposome that enhances drug encapsulation efficiency, drug stability, and skin permeability. A preparation method of liposomes that encapsulate hydrophobic drugs, which is selected from the group comprises of all-trans retinoic acid (RA), 4-phenylbutyric acid (PBA), and diclofenac diethylamine.
2. Description of the Related Prior Art
All-Trans Retinoic Acid (abbreviated as RA) is one of the most effective drugs for acne treatment currently. Its functional mechanism is that the synthesis of an active substance after RA clearance in the system and this active substance will induce the epithelial cell proliferation. Due to the proliferation of epithelial cells, keratosis becomes abnormal whereby keratinocytes tend to become loose and less intact, resulting in acne tissue falling off the treated area and thus excellent acne treatment can be achieved. However, some side effects in the course of RA therapeutic session, skin has tendency to peel off, inflame, swell, and also has other unpleasant consequence. The application of using liposomes as a carrier to encapsulate RA may prevent RA in direct interaction with skin, thereby reduce skin irritation. RA liposomes also extend the residence time of drug in the dermal layer and reduce the systemic effect to enhance the drug efficacy in the skin. RA is poorly soluble in water, therefore a non-irritating solubilizer is necessary to apply to increase both RA solubility and the drug concentration in liposomes. Increasing potential risk of fetal deformity in pregnancy while RA is in systemic circulation. The use of RA liposome tends to reduce the amount of RA in systemic circulation by retaining most RA under both the dermis and epidermis of the skin.
Both of 4-phenylbutyric acid, a skin cancer and wound healing drug for treating skin cancer, and diclofenac, a non steroid anti-inflammatory and allergestic drug, may cause potential side effects on skin such as allergic or non-allergic dermatitis, pimples, skin blush, dropsy, scaling, and other symptoms such as the stomach irritation. Therefore, it is desirable to use a liposomes carrier to diminish skin irritation and allow the drugs to remain in the skin (local area) longer for better therapeutic effect and to reduce systemic side effect in the body.
Liposomes are one of the most potential drug carriers available currently. The composition of liposomes contains both hydrophobic bilayer, which may encapsulate hydrophobic substance, and an aqueous core, which may encapsulate hydrophilic substance. The uniqueness for the present invention is that with the use of liposome to encapsulate both hydrophobic and hydrophilic substances. Therapeutic effect is enhanced due to the skin permeability is also enhanced. To prolong RA liposome retention in the dermis and epidermis in the skin will minimize the side effect from systemic circulation. To achieve this goal, the encapsulation efficiency and stability of drug address a major role in present invention.
In according to the past reviews, an amphiphilic Vitamin E derivative (d- tocopheryl polyethylene glycol 1000 succinate abbreviated here as TPGS) is used as the solubilizer for Paclitaxel in the oral delivery, or as an ingredient in cosmetic preparation, skin medication, and blood clotting. However, the most TPGS applications are irrelevant to the present invention. In the present invention, TPGS hereinafter is solely functioned for active ingredient of topical-used liposome, drug absorption enhancement, or functioned as a solubilizer for insoluble drugs. Due to amphiphilic characteristic of TPGS, that is one hydrophobic portion, Vitamin E, and other hydrophilic portion, polyethylene glycol 1000, liposomes that consist of TPGS which is utilized as a surfactant between the liposome bilayer and core, and will enhance the stability for both hydrophilic or hydrophobic drugs. Furthermore, it will increase the amount of drug encapsulated in the liposome for clinical application.
The main objective of the present invention is to provide a composition of a liposome and a method for preparing liposomes. It is able to encapsulate either hydrophobic or hydrophilic drugs within liposomes and increase solubility of hydrophobic drugs. The long-term stability for encapsulating either hydrophobic or hydrophilic drugs in the liposome is also enhanced. Another objective of the present invention is to provide a novel type of liposomes, which are encapsulating either hydrophobic or hydrophilic drug, that skin permeation, encapsulation efficiency, and long term stability are enhanced. Liposomes, that comprises with TPGS, encapsulate either the hydrophobic or hydrophilic drug, that the localized administration of the drug and reduction of skin irritation can be achieved.
To accomplish prescribe objectives, the present invention describes a liposome composition and a preparation method, which includes phospholipids bilayer and an aqueous core; the liposome comprise either a hydrophobic or hydrophilic drug and TPGS (d- tocopheryl polyethylene glycol 1000 succinate), and the protocols for liposomes preparation is in the following:
First the prescribed TPGS is dissolved in a solvent, such as water, ethanol, methanol, or 2-propanol to obtain a TPGS solution. In addition, a hydrophobic drug (All-Trans Retinoic Acid) is added to the TPGS solution and stirred until dissolved. Phosphatidyl choline as well as other ingredients, such as cholesterol and Vitamin E, (an antioxidant which may prevent accidental oxidation of the liposome or drug), is added in a pre-determined amount and followed by hydration and sonication to produce the liposomes that is comprising of TPGS.
