WO1996014057A1 - Tangential filtration preparation of liposomal drugs and liposome product thereof - Google Patents

Abstract

Liposomes are formed by the constant volume tangential filtration of a starting aqueous solution of bilayer-forming material and detergent to reduce the molar ratio of detergent to bilayer-forming material in the starting mixed micelle or lipid-detergent associates solution, while simultaneously avoiding dilution of the volume of the mixed micelle solution or of the concentration of the bilayer-forming material therein by replacing the detergent solution removed by the tangential filtration simultaneously with its removal with aqueous non-solubilizing agent-containing solution which may contain buffer, electrolyte, or dissolved drug, medicament, or cosmetic as desired. The method enables the production of small particle size vesicles or liposomes which are unilamellar and of homogeneous particle size distribution using relatively large bilayer-forming material concentrations in a relatively short time and with relatively small waste detergent filtrate volumes, and thus allows greater production of acceptable liposomes per unit time, making the disclosed constant volume tangential filtration method a highly-advantageous procedure which may be conducted rapidly, continuously, commercially, and economically. Liposomes produced by the constant volume tangential filtration method disclosed.

Description

TANGENTIAL FILTRATION PREPARATION OF LIPOSOMAL DRUGS AND LIPOSOME PRODUCT THEREOF

FIELD OF THE INVENTION

The field of the invention is the preparation of liposomes and active-ingredient-containing, e.g., medicament- containing, liposomes. The present invention provides the first and highly-advantageous method for the preparation of liposomes directly by constant volume tangential filtration and without the previously-required "dilution" or "concentration-dilution" procedures of the prior art.

According to the present invention, liposomes are produced rapidly, conveniently, economically, and optionally continuously by tangential filtration of an aqueous solution of aggregates of one or more membrane-forming substances and one or more solubilizing agents forming water-soluble aggregates referred to as mixed micelles, which are in equilibrium with solubilizing agent, e.g., detergent monomers. When the concentration of solubilizing agent is reduced by tangential filtration under pressure, with simultaneous introduction of an equal volume of aqueous non- solubilizing agent solution as solubilizing-agent solution is filtered off, so as not to dilute the volume of the aqueous solution of the mixed micelles or the concentration of the membrane-forming lipids in the aqueous solution, the mixed micelles are forced to grow in size due to reduction in solubilizing agent concentration and finally to form liposomes which will contain the drug or other active ingredient present in the aqueous solution of water-soluble aggregates (mixed micelles).

BACKGROUND OF THE INVENTION AND PRIOR ART The present invention relates to a method for the preparation of liposomes and liposomal drugs, which are aqueous dispersions of artificial hollow sphere-like aggre¬ gates (vesicles) of suitable amphiphilic membrane-forming substances, preferably associated with drug or other active ingredients, and optionally in gel form and/or mixed with pharmaceutical auxiliaries, e.g., excipients or carriers. Amphiphilic membrane-forming substances are molecules, which are poorly soluble in water and have separate hydro- philic (polar) and lipophilic (apolar) domains. Such molecules in most cases form double layer membranes, but also membrane-spanning monolayer-forming amphiphiles are known. Liposomes consist of at least one membrane enclosing an aqueous compartment and have a size range of 0.02 μm up to several μm in diameter. There are various types of liposomes: Small unilamellar vesicles (SUVs) have one membrane and a size range between 0.02 and 0.05 μm. Large unilamellar vesicles (LUVs) have one membrane and a diameter larger than 0.05 μm. Oligolamellar large vesicles (OLVs) and multilamellar large vesicles (MLVs) are larger than 0.1 μm and have several or many, in most cases concentric, membranes. Liposomes with several nonconcentric membranes, i.e., several smaller vesicles side by side in a larger vesicle, are called multivesicular vesicles ( Ws).

Drugs, medicaments, and cosmetics, depending on their physicochemical properties, can be associated with liposomes by encapsulation into their inner aqueous compartment, by incorporation into their nonpolar membranous interior, or by adsorption to their membrane/water interfaces. Such association to liposomes can result in improved solubility, enhanced stability, in well-directed biodistribution (targeting), reduced unwanted side-effects, and in controlled drug release.

Hydrophobic drugs, other medicaments, or cosmetics can be associated with liposomes during their preparation or by mixing with preformed liposomes. Hydrophilic materials, e.g., drugs, can be encapsulated in preformed liposomes by a difference in pH-values between the inner and outer aqueous compartment orbyproducingmembranedisturbances, which make a penetration of drug from the outside to the inside possible.

Methods used to date for the preparation of liposomes involve increasing the ratio between the bilayer-forming substance to solubilizing agent in the associates-containing aqueous phase in one of several ways, such as reducing the total concentration of solubilizing agent in the aqueous phase by dilution of the aqueous phase, with or without intermittent concentration removal of solubilizing agent from the associates by chemical and/or physico-chemical reactions of the solubilizing agent in the aqueous phase, reducing the total concentration of solubilizing agent by countercurrent dialysis of the aqueous phase, or increasing the total concentration of bilayer-forming substance in the aqueous phase by adding additional bilayer-forming substance. In this context the term "total concentration of the solubilizing agent" means the total content, based on the aqueous phase, of solubilizing agent in the aqueous phase and in the associates. As far as physico-chemical inactivation of the solubi¬ lizing agent in the aqueous phase containing the associates, the usual ways for effecting such an inactivation is by a sudden change in temperature (temperature jump) in the aqueous phase, addition of suitable adsorbents which remove solubilizing agent present in the aqueous phase and in the associates when coming into contact therewith, a sudden change in pH value (pH jump) in the aqueous phase, and addition of a further substance to the aqueous phase to complex or precipitate solubilizing agent contained therein. Each method presents its own problems and shortcomings as does dilution or concentration-dilution of the aqueous phase containing the associates or the employment of countercurrent dialysis, as will be pointed out hereinafter. Flow-through dialysis using a stationary phase can also be employed, as represented by USP 4,438,052. Other liposome-forming procedures mentioned in the foregoing are well set forth and represented by USP 4,731,210, and Aitcheson and Tenzel, USP 4,994,213, disclose a reverse osmosis process for forming lipid structures including liposomes using organic solvents as solubilizing agents.

Weder USP 4,731,210 at Column 1, line 48 and following, reviews the state of the art. As already stated, there are, in principle, several methods for the production of liposomes. Detergent-mediated liposome production is based upon the solubilization of an amphiphilic bilayer-forming substance, e.g., a lipid such as lecithin, with the aid of a solubilizing agent, e.g., a detergent such as sodium cholate, octyl glucoside, or the like, followed by a controlled removal of the solubilizing agent by dialysis. During the process, liposomes are formed from the initial lipid-detergent associates (mixed micelles) in direct relation to a reduction of the solubilizing agent, e.g. , detergent, concentration. In USP 4,731,210, at Column 4, line 57 following, but as is also well known in the art, several methods are disclosed as presently available for reduction of the solubilizing agent, e.g., detergent. concentration. The method most frequently employed is the so-called flow-through dialysis (Weder's Point C) and this may be conducted in a countercurrent manner as in USP 4,731,210 or using a stationary phase micelle-containing colloidal solution as in Weder USP 4,438,052.

Problems inherent in the presently most advantageous specific techniques, i.e., the dilution technique and the countercurrent dialysis technique, are clear.

Weder USP 4,731,210 provides a good starting point for the identification of problems with the most widely-accept¬ able methods for liposome production. For example, when the "dilution" method is employed, the aqueous phase with regard to the solubilizing agent, e.g., detergent, content thereof, is simply diluted by adding an additional amount of the aqueous phase. In Column 5, line 5 and following of this patent, it is stated: "— the aqueous phase — can be diluted with an additional amount of aqueous phase —", for example, in a ratio of 4:1 (line 9) and, in line 18: the composition of the "aqueous phase to be added—" is described in more detail. In lines 