JP2634047B2 - ER based on alpha-tocopherol - Google Patents

Description

Description: BACKGROUND OF THE INVENTION The present invention relates to a method of incorporating a compound into an endoplasmic reticulum composed of a salt of an organic acid derivative of alpha-tocopherol (vitamin E) capable of forming bilayers. And compositions. Alpha-tocopherol, which has a hydrophilic moiety attached to it, such as a salt of an organic acid, is known as a multilamellar endoplasmic reticulum (vesic).
les) or small unilamellar ER suspensions. These vesicles can be made with or without organic solvents and can incorporate or associate with water-soluble compounds, partially water-soluble compounds and water-insoluble compounds. For convenience, the endoplasmic reticulum of the present invention will be referred to simply as "alpha-tocopherol endoplasmic reticulum", but it should be understood that salts of alpha-tocopherol organic acids are always used in making the endoplasmic reticulum.

The alpha tocopherol vesicles described herein are particularly useful for incorporating or associating biologically active or pharmaceutical compounds that can be administered in vivo. On the other hand, the endoplasmic reticulum of the present invention may be used in a test tube. For example, the hemi-succinate of alpha-tocopherol described herein may be used in vitro in a divalent cation-dependent assay.

The alpha-tocopherol vesicle of the present invention is a liposome. Liposomes are completely closed bilayer membranes containing an encapsulated aqueous phase.
Liposomes can be of any type, multilamellar vesicles (an onion-like structure characterized by a bilayer membrane separated by an aqueous layer) or unilamellar vesicles (having a single bilayer membrane). There may be.

Two parameters of liposome production are the relationship between endoplasmic reticulum size and lipid concentration: (1) The capture volume, defined as the volume entrapped by a fixed amount of lipid, is the liters incorporated per mole of total lipid. Expressed as units (1 mol ^-1 ). The uptake capacity depends on the number of lamellae and the radius of the liposomes and is further influenced by the lipid composition of the endoplasmic reticulum and the ionic composition of the medium. (2) Encapsulation efficiency is defined as the percentage of the initial aqueous phase separated by the bilayer membrane. [Deamer and Astor (Deam
er and Uster), 1983, Production of liposomes: methods and mechanisms, liposomes, M. Ostro
Editing, Marcel, Decker, Incorporated (Ma
rcel Dekker, Inc.), New York, pp. 27-51] First liposome production method [Bangham et al.
l.) Journal of Molecular Biology (J. Mol. Biol.) 13: 228 (1965)] suspends phospholipids in an organic solvent, then evaporates the solvent to dryness,
A waxy phospholipid was precipitated on the reaction vessel. Then, an appropriate amount of aqueous phase was added, the mixture was "swelled" and the resulting liposomes composed of multilamellar vesicles (MLV) were dispersed by mechanical means. The structure of the resulting bilayer membrane is such that the hydrophobic (non-polar) “tail” of the lipid is oriented toward the center of the bilayer membrane and the hydrophilic (polar) “head” is oriented toward the aqueous phase. ing. This technique is based on the sonicated small unilamellar vesicles described by Papahadjopoulos and Miller (Bioehim. Biophys. Acta. 135: 624 (1967)). (Hereinafter SUV
) Became the basis of the development.

In order to increase the encapsulation efficiency, it contains liposome precursors or micelles, ie an aqueous phase wrapped by one layer of lipid molecules (monolayer monolayar) oriented such that the polar head group is oriented towards the aqueous phase ER formation was first performed. The liposome precursor is formed by adding an aqueous solution to be encapsulated to an organic solvent solution of a polar lipid and sonicating. The liposome precursor is then emulsified in a second aqueous phase in the presence of excess lipid and evaporated. The resulting liposomes consist of an aqueous phase encapsulated by a lipid bilayer membrane and are dispersed in the aqueous phase [M. Schneide on September 23, 1980.
r) US Pat. No. 4,224,179].

In another attempt to maximize encapsulation efficiency, Papahadjopoulos (U.S. Pat. No. 4,235,871 issued Nov. 25, 1980) uses reverse-phase evaporative vesicles (hereinafter referred to as REVs). ) Describes "reverse-phase evaporation," which produces oligolamellar lipid vesicles, also known as. According to this method, an aqueous substance to be encapsulated is added to a polar lipid mixture in an organic solvent. The organic solvent is then evaporated until a uniform water-in-oil emulsion is formed and a gel is formed. Thereafter, the gel-like mixture is dispersed in an aqueous medium to make the gel into a suspension. The generated REV is
It consists mostly of unilamellar vesicles (large unilamellar vesicles, LUV), and also consists of some oligolamellar vesicles, characterized by only a few concentric bilayer membranes with a large internal aqueous space.

In addition, liposomes can be produced in the following manner. (A) Lenk et al., US Patent No. 4,52.
Stable multilamellar endoplasmic reticulum (SPLV), (b) Fontin et al. (Fountain et.a.
l.), a single phase (mon) by the method of U.S. Pat. No. 4,588,578.
ophasic) endoplasmic reticulum (MPV) and (c) Barry et al.
t.al.), U.S. Patent Application No. 80, filed November 21, 1985.
Ligated-thawed multilamellar endoplasmic reticulum (FATMLV) according to the method of US Pat. No. 0,545 (US Pat. No. 4,975,282).

Liposomes can be dehydrated and rehydrated. See Janoff et al., "Dehydrated Liposomes," PCT Application No. 8601103, published February 27, 1986 (European Patent No. 0190315).

Much has been said about the potential use of liposomes in drug delivery systems. For example, 19
On November 23, 76, Yuch-Erh Rahman and Elizabeth
U.S. Patent Nos. 3,993,754 and 1979 issued to A. Cerny)
Barry D. Sear on March 20, 2013
See U.S. Pat. No. 4,145,410 issued to s). In liposome drug delivery systems, the drug is taken up during liposome formation and then administered to the patient to be treated. The drug may be soluble in water or a non-polar solvent. A typical example of such a statement is 1980
On November 25, Papajajapoulos and Soca (Papahadjapou)
U.S. Pat. No. 4,235,871 issued to M. Schnerde on September 23, 1980.
No. 4,224,179 issued to r). When making liposomes for use in vivo, (1) eliminate the need to use organic solvents during liposome formation; and (2) incorporate larger volumes and higher concentrations per dose. It may be advantageous to maximize the encapsulation efficiency and the capture capacity so that the material can be supplied.

SUMMARY OF THE INVENTION The present invention includes methods and compositions for incorporating and administering various substances to the endoplasmic reticulum where the bilayer membrane contains salts of organic acid derivatives of alpha-tocopherol.
The endoplasmic reticulum of the present invention is particularly useful for administering an incorporated compound into a living body, in which case a biocompatible salt of an organic acid derivative of D-alpha-tocopherol is used to form the endoplasmic reticulum. Should be used. In fact, for in vivo administration, the tris (hydroxymethyl) aminomethane salt of an organic acid derivative of alpha-tocopherol (Tris-
Salts) are particularly useful as ER bilayer membrane components. Such derivatives may be esters or hemiesters of succinic acid, and the endoplasmic reticulum may incorporate bioactive agents such as, but not limited to, hormones, antifungals, antiglaucoma agents. The endoplasmic reticulum is administered by a variety of routes, including, but not limited to, topical, parenteral, oral and vaginal.

A method for producing alpha tocopherol ER comprises:
Adding to the buffered aqueous solution a salt of an organic acid derivative of alpha-tocopherol that forms a closed bilayer membrane in an amount sufficient to form a fully closed bilayer membrane incorporating the aqueous compartment. A suspension of ER is made by shaking the mixture. When the buffered aqueous solution also contains a counter ion of the salt in the solution, the formation of endoplasmic reticulum is promoted. Furthermore, if the dissociated salt of the organic acid derivative of alpha-tocopherol is negatively charged at neutral pH, the aqueous buffer solution should be substantially free of divalent cations. Similarly, dissociated salts of organic acid derivatives of alpha-tocopherol are neutral
If positively charged at pH, the aqueous buffer solution should be substantially free of polyvalent anions. By the application of suspension energy, ie sonication, the multilamellar vesicles are transformed into unilamellar vesicles.

Numerous approaches are possible for incorporating water-soluble, partially water-soluble and water-insoluble compounds into the alpha-tocopherol vesicles of the present invention. A compound (for example, a water-insoluble compound) or a water-soluble compound that partitions into the alpha-tocopherol bilayer membrane may be added to the aqueous phase before the formation of the endoplasmic reticulum, and incorporated into the endoplasmic reticulum during the formation. On the other hand, a water-insoluble or lipid-soluble compound may be added to the suspension of vesicles after vesicle formation,
In this case, the compound partitions into the alpha-tocopherol bilayer membrane. In another example, a water-insoluble compound and a salt of an organic acid derivative of alpha-tocopherol can be added to an organic solvent so that both are soluble (both are solubilized). The organic solvent is then evaporated, leaving a film in which the water-insoluble compound and the alpha-tocopherol derivative are evenly distributed. When an aqueous buffer solution is added to the film with stirring, alpha-tocopherol vesicles incorporating the water-insoluble compound are formed. Such vesicles can then be sonicated to form unilamellar vesicles.

The alpha-tocopherol vesicles of the present invention are particularly advantageous when used to incorporate water-insoluble or only sparingly soluble bioactive agents in water. This allows for the in vivo administration of a water-insoluble bioactive agent, such as a drug. Furthermore, since the dose: volume ratio can be changed, the water-insoluble compound is administered in vivo at a higher concentration. The alpha-tocopherol vesicles of the present invention show similar advantages when used to incorporate water-soluble bioactive agents. The alpha-tocopherol vesicle may be an ester-formed ester or hemiester of an organic acid,
These organic acid derivatives may be salts of bioactive agents, such as salts with pilocarpine. The endoplasmic reticulum of the present invention can also be used for in vitro diagnostic tests.

The present invention includes compositions comprising salts of organic acid derivatives of alpha-tocopherol, particularly those having a bioactive agent. Salts can be derived from ionizable bioactive agents. In the case of pilocarpine alpha-tocopherol vesicles, a pilocarpine salt of an organic acid derivative of alpha-tocopherol is used, and is preferably used in a molar ratio of 1: 1.

 The invention offers many advantages in the following respects.

 Alpha tocopherol vesicles are (1) easily and quickly formed.

(2) It has a higher encapsulation efficiency than the phospholipid MLV.

(3) The use of an organic solvent is not required for their production (although the alphatocopherol vesicle of the present invention can be produced using an organic solvent).

(4) It has a high capture capacity.

(5) It can take up bioactive agents or drugs that are released and metabolized when administered in vivo. Attribution of the incorporated agent in vivo depends on the mode of administration.

Furthermore, the alpha-tocopherol vesicle of the present invention
It can be used in a two-step method in which the substance to be taken up is first solubilized by incorporation into the alpha-tocopherol vesicles, and then the alpha-tocopherol vesicles containing the incorporated substance are taken up in ordinary liposomes. .

BRIEF DESCRIPTION OF THE DRAWINGS The invention can be more readily understood with reference to the following drawings.

FIGS. 1 and 2 graphically illustrate the effect of component concentration on the formation of complex vesicles containing alpha-tocopherol succinate hemiester tris salt, cholesterol succinate hemiester tris salt and associated cyclosporine. At 70 ° C. and at 60 ° C., respectively.

FIG. 3 is a graph showing the effect of the component concentration on the formation of a composite vesicle containing alpha-tocopherol succinate hemiester tris salt, cholesterol succinate hemiester tris salt and associated miconazole.

FIG. 4 graphically illustrates the effect of free and endoplasmic reticulum-incorporated bovine growth hormone on the growth of hypophysectomized rats, measuring growth as a weight change as a function of time. Things.

DETAILED DESCRIPTION OF THE INVENTION Alphatocopherol vesicles can be used to incorporate water-soluble, partially aqueous or water-insoluble compounds into the endoplasmic reticulum, the bilayer membrane of which is capable of forming a closed bilayer membrane. Contains salts of organic acid derivatives of tocopherol. Thus, the alpha tocopherol vesicles of the present invention may be prepared to (1) incorporate a water-soluble compound into the aqueous compartment, (2) incorporate a water-insoluble compound that partitions into the ER bilayer membrane, or (3) ) Both water-soluble and water-insoluble compounds can be incorporated into one ER preparation.

In the practice of the present invention, any salt of an organic acid derivative of alpha-tocopherol that can form a completely closed bilayer membrane in an aqueous solution similar to liposomes can be used. The suitability of certain salts of organic acid derivatives of alpha-tocopherol depends on the ability to sequester the water-soluble compounds from contact with the outside world.

The following criteria have been established for liposomes, which may be applied by analogy, to clearly determine that uptake of the endoplasmic reticulum into the aqueous compartment has taken place. [See Sessa and Weissmann, Biol. Chem. 245: 3295 (1970)].

(A) A clear separation free of sequestered compounds must be obtained by gel filtration.
(B) There must be no hydrophobic or charge-charge interactions between the outermost ER bilayer membrane and the incorporated compound. This results in failure to separate free compounds from the endoplasmic reticulum by molecular sieving operations, thereby artificially increasing the apparent isolation efficiency. To exclude this possibility, it must be shown that the water-soluble compounds added to the previously formed ER suspension do not elute with the ER. (C) the location at which the gel filtration pattern of the isolated molecule elutes with free molecules from the location that coincides with the ER peak by disruption of the gel-filtered vesicle by use of a surfactant or other membrane perturbant. Have to shift to

Organic acids that can be used to derivatize alpha-tocopherol include, but are not limited to, dicarboxylic acids, polycarboxylic acids, amino acids, and polyamino acids. Such a derivative may be an ester or a hemiester. Any organic acid can be used to derivatize alpha-tocopherol since salts increase the water solubility of the organic acid. However, it would be advantageous if the organic acid moiety itself was water soluble. Such water-soluble organic acid moieties include
Water-soluble aliphatic dicarboxylic acids such as malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, maleic acid, etc. (Note: It is recognized that the shorter the chain length, the easier it is to dissolve in water. the solubility of the boundary line is in the C ₆ ~C _7);
Water-soluble aromatic dicarboxylic acids such as hemimelitic acid, trimesic acid, succinimide and the like; polycarboxylic acids and any amino acids and polyamino acids, but are not limited thereto.

The derivatized salt of alpha-tocopherol is obtained by dissolving both the organic acid derivative of alpha-tocopherol and the counterion of the salt (eg, the free base of the salt) in a suitable volatile solvent and removing the solvent by evaporation or a similar technique. And leaving a residue consisting of a salt of an organic acid derivative of alpha-tocopherol. Counterions which can be used include Tris, which forms the corresponding salt, 2
-Amino-2-methyl-1-propanediol, 2
-Aminoethanol, bistrispropane, triethanolamine and the like, but are not limited thereto. In fact, the free base of an ionizable bioactive agent, such as miconazole free base, can be used as a counterion.

Generally, a 1: 1 molar ratio of the free base of the bioactive agent to the dicarboxylic acid derivative of alpha-tocopherol is used to make the corresponding bioactive agent salt. Organic solvents that dissolve both starting materials include those such as methanol, ethanol, chloroform, dimethylsulfamide.
The starting material is preferably at about 20-50 ° C, more preferably at about 20 ° C.
Added to the solvent at 20-30 ° C. Following the reaction, the resulting bioactive agent salt can be isolated by solvent removal under reduced pressure, evaporation, crystallization, or other methods known in the art. Preferred dicarboxylic acid derivatives are succinic acid derivatives. A preferred alpha-tocopherol is D-alpha-tocopherol.

When the bioactive agent free base is pilocarpine and the dicarboxylic acid is succinic acid, the pilocarpine: D-alpha-tocopherol molar ratio can be used in the range of 1: 0.5 to 1: 1. The most preferred equimolar amount of the starting material is dissolved in a polar organic solvent such as chloroform or methylene chloride. For methylene chloride, preferably about
30-60 ° C, more preferably about 40-60 ° C, most preferably
Add starting material at 55 ° C. and heat to reflux. After the reaction is completed
The solvent was removed under reduced pressure to give a product consisting of the pilocarpine salt of alpha-tocopherol succinic acid hemiester.

Miconazole, terconazole, econazole, isoconazole, thioconazole, bifonazole, clotrimazole, ketoconazole, butaconazole, itraconazole, oxyconazole, fenticonazole,
Antifungal agents such as nystatin, naftifine, amphotericin B, dinoconazole and ciclopirox olamine, especially miconazole or terconazole, are used as ionizable bioactive agents.

The alpha-tocopherol vesicles of the present invention are prepared so that the derivatized alpha-tocopherol is present in an amount sufficient to form an endoplasmic reticulum (ie, a fully closed bilayer membrane containing an incorporated aqueous compartment). May be added to the aqueous phase. The product is then shaken until an ER, usually a multilamellar milky suspension, is formed. In a preferred embodiment, the aqueous phase should contain salts in solution to promote vesicle formation. Furthermore, if the dissociated salt of the organic acid derivative of alpha-tocopherol is negatively charged at neutral pH, the aqueous buffer solution should be substantially free of polyvalent cations. Similarly, if the dissociated salt of the organic acid derivative of alpha-tocopherol is positively charged at neutral pH, the aqueous buffer solution cannot contain substantially multivalent anions.

The vesicles of the present invention may be used as multilamellar vesicles and may be reduced in size using many techniques known in the art, such as filtration and sonication. Also, Hope et al., BBA 812, 1985, pp.5
Cullis et al., Filed on Oct. 5-56 and Oct. 16, 1985, the subject of which is the "extrusion process for producing unilamellar vesicles".
et.al.), co-pending U.S. Patent Application No. 788,017 (related U.S. Patent No. 5,008,050).
The size of the endoplasmic reticulum may be reduced using the ET method (endoplasmic reticulum extrusion technique). Other techniques for matching the size of the ER
There is a CSR method (continuous size reduction) in which an ER pump continuously extrudes from a filter unit.

Methods reported for multilamellar endoplasmic reticulum formation [eg Brockerhof and Brockerhof
f and Ramsammy), Bio-Himika und Bio-Physica of Acta (Biochim, Biophys, Acta.) 691: 227
(1982), and the method for forming alpha-tocopherol multilamellar vesicles of the present invention does not require the use of an organic solvent. Further, unlike the blocker Hoff and Ramsamii methods, no sonication is required to form multilamellar vesicles. However, sonication of the milky suspension of the alpha-tocopherol multilamellar vesicles of the present invention or Flempress (SLM-Aminco), Ill.,
Subsequent sonication following the use of Urbana may be performed to convert the emulsion suspension of multilamellar alpha-tocopherol vesicles into a clear suspension of unilamellar vesicles. Using a French press without sonication often results in unilamellar vesicles.

As previously described, tris-salts of any organic acid derivative of alpha-tocopherol may be advantageously used in the practice of the present invention, e.g., alpha-tocopherol, such as alpha-tocopherol succinate hemiester or a mixture of succinate hemiesters. Tris-salts of dicarboxylic acid hemiesters are particularly useful for forming ER bilayer membranes of alpha-tocopherol vesicles for in vivo administration. For example, when using a hemiester succinate of alpha-tocopherol, about 5-70
0 micromolar of the tris-salt, including Tris-HCl (tris (hydroxymethyl) aminomethane hydrochloride) at about 5.
It can be added to 0 ml of buffered aqueous solution to form vesicles. In this case, the aqueous buffer solution should be substantially free of divalent cations.

According to the invention, water-soluble, water-insoluble or slightly soluble compounds can be incorporated or associated with liposomes composed of salts of organic acid derivatives of alpha-tocopherol in a number of ways.

(1) The water-insoluble compound can be added to a suspension of alpha-tocopherol vesicles (either multilamella or unilamella) made as described above using a suitable salt of an organic acid derivative of alpha-tocopherol. .
This compound is incorporated into the endoplasmic reticulum for distribution within the alpha-tocopherol bilayer membrane. This embodiment is conveniently implemented as follows. The water-insoluble compound is dissolved in a suitable organic solvent and the solvent is evaporated, leaving a film or residue of the compound. If an aqueous dispersion of preformed alpha-tocopherol vesicles is added to the residue, the residue will be incorporated into the ER bilayer membrane. In a preferred embodiment, unilamellar vesicles should be used. When multilamellar vesicles are used instead, the water-insoluble compounds are incorporated only into the outermost bilayer of the vesicles, leaving the innermost bilayer membrane unchanged, wasting derivatized alpha-tocopherol. May be used.

(2) solubilizing the water-insoluble compound and the salt of the organic acid derivative of alpha-tocopherol together in an organic solvent, and then evaporating the solvent to leave a film containing the water-insoluble compound and alpha-tocopherol evenly distributed; Can be. When the aqueous phase is added to the film with shaking, a suspension of alpha-tocopherol vesicles containing the incorporated compound is formed. As mentioned earlier, multilamellar vesicles can be turned into unilamellar vesicles.

(3) By adding a water-soluble compound or a water-soluble compound to the aqueous phase used for the production of the endoplasmic reticulum, the compound can be incorporated into alphatocopherol vesicles. That is, the compound can be added to the aqueous phase before or simultaneously with the addition of the salt of the organic acid derivative of alpha-tocopherol. In this case, the water-insoluble compound is
As it partitions into the bilayer membrane during ER formation, it becomes incorporated, while water-soluble compounds become incorporated into the aqueous compartment of the ER. In either case,
As mentioned earlier, multilamellar vesicles can be turned into unilamellar vesicles.

(4) If the bioactive agent is ionizable, the free base of the bioactive agent can be used as a counter ion for forming a salt of an organic acid derivative of alpha-tocopherol. The resulting composition can have increased solubility or stability. Further, alpha-tocopherol vesicles may be produced by any of the methods described above using a bioactive agent salt of an organic acid derivative of alpha-tocopherol. For example, miconazole, terconazole, econazole, isoconazole, thioconazole, bifonazole, clotrimazole, ketoconazole, butaconazole, itraconazole, oxyconazole, fenticonazole, nystatin, naftifine, amphotericin B, ziconazole and cyclopyroxolamine The antifungal free base can be used to make salt derivatives in one embodiment of the present invention. Also, free salts of pilocarpine can be used to make salt derivatives in one embodiment of the present invention. Liposomes using pilocarpine salt derivatives of alpha-tocopherol succinic acid hemiester can be made by adding an aqueous solution to dried pilocarpine salt of alpha-tocopherol succinic acid hemiester at a temperature of 30-40 ° C and stirring the suspension. . Examples of aqueous solutions that can work include USP, Distilled Water for Injection, and any of the following, alone or in combination: 0.01% (w / v) benzalkonium chloride, 0.025-0.1% (w / v) sorbic acid EDTA (ethylenediaminetetraacetic acid), 1.4% (w / v) polyvinyl alcohol and 0.05-0.50% (w / v) hydroxy Propyl methylcellulose. Other ionizable bioactive agents named herein may be used.

The compositions of the present invention may contain, in addition to the salt of the organic acid of alpha-tocopherol, a bioactive agent incorporated within or between the bilayer membranes of the endoplasmic reticulum, or wherein the bioactive agent is a bilayer membrane. You may have met with Such association results in the bioactive agent being located outside the endoplasmic reticulum.

The compositions of the present invention can be used for administration to the eye in the treatment of eye diseases such as glaucoma. In such applications, the composition may be administered by an art-known delivery system such as an eye dropper or applicator. The composition may further comprise a cohesive mucus such as hyaluronic acid, chondroitin sulfate, hydroxylpropyl methylcellulose or polyvinyl alcohol.
tics), a preservative, such as EDTA or benzalkonium chloride, in the above percentages, the usual amount of diluent and / or
Or it may contain a carrier material.

When administering to a person in the treatment of an eye disease such as glaucoma, the prescribing physician will ultimately determine the appropriate dose for a given patient, which, along with the nature and severity of the patient's symptoms, will be determined by the individual. It can be expected to change depending on age, weight and response. Typically, the ophthalmic dosage of the composition will be in the form of a 4% pilocarpine solution administered to the average adult patient once or twice daily in a suitable pharmaceutically acceptable diluent or carrier, from 25 to 50 μl. Would be in range. However, these numbers are merely illustrative and in some cases it may be necessary to use dosages outside this limit.

Using any of the above four methods, both water-soluble and water-insoluble compounds can be incorporated into a single alpha-tocopherol ER product.

According to the above method for incorporating a water-insoluble compound using the alpha-tocopherol vesicle of the present invention, once the water-insoluble compound is distributed to the bilayer membrane, the vesicle does not need to remain as it is. In fact, once the compound partitions into the bilayer membrane, the endoplasmic reticulum is disturbed or destroyed, thereby leaking or releasing the entrapped aqueous compound. These "leaky" vesicles can be used to deliver entrapped water-insoluble compounds, but should not be used to encapsulate or deliver water-soluble materials.

According to one embodiment of the present invention, the following liposomes can be produced using the tris salt of alpha-tocopherol succinic acid hemiester. About 1-400 mg of alpha-tocopherol succinic acid hemiester tris salt is added per 1 ml of a buffered aqueous solution containing 0.01 M Tris-HCl and 0.14 M NaCl. The mixture is shaken to form a milky suspension of alpha-tocopherol succinic acid hemiester. The endoplasmic reticulum may be pelletized by centrifugation, and the buffered aqueous solution may be repeatedly washed. Alpha-tocopherol succinate hemiester multilamellar vesicles (AHS-MLV) can also be sonicated to form small unilamellar vesicles of alpha-tocopherol succinate hemiester (AHS-SUV). The endoplasmic reticulum is unstable in the presence of divalent cations. That is, upon exposure to divalent cations, the entrapped aqueous fraction and water-soluble compounds are released. Thus, the aqueous medium used during storage during production of the endoplasmic reticulum should be substantially free of divalent cations.

The compounds incorporated by the method of the present invention can be used in various ways. For example, if the compound is a bioactive agent, the compound incorporated into alpha-tocopherol vesicles can be administered in vivo. This facilitates the in vivo delivery of bioactive agents that are usually insoluble or slightly soluble in aqueous solutions. Incorporation into the endoplasmic reticulum composed of salts of organic acid derivatives of alpha-tocopherol can facilitate administration of such insoluble compounds at higher dose: volume ratios. In fact, since the vesicles are used to incorporate one or more bioactive agents for delivery into the body, the alpha-tocopherol vesicles of the invention
It is particularly advantageously used. Furthermore, since the vesicles of the present invention can be produced without using an organic solvent, they show advantages over ordinary lipid vesicles or liposomes when used in vivo.

Compounds that are bioactive agents can be incorporated into the alpha-tocopherol vesicles of the invention. Such compounds include antimicrobial compounds such as gentamicin,
Antiviral compounds such as rifampin, antifungal compounds such as amphotericin B, anthelmintic compounds such as antimony derivatives, tumoricidal compounds such as adriamycin, antimetabolites, peptides, proteins such as albumin, diphtheria Toxins such as toxins, enzymes such as catalase, polypeptides such as cyclosporin A, hormones such as estrogen, hormone antagonists, neurotransmission antagonists such as acetylcholine, glycoproteins such as hyaluronic acid, alpha Lipoproteins such as trypoproteins, immunoglobulins such as IgG, immunomodulators such as interferon or interleukin, dyes such as arsenazo III, radiolabels such as ¹⁴ C, radiopaque such as ⁹⁰ Te Compound, carboxyfluorescein Such as fluorescent compounds, receptor binding molecules such as estrogen receptor proteins, anti-inflammatory compounds such as indomethacin, anti-glaucoma agents such as pilocarpine, mydriatic compounds, local anesthetics such as lidocaine, and codeine Various anesthetics, vitamins such as alpha-tocopherol, nucleic acids such as thymine, polynucleotides such as RNA polymers, psychoactive or anti-stabilizing agents such as diazepam, monosaccharides, disaccharides or polysaccharides, etc. It is not limited to these. Some of the many specific compounds that can be incorporated are pilocarpine; polypeptide growth hormones such as human growth hormone, bovine growth hormone and pig growth hormone; indomethacin; diazepam; alpha tocopherol itself and There is tyrosine. Antifungal compounds include miconazole, terconazole, econazole,
There are isoconazole, thioconazole, bifonazole, clotrimazole, ketoconazole, butaconazole, itraconazole, oxyconazole, fenticonazole, nystatin, naphthyfine, amphotericin B, dinoconazole and ciclopirox olamine, with miconazole or terconazole being preferred. The simultaneous incorporation of two or more compounds may be particularly desirable where those compounds exert a captive or synergistic effect. The amount of drug administered in the liposome will generally be the same as for the free drug, but may be administered less frequently.

Drugs that are taken up or associated with alpha-tocopherol vesicles can be injected parenterally or injected (eg, intravenous, peritoneal, intramuscular, subcutaneous, intra-aural, intramammary, etc.), topically ( Absorption from the epithelial or mucosal skin layers (eg, nose, mouth, vagina, rectum, gastrointestinal mucosa), for example on parts like eyes, skin, in ears or on affected areas such as wounds and burns And the like, can be administered in vivo by any suitable route, including, but not limited to.

In another example of these uses, compounds incorporated into alpha-tocopherol vesicles can be incorporated into lipid vesicles or a wide range of substances including, but not limited to, liposomes, gels, oils, emulsions, and the like. Not something. For example, any type of liposome product (eg, phospholipids SPLV, MPV, FATMLV, MLV,
As a component in SUVs, LUVs, REVs and others), a suspension containing the incorporated compound may be added to the aqueous phase.
Thereby, the water-insoluble compound is incorporated into the phospholipid liposome.

The alpha-tocopherol vesicles of the present invention are described in co-pending U.S. Patent Application No. 773,429, filed September 10, 1985, entitled "Steroidal Liposomes" (Related U.S. Patent No. 4,891,208). It can be used advantageously with the vesicles of salts of organic acid derivatives of sterols, such as the vesicles of the cholesterol succinic acid hemiester tris salts. Such steroid liposomes form bilayer vesicles, are probably unilamellar or multilamellar, and can incorporate compounds that are bioactive agents.

Generally, any sterol that can be modified by the binding of an organic acid can be used. For example, such sterols include, but are not limited to, cholesterol, vitamin D, phytosalol (including, but not limited to, sitosterol, campesterol, stigmasterol, and the like), steroid hormones, and the like. It is not limited.

Organic acids that can be used to derivatize sterols include the organic acids used to derivatize alpha-tocopherol as described above.

The organic acid can be attached to the sterol hydroxyl group via an ester or ether linkage using conventional methods (e.g., U.S. Patent No. 3,859,047, U.S. Patent No. 4,040,784, U.S. Patent No. 4,042,330, See U.S. Pat. Nos. 4,183,847 and 4,189,400). Derivatized sterol salts are prepared by dissolving both the sterol organic acid derivative and the salt counterion (eg, the free base of the salt) in a suitable volatile solvent and removing the solvent by evaporation or a similar technique. Can be made by leaving a residue consisting of salts of organic acid derivatives of sterols. Counterions which can be used include tris, 2-amino-2-methyl-1, 3-propanediol, 2-aminoethanol, bis-trispropane, triethanolamine, etc. which form the corresponding salt, It is not limited to these. In fact, the free base of an ionizable bioactive agent, such as miconazole free base, can be used as a counterion. Thus, a bioactive agent can be used as a counterion.

In the endoplasmic reticulum of the present invention, when an organic acid derivative of sterol is used together with alpha-tocopherol, the ratio of sterol to alpha-tocopherol is 0:10.
It is used at 0 to 100: 0 mol%.

The alpha-tocopherol vesicles of the present invention can also be used with conventional liposomes to solubilize various substances. The substance is first taken into the alphatocopherol vesicles,
It can be incorporated into liposomes. On the other hand, the substance to be solubilized may initially be present, together with all the substances required to make the two vesicles.

Other uses for the particular properties of the product will occur to those skilled in the art. For example, the alpha-tocopherol succinic acid hemiester vesicles of the present invention are highly sensitive to divalent cations, and thus incorporate a support dye sensitive to divalent cations for use in in vivo colorimetric diagnostics. Can be made.

The following examples are provided for illustrative purposes and are not intended to limit the scope of the invention.

Example 1 Preparation of Tris Salt of Alcotocopherol Hemisuccinate Alphatocopherol hydrogen succinate [Sigma Chemical Co.,
5 g of St. Louis, Mo.) was dissolved in 100 ml of diethyl ether. Tris base (Fisher, Fairlawn, NJ) (1.14 g) dissolved in about 5 ml of water was then added to the ether solution with stirring or shaking in 0.5 ml portions. The solution was rotary evaporated to dryness and then further dried under high vacuum to yield the title compound in the form of a gummy yellow residue.

Example 2 Preparation of Tris Salt of Alpha-Tocopherol Succinic Acid Hemiester 2.28 g of tris [hydroxymethyl] aminomethane in 3 ml of hot water was mixed with 10 ml of methylene chloride in a 500 ml round bottom flask.
To a solution of 10 g of D-alpha-tocopherol acidic succinate in 0 ml at 25 ° C. was added with stirring. The reaction mixture was rotated on a rotary evaporator while maintaining at 55 ° C. for 15 minutes in a thermostat.

The solvent was removed under reduced pressure and the product was frozen for 24 hours. The material was removed, triturated with a mortar and pestle, and excess solvent was removed under vacuum for 24 hours. Generated title compound (4.8
g) was stored in a closed vial and protected from light.

 Alternatively, this material was frozen for 48 hours.

In another manufacturing method, the solvent was removed by lyophilization.

Example 3 Arsenazo III (A) to alpha-tocopherol ER
Incorporation of 100 mg of Tris salt of alpha-tocopherol succinic acid hemiester prepared as above
1M Tris-HCl, 0.14M NaCl and 4.5mM Arsenazo III (A
rsenazo III) was added to 1 ml of a solution containing all at pH 7.3 and the suspension was swirled in the presence of 3 mm glass beads. The resulting Tris salt vesicles of alpha-tocopherol succinic acid succinate are pelleted by centrifugation at 10,000 xg for 15 minutes, after which the pellet is washed and pH 7.3 containing 0.01 M Tris-HCl and 0.14 M NaCl.
Was re-centrifuged three times in 10 ml of the solution. The resulting pellet is
Red color was exhibited by the incorporation of Arsenazo III. The degree of incorporation was measured as 30%.

Example 4 Solubilization of Prognanolone 50 mg of alpha-tocopherol succinic acid hemiester tris salt, 50 mg of cholesterol succinic acid hemiester tris salt and 5-β-pregnan-3-ol-20-
20 mg of ON (KabiVitrum, Stockholm, Sweden) was added to excess methanol and dried in a round bottom flask under vacuum. The resulting film was then resuspended in 1.0 ml of 10 mM Tris-HCl buffer, pH 7.4, containing 140 mM NaCl, in the presence of glass beads, with shaking until a firm gel was formed. Extensive sonication with a Branson E-Module (40 KHZ) 5 gallon water bath sonicator reduced the gel viscosity and produced vesicles 0.2-0.4 microns in diameter.

A tris salt of cholesterol succinic acid hemiester was prepared as follows. Ohio, Cleveland
Cholesterol hydrogen succinate (50.3) manufactured by ICN
g, 0.11 mol) was dissolved in 1.5 diethyl ether.
Fairlawn Fisher, New Jersey
Sher) (12.1 g, 0.1 mol) was dissolved in 30 ml of water. The Tris solution was then added to the cholesterol solution and the resulting solution was rotary evaporated to a milky wet residue. The milky residue was lyophilized for 12 hours, after which the tris salt product of cholesterol succinic acid hemiester was recrystallized three times from a volume of about 5 liters of boiling ethyl acetate.

The boiling ethyl acetate solution was filtered while hot and cooled to room temperature. A gel-like cholesterol succinic acid hemiester tris salt formed and a 100 ml sinter filtered it through a glass funnel to remove ethyl acetate. Initial removal of the solvent was by squeezing. In another manufacturing method, the initial solvent removal was performed by mechanical compression. Further, the solvent was removed under a vacuum of 0.1 mmHg for 12 hours. At that time, a hard, brittle white substance in the form of a 23-g disk of silver coin size was observed.

Powder the white disc with a mortar and pestle and bring the substance to 50 ° C.
And the last traces of ethyl acetate were removed by applying a vacuum of 0.1 mmHg. 55 mg of the resulting powder, 0.14M
The suspension was suspended in 1.0 ml of 0.01 M Tris-HCl buffer containing NaCl at pH 7.4. A milky suspension formed, which was sonicated with a water bath sonicator to produce a clear cholesterol succinate hemiester tris salt vesicle solution.

Example 5 Solubilization of Cyclosporin A Cyclosporin A (Sandoz, Inc., East Hanover, NJ), Tris salt of cholesterol succinic hemiester and alpha tocopherol succinic acid Hemiester tris salts were dissolved in methanol in the relative proportions shown in Table 1.

A portion of the solution, sufficient to bring the final aqueous suspension volume to 0.25 ml, was placed in a 13 x 100 mm test tube,
Placed in a 500 ml round bottom flask in a water bath at 0 ° C. and dried to a thin film by removing the solvent by rotary evaporation.

The film obtained in this manner is adjusted to pH containing 0.14 M NaCl.
0.25 ml of 7.3 0.01 M Tris-HCl buffer was added and the film was rehydrated while swirling the suspension in the presence of glass beads.

Under the various conditions listed in Table 1, the presence or absence of cyclosporin A crystals was examined by microscopic observation, and the results are shown in FIG. As shown in FIG. 1, when the concentration of the tris salt of cholesterol succinic acid hemiester and / or the tris salt of alpha-tocopherol succinic acid hemiester was too low, crystals of cyclosporin A were observed.

The experiment was repeated using a 60 ° C. water bath. The result is shown in FIG.

Example 6 Solubilization of miconazole Miconazole free base (MCZ), cholesterol succinic hemiester tris salt (CHS) and D-alpha-tocopherol succinic hemiester tris salt (TH
A stock solution of S) was prepared as a 50 ml solution of absolute ethanol.

An appropriate amount of the stock solution was pipetted into a 50 ml round bottom flask. Each flask has the relative proportions shown in Table 2,
A total of 0.1 mmol of material was included.

Solvent was removed by rotary evaporation at 60 ° C. Then
The material was resuspended in 1.0 ml of 0.15 M NaCl, pH 7.4, containing 0.01 M Tris-HCl. For more than 2 weeks, the product was observed microscopically for evidence of miconazole crystals and macroscopically for evidence of phase separation. I checked in. If no crystals or phase separation were observed, the product was deemed satisfactory (see FIG. 3).

Example 7 Sustained Supply of Bovine Growth Hormone to Hypophysectomized Rats To demonstrate the solubilizing and sustained release properties of the present invention,
Two types of endoplasmic reticulum associated with bovine growth hormone (BGH) were prepared. One type of endoplasmic reticulum contained egg phosphatidylcholine (EPC or lecithin) and the tris salt of alpha-tocopherol succinate hemiester associated with BGH. The other endoplasmic reticulum contained egg phosphatidylethanolamine (EPE) in addition to the same components.

The endoplasmic reticulum without EPE was prepared as follows. The solvent phase was prepared by removing the solvent from a chloroform solution containing 400 mg [EPC Sigma Chemical Co., St. Louis, Mo.] by rotary evaporation at 37 ° C. Then the residue was dissolved in 5 ml of diethyl ether. The aqueous phase containing BGH associated with alpha-tocopherol vesicles is pH adjusted with 0.14M NaCl.
Prepared by adding 25 mg of succinic acid hemiester of alpha-tocopherol to 1 ml of 0.01 M Tris-HCl at 7.4. An opaque suspension forms, which at 500 nm
Sonication was performed as described above until an optical density between 0.3 and 0.6 was obtained, which was clarified. A reading of 0.3 is preferred.
28 mg of powdered BGH (Eli Lilly, Indianapolis, IN) was added to 0.3 ml of endoplasmic reticulum product by swirling with brief sonication to disperse the powder. A milky suspension resulted, which did not show any evidence of a powder as such. This aqueous phase suspension was dropped into the solvent phase. If not disturbed, the droplets would sink and create an opaque bottom layer.

However, regarding the addition of the aqueous phase to the solvent phase, stable multilamellar vesicles (SPL) can be obtained by sonicating water droplets in the ether phase while flowing a nitrogen stream over the vessel.
V, Lenk et al., US Patent No. 4.522.803.
No.] was promoted. The solvent was evaporated, leaving a paste, was it rehydrated the above Tris -HCl buffer 10ml containing 10 mM CaCl ₂ final concentration. Calcium neutralized the charge imparted by the tris salt of alpha-tocopherol succinate hemiester and promoted strong liposome pelleting on centrifugation. Slow vortexing promoted rehydration.

The resulting SPLVs were pelleted by centrifugation at 10,000 xg for 10 minutes, and the pellets from the four batches were pooled and washed twice more with Tris-HCl / calcium buffer. The supernatant was decanted to leave a viscous pellet, about 0.5 ml of which was used for the following studies.

The endoplasmic reticulum further containing EPE is a chloroform solution containing EPC 1.54 and EPE [Avanti Polar Lipids,
Inc. (Avanti Polar Lipids, Inc.),
Birmingham, Alabama] was prepared by mixing a chloroform solution containing 52.8 mg in a round bottom flask and removing the chloroform by rotary evaporation at 37 ° C. The resulting dried lipid film was dissolved in 20 ml of diethyl ether and an aqueous phase containing BGM associated with tris salt vesicles of alpha-tocopherol succinic acid hemiester was added.

The aqueous phase was 0.01 M Tris-HCl at pH 7.4 containing 0.14 M NaCl.
Prepared by solubilizing 112 mg of BGH in 1.2 ml of 25 mg / ml alphatocopherol succinic acid hemiester tris salt in buffer. The alphatocopherol suspension was previously prepared at a pressure of 40,000 pounds / parallel inch by a French pressure cell press [SML Instruments, Inc. (SLM Instruments, Incorporated).
c.), Urbana, Illinois] to give an optical density at 550 nm of 0.3-0.6.

As described above, while sonicated under nitrogen for, by adding water phase in ether phase, the viscosity of the SPLV paste made, buffering pH7.4 containing 0.01M Tris, 0.14 M NaCl and 10 mM CaCl ₂ It was rehydrated with 20 ml of liquid. Rehydration was performed with orbital mixing in the presence of glass beads. The resulting liposomes were washed three times with a total of 20 ml of the above buffer solution, and then washed with a Backman J2-21 using a JA-14 rotor.
Centrifugation was performed at 10,000 rpm for 45 minutes in a centrifuge. Approximately a 0.5 ml portion of the resulting viscous pellet was used for the following studies.

Sustained release of the endoplasmic reticulum was confirmed by Charles River Breeze, Inc., of Charles River Breeding Laboratories, Inc., Wilmington, Mass.
ding Laboratories, nc.) in female hypophysectomized rats at 25 days of age. Upon arrival, animals were weighed and fed a 5% glucose diet for 2 days, after which they were switched to water and rat diet ad libitum. Animal weight 32 days and 39
Measured daily and those that gained more than 10 g during this period were excluded as incomplete pituitary resections. Groups of eight animals were then injected intramuscularly (IM) or subcutaneously (SC) with free BGH or BGH incorporated into one of the ER preparations. Animals receiving free BGH receive SC daily.
Was done. On the other hand, for animals receiving endoplasmic reticulum-associated BGH, the route shown in FIG.
Association hormone (estimated 70% associated during ER production)
5.6mg (assumed to be 40% of association for EPC products)
Was injected only once on the first day. Control animals received no treatment. The data shows the average weight change value for 8 animals in each group.

The abbreviations shown in FIG. 4 are as follows. (1) EPC: EPE /
-THS-BGH SC means that bovine growth hormone associated with the endoplasmic reticulum containing egg phosphatidylcholine, egg phosphatidylethanolamine and the tris salt of alpha-tocopherol succinate hemiester was administered subcutaneously. (2) EPC / -THS-BGH SC was administered subcutaneously with bovine growth hormone associated with endoplasmic reticulum containing egg phosphatidylcholine and tris salt of alpha-tocopherol succinic acid hemiester (egg phosphatidylethanolamine is not present). Means (3) EPC / −
THS-BGH IM, except for intramuscular administration,
Same as (2).

As shown in FIG. 4, the treated animals did not show significant weight gain during the course of the experiment. By free BGH,
Administration of free BGH occurred daily, although significant growth occurred. The growth promotion produced by the various associated BGH-ER preparations, although somewhat lower, was significant given the fact that only one injection of these products was made. The best results were seen when BGH associated with EPC: EPE / THS ER was administered subcutaneously.

Example 8 Solute Incorporation of Tris Salt of Alpha-Tocopherol Succinate Hemiester Various amounts of the D-alpha-tocopherol succinate hemiester tris salt ("Tris salt") powder shown in Table 3 were added with 5 microliters of ⁵¹ Cr. 5.0 ml of the 0.01 M Tris / 0.14 M NaCl buffer (pH 7.4) was added in a round bottom flask. The suspension was stirred for 2 minutes to dissolve the Tris salt and allowed to stand for 2 hours to form MLV. on the other hand,
Thomas Scientific Spectra (Th
omas Scientific Spectrapor) 12,000 MWCO 1/4 "dialysis tubing was cut into 7 inch lengths and boiled for 1 hour with 2 changes of distilled water.
1.0 ml from the product was pipetted into the bag. The bag was closed at the top with a plastic zipper and the radioactivity was counted for 1 minute on a TMA nalytic model 1191 gamma counter. Then 10
Each bag was dialyzed in 0 ml of Tris / NaCl buffer with stirring. After 6 hours, the dialysate was replaced with fresh buffer. Dialysis continued for 20 hours. The radioactivity of the bag was then measured and the percentage of incorporated ⁵¹ Cr was calculated as follows.

The captured volume (solute) value was calculated as follows. The counts from three tests per sample volume of Tris salt were averaged, and the percent uptake calculated (above) and the sample volume expressed in known microliters (5.0 ml = 5000μ) were used to calculate the lipid microparticles. 1 / mol ⁵¹ Cr per mole was calculated as follows.

The results are shown in Table 3.

Example 9 Pilocarpine-Alphatocopherol succinic acid hemiester 5 g of pilocarpine base was placed in a weighed 500 ml round-bottomed flask with a stopper, and the total mass was recorded in grams. D-alpha-tocopherol acid succinic acid sul (12.75 g,
(Corresponding to a 1: 1 molar ratio of pilocarpine: D-alpha-tocopherol)) and the contents weighed again.
Methylene chloride (50 ml) was added, the flask was stirred, the solids dissolved, and the flask was weighed again. Place the flask on a rotary evaporator in a 55 ° C water bath,
Spin for minutes (no vacuum applied). After 30 minutes, the flask and contents were weighed again, then rotary evaporated in vacuo at 55 ° C. Thereafter, the weight of the flask was recorded every 30 minutes until two consecutive weighings were within 0.1 g. Then
The product was cooled to room temperature (25 ° C), stoppered and stored at 4 ° C.

Example 10 Pilocarpine-Alphatocopherol succinic acid hemiester 5.0 liter stoppered round bottom flask, pilocarpine base 3
Using the materials and methods of Example 9, using 0 g, D-alpha-tocopherol succinate (corresponding to a molar ratio of pilocarpine: D-alpha-tocopherol 1: 1), and 300 ml of methylene chloride.

Example 11 Preparation of pilocarpine-alpha-tocopherol succinic acid hemiester liposome A 500 ml round bottom flask containing the product of Example 9 above was placed on a rotary evaporator, and the water bath temperature was set at 55 ° C. After warming the salt for 30 minutes, the water bath temperature was reduced to 35 ° C. An aqueous solution (92 ml) of 0.1% (w / v) sorbic acid and 0.1% (w / v) sodium EDTA dihydrate was added and the suspension was swirled. The final volume was adjusted to 125 ml by adding the aqueous phase.
The resulting liposomes were treated with CSR (continuous size reduction), a method and apparatus for continuously treating large volumes of liposomes to produce small liposomes having a uniform average diameter.

The size reduction device is (a) a high pressure piston pump,
It consists of (b) an in-line filter element with a predetermined pore size, (c) a reservoir for the supply stock (liposome suspension) and (d) a reservoir for collecting the treated supply stock. The system may include a valve unit that directs the feed back to the supply vessel for recirculation or to the process collection vessel. Supplying the supply stock to the pump by any conventional means, including but not limited to pumping the pump head by the piston action of the pump head itself and / or externally pumping the supply stock by an external device. Can be. The pump head provides energy to circulate the feed stock to the filter unit.

In the above example, the liposomes were passed 10 times through a stainless steel scale filter with a nominal pore size of 500 nm.

The above method was carried out using the aqueous phase composition according to Table 4.

Example 12 Investigation of sizing of pilocarpine alfa-tocopherol succinic acid hemiester vesicles The product of Example 11 was examined by freeze fracture electron microscopy. The result is a predominantly unilamellar liposome population in the size range of about 30-225 nm. The vesicles prepared as in Example 11 were also measured using quasi-elastic light scattering.

Example 13 Pilocarpine-Alphatocopherol succinic acid hemiester Two nucleopores with a pore size of 400 nm (Nucleopor
e) Using the material and method of Example 11, using the VET400 method (ER extrusion technique) to reduce the size through a filter. The VET400 process is described in co-pending U.S. Patent Application No. 788,017, filed October 16, 1985, entitled "Extrusion Process for Producing Unilamellar Endoplasmic Reticulum" by Cullis et al. U.S. Pat. No. 5,008,050).

0.1% (w / v) with 0.1% (w / v) EDTA as aqueous phase
The above method was performed using sorbic acid.

Example 14 Preparation of pilocarpine-alpha-tocopherol succinic acid hemiester conjugated batch liposomes The materials and methods of Example 11 were used and the sized batches were combined to a total volume of 750 ml. Then
The liposome was treated in the same manner as in Example 11.

The above method was also carried out using the aqueous phase composition according to Table 6.

Example 15 Preparation of pilocarpine-alpha-tocopherol succinic hemiester liposome A round bottom flask containing the product of Example 10 above was placed on a rotary evaporator, and the water bath temperature was set at 55 ° C. After warming the salt for 30 minutes, the water bath temperature was reduced to 35 ° C. An aqueous solution (550 ml) of 0.01% (w / v) sorbic acid 0.01% (w / v) EDTA-sodium dihydrate was added and the suspension was mixed for 1-1.5 hours using a stirring blade. The final volume was adjusted to 750 ml by adding the aqueous phase. The resulting liposomes were treated with CSR (continuous size reduction). That is, the nominal pore size is 500n
The liposomes were passed 10 times through a stainless steel filter, m.

The above method was carried out using the aqueous phase shown in Table 7.

Example 16 Pilocarpine-alpha-tocopherol succinic acid hemiester 1.0 g of pilocarpine, D-alpha-tocopherol,
The method and materials of Example 11 were repeated using a pilocarpine: D-alpha-tocopherol molar ratio of 1: 0.5, equivalent to 1.28 g. Liposomes were formed by the method of Example 12. In this case, the added aqueous phase was distilled water for injection according to the United States Pharmacopeia, so that the final concentration of pilocarpine was 4%. The results regarding the viscosity of the liposome solution,
It is listed in Table 8.

Using distilled water for injection of 0.1% (w / v) sorbic acid and 0.1% (w / v) EDTA-sodium dihydrate in the United States Patent
The above method was repeated.

Example 17 1.0 g of pilocarpine, D-alpha-tocopherol 5.
The method and materials of Example 16 were repeated using a molar ratio of pilocarpine: D-alpha-tocopherol of 1: 2 equivalent to 1 g. Liposomes were formed by the method of Example 12.
In this case, the added aqueous phase was distilled water for injection in the United States Pharmacopeia, so that the final concentration of pilocarpine was 4%. Table 8 lists the concentration results for the liposome solution.

Using distilled water solution of 0.1 (w / v) sorbic acid and 0.1% (w / v) EDTA-sodium dihydrate in the United States Pharmacopeia for injection,
The above method was repeated.

Example 18 1.0 g of pilocarpine, D-alpha-tocopherol 1
The method and materials of Example 16 were repeated using a molar ratio of pilocarpine: D-alpha-tocopherol of 1: 4 corresponding to 0.2 g. Liposomes were formed by the method of Example 12. In this case, the added aqueous phase was distilled water for injection in the United States Pharmacopeia, so that the final concentration of pilocarpine was 4%. Table 8 lists the concentration results for the liposome solution.

The above procedure was repeated using 0.1% (w / v) sorbic acid and 0.1% (w / v) EDTA-sodium dihydrate in distilled water for injection in the United States Pharmacopeia.

Table 5 Measurement of pilocarpine- THS endoplasmic reticulum by quasi-elastic light scattering method Pillow THS: artificial tear diameter (nm) without mixing 205 4: 1 202 1:11 206 1:10 233

 ──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Volksack, Lois, E. United States New Jersey 08648, Lawrenceville, Tower Place 11 (72) Inventor Weiner, Alan, El United States of America New Jersey State 08648, Lawrenceville, Oacrin-Terrace 117 (72) Inventor Trenbraye, Paul, A. Nottingham Way, Nottingham Way, Hamilton, New York, 08619, USA 2633 (72) Inventor Bergamini, Michael, Vi. Sullivan Trail, Easton, 18942, Pennsylvania, USA 941 (56) References Biochemistry, Vol. 7 (1985) p. 1646-1653

Claims (31)

(57) [Claims] An alpha-tocopherol vesicle comprising a completely closed bilayer membrane comprising an ionizable bioactive agent salt of an alpha-tocopherol organic acid derivative. 2. The alpha-tocopherol vesicle according to claim 1, wherein the ionizable bioactive agent comprises an antifungal compound. 3. An antifungal compound comprising miconazole, terconazole, econazole, isoconazole, thioconazole, bifonazole, clotrimazole, ketoconazole, butaconazole, itraconazole, oxyconazole, fenticonazole, nice titanium, naftifine, amphotericin B, The alphatocopherol vesicle according to claim 2, which is dinoconazole or ciclopirox olamine. 4. The method of claim 1, wherein the ionizable bioactive agent is a peptide,
2. The alphatocopherol vesicle according to claim 1, comprising a protein, a glycoprotein or a lipoprotein. 5. The alphatocopherol vesicle according to claim 1, wherein the ionizable bioactive agent is pilocarpine. 6. The alpha-tocopherol vesicle according to claim 1, wherein the organic acid derivative is a succinic hemiester. 7. The alpha-tocopherol vesicle of claim 5, wherein the molar ratio of pilocarpine to alpha-tocopherol is between about 1: 0.5 and 1: 1. 8. The alphatocopherol vesicle according to claim 1, wherein the organic acid is a dicarboxylic acid or a polycarboxylic acid. 9. The alpha-tocopherol vesicle according to claim 8, wherein the dicarboxylic acid is an aliphatic dicarboxylic acid. 10. The alpha-tocopherol vesicle according to claim 9, wherein the aliphatic dicarboxylic acid has 7 or less carbon atoms. 11. The alphatocopherol vesicle according to claim 10, wherein the aliphatic dicarboxylic acid is succinic acid. 12. The alpha-tocopherol vesicle according to claim 1, wherein the organic acid is an amino acid or a polyamino acid. 13. The alpha-tocopherol vesicle according to claim 1, wherein the alpha-tocopherol is D-alpha-tocopherol. 14. Tris (hydroxymethyl) aminomethane salt of an organic acid derivative of alpha-tocopherol, 2-
Amino-2-methyl-1,3-propanediol salt, 2
2. The alpha-tocopherol vesicle according to claim 1, comprising a complete bilayer membrane comprising -aminoetalol salt, bis-trispropane salt or triethanolamine salt. 15. The alpha-tocopherol vesicle according to claim 14, comprising a tris (hydroxymethyl) aminomethane salt of a succinic acid hemiester derivative of alpha-tocopherol. 16. An alpha-tocopherol vesicle comprising a completely closed bilayer membrane comprising an ionizable bioactive agent salt of an organic acid derivative of alpha-tocopherol, and further comprising a bioactive agent. 17. The alpha-tocopherol vesicle according to claim 16, wherein the bioactive agent further comprises a compound selected from the group consisting of an antibacterial compound, an antifungal compound, an antiviral compound and an anthelmintic compound. 18. The method according to claim 18, wherein the antitumor compound is tylosin.
18. The alpha tocopherol vesicle according to claim 17, which is: 19. The antifungal compound may be miconazole, terconazole, econazole, isoconazole, thioconazole, bifonazole, clotrimazole, ketoconazole, butaconazole, itraconazole, oxyconazole, fenticonazole, nice titanium, naftifine, amphotericin B, 18. The alpha tocopherol vesicle according to claim 17, which is dinoconazole or ciclopirox olamine. (20) a bioactive agent further comprising a peptide,
17. The alphatocopherol vesicle according to claim 16, which is selected from the group consisting of a protein, a glycoprotein and a lipoprotein. 21. The alpha-tocopherol vesicle according to claim 16, wherein the bioactive agent further contains a growth hormone. 22. The alpha-tocopherol vesicle according to claim 21, wherein the growth hormone comprises bovine growth hormone. 23. The alpha-tocopherol vesicle according to claim 20, wherein the bioactive agent further comprises insulin. 24. The alphatocopherol vesicle according to claim 16, wherein the bioactive agent further contained is selected from the group consisting of a dye, a radiolabel, a radiopaque compound and a fluorescent compound. 25. The method according to claim 16, wherein the bioactive agent is further selected from the group consisting of an anti-inflammatory compound, an anti-glaucoma compound, a mydriatic compound, an analgesic compound and an anesthetic compound.
Item 7. The alpha-tocopherol vesicle according to Item. 26. The alpha-tocopherol vesicle according to claim 25, wherein the bioactive agent comprises indomethacin. 27. The alpha-tocopherol vesicle according to claim 25, wherein the bioactive agent comprises pilocarpine. 28. The alpha-tocopherol vesicle according to claim 16, wherein the bioactive agent comprises an anesthetic, a psychoactive agent or an anxiolytic. 29. The alphatocopherol vesicle according to claim 28, wherein the bioactive agent comprises diazepam. 30. The alpha-tocopherol vesicle according to claim 16, wherein the bioactive agent comprises a vitamin. 31. The alpha tocopherol vesicle according to claim 16, wherein the alpha tocopherol is D-alpha tocopherol.