TPGS is obtained by esterification of d--tocopheryl acid succinate with polyethyleneglycol 1000. The HLB value of TPGS (hydrophile-lipophile balance) lies between 15-19, indicating that TPGS is a surfactant, which is well soluble and capable of emulsifying hydrophobic drugs. Therefore, TPGS is added with ethanol to increase the solubility of the hydrophobic drug in according to the present invention. For example, All-Trans Retinoic Acid (abbreviated as RA), the results that are indicate in Table 1, the solubility of RA in ethanol increases 10 times when 20% (w/w) TPGS is added.
The purpose of this invention is to increase the TPGS concentration from 20% to 40%, thus it increase RA solubility in ethanol from 1 mg/mL to 16.7 mg/mL. Without TPGS addition, the solubility of RA is less than 1 mg/mL.
There are eighteen examples to demonstrate the technical breakthrough for this invention.
Formulation of the present example is illustrated in the following:
First, 1 g of SPC, 1.12 g of Cholesterol and 0.05 g of Vitamin E are dissolved in ethanol and the solution is stirred until dissolved completely.
In addition, 0.2 g of TPGS and 0.01 g of RA are also mixed and stirred until dissolved completely in ethanol.
8.4 mL of 2.25% glycerin is pipetted into a hydration cell, while the internal temperature is controlled at 25 C. by water circulation, then the resultant solution of SPC, cholesterol, Vitamin E, TPGS, and RA is injected and hydration for an hour.
Finally sonication is performed with the prepared multi-lamellar vesicles (abbreviate as MLVs) liposome. The solution is slightly transparent yellow, which is the target RA liposome.
The results of table 3 indicate that the formulation, without addition of TPGS, will result in either failure of liposomes formation or decomposed within seven days.
The result of Table 4 shows, in comparison between comparative examples, 6 and 7, 8 and 11, that is, without added TPGS in the formulation, the failure of liposomes formation and decomposed in seven days.
For example 2, the liposomes preparation can be achieved due to the addition of TPGS in the formulation. The drug concentration is increased to 0.085%.
For example 3, the stability of liposomes has enhanced due to the addition of TPGS in the formulation. The RA concentration remains the same after seven days.
In according to the method of example 1 preparation. It follows the same method of formulation preparation in Table 3.
FIG. 1 illustrates the encapsulation efficiency of RA versus storage time of examples 5 and 7. As shown in FIG. 1, the present invention employing TPGS as composition of RA liposome prolong the stability of RA within the liposome to 180 days while greatly enhancing the encapsulation efficiency of RA.
The method of liposome preparation for examples 10 to 13 is using the same method as in example 1. The only difference is that SPC is being replaced by E60 (EPC of 60% purity). The composition and properties for previous 4 formulations are listed in Table 6.
The results of the above examples are shown in FIG. 2, which illustrates the encapsulation efficiency versus storage time of examples 10 to 13. As shown in the FIG. 2, the present invention employing TPGS as composition of RA liposome prolong the stability of RA within the liposome to 180 days while greatly enhancing the encapsulation efficiency of RA.
The method of liposome preparation of examples 14 to 15 is the identical method as in example 1, only soybean PC is being replaced by hydrogenated soy phosphatidyl choline (abbreviated as HSPC) and soybean PC together, or employing both SPC and HSPC without the use of Vitamin E. Formulation compositions and liposome properties of the above mentioned examples are shown in Table 7.
FIG. 3 illustrates the encapsulation efficiency versus storage time graph of examples 14 to 15. As shown in the FIG. 3, the present invention employing TPGS as composition of RA liposome prolong the stability of RA within the liposome to 70 days while greatly enhancing the encapsulation efficiency of RA.
Formulation of soybean PC liposome encapsulated with 4-phenylbutyric acid (abbreviated as PBA). As in the preparation method of example 1, and in accordance with liposome formulation preparation of Table 8, compositions and properties of all examples are illustrated in Table 8.
PBA encapsulated efficiency versus storage time of examples 16 to 21 is illustrated in FIG. 4. From the graph interpretation, the present invention employs TPGS as the composition of PBA liposome, which enhances encapsulation efficiency while promote the stability of PBA liposome.
Formulation of soybean PC liposome encapsulated with Diclofenac diethylamine.
Formulation of the present example is shown in Table 9:
First, 0.42 g of SPC, 0.02 g of cholesterol, and 0.0653 g of TPGS are dissolved in 0.5 mL of 1% Diclofenac diethylamine solution. Following by a grinding technique, the solution is to dissolve completely to a paste-like mixture.
Hydration is processed by adding PBS solution with paste-like mixture for an hour at room temperature with the volumetric ratio of 1:1 for solute to water. When the hydration is terminated, the yield product is a milky yellow solution, which is Diclofenac diethylamine liposome.
As in the manufacture method of example 22, and in accordance with liposome formulation preparation of Table 10, all compositions and properties of examples are listed below in Table 10.
With the addition of TPGS as described in the above Table 8, solubility of RA can be increased; moreover TPGS can also increase the encapsulation efficiency of liposomes. Other than enhancing the encapsulated stability of RA in the liposome, the above mentioned effects can still be obtained when preparing liposomes with different sources of phosphatidyl choline. Therefore, the technique of adding TPGS in present invented liposomes can be applied widely in formulating liposomes, and it is not limited to phosphatidyl choline of the present example.
It is to be noted that the concentration of TPGS in the TPGS solution is not restricted, however, between 1%-50% (by weight percent) is preferable. Depending on the need, 0.120% (by weight percent) of Vitamin E can also be added to the TPGS solution. The amount of Vitamin E in the TPGS-Vitamin E solution is not restricted, but preferably is 1%-20% of the total solution by weight. Preferable phospholipid that is used in the liposome of the present invention includes, but is not limited to, saturated phosphatidyl choline or unsaturated phosphatidyl choline, for example, hydrogenated natural phospholipid or long chain saturated phospholipid, unsaturated phospholipid or short chain saturated phospholipid. Preferable long chain saturated phospholipid includes, but is not limited to, phosphatidyl choline (PC), phosphatidyl glycerol (PG), phosphatidyl serine (PS), or phosphatidyl ethanolamine (PE). Preferable phosphatidyl choline includes, but is not limited to, Hydrogenated egg phosphatidyl choline (HEPC), and hydrogenated soy phosphatidyl choline (HSPC). Preferable long chain saturated phosphatidyl choline includes, but is not limited to, dipalmitoyl phosphatidyl choline (DPPC), distearyloyl phosphatidyl choline (DSPC), or the combination thereof. Examples of unsaturated phophatidyl choline include, but are not limited to, egg phosphatidyl choline (EPC), soy phosphatidyl choline (SPC), and other synthetic unsaturated PC or natural unsaturated PC. Preferable short chain saturated phosphatidyl choline includes, but not limited to, dilauroyl phosphatidyl choline (DLPC).
In vivo test of skin irritation and hypersensitivity of example 5 are obtained in courtesy of US Northview Pacific Laboratories, Inc. The results are shown as the following.
(1) Dermal Sensitization Test
The experimental protocol is performed in according to Northview Standard Operation Procedure 16G-60. Using Buehler method for animal studies, that is, in observation of 10 six-weeks old Albino (guinea pig), each weight is between 300 to 500 g, for seven days. The purpose is to determine any skin allergic reaction in Albino (guinea pig) in contacting with the RA liposome. The result indicates that there is no hypersensitive effect.
(2) Skin Irritation Test
The experimental protocol is performed in according to Northview Standard Operation Procedure 16F-03. Six New Zealand White rabbits, each weight is between 2.5 to 2.8 kg, that each has been treated with 0.5 g/site RA liposomes gel for seven days. Any sign of skin irritation has been observed in 24 and 48 hours time period after the removal of drug patches. The results indicate that there is no skin irritation due to low PIS value. Low PIS values represent low irritation reaction.
No erythema: 0
Very slight erythema (barely perceptible): 1
Well-defined erythema: 2
Moderate erythema: 3
Severe erythema to eschar formation: 4
In order to evaluate the efficacy of present invention, some in vitro skin permeation studies have been performed.
Methods of In Vitro Skin Permeation Study
1. Materials and Reagents:
According to the methods mentioned above, we proceed to the in vitro skin permeation test of example 3, 10, 11, and 18. The results are showed in table 12.
As result of table 12, we find the skin permeability differs with various formulations. In these four examples in table 12, the permeability achieve to 50% in 24 hours. We are able to verify our present invention that will enhance the permeation of RA into the skin.
The in vitro skin permeability of example 5 and commercial RA cream are showed in table 13.
According to the results in table 13 that indicate the present invention is able to improve the skin permeation efficiency to 1-fold. Due to the RA encapsulated in liposome could enhance the interaction between the skin and the liposome, liposome is capable of increasing the penetration of RA. As a result of TPGS in our invention, there is a clear evidence of penetration enhancement.
Although the present invention has been explained in relation to its preferred embodiment, it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the invention as hereinafter claimed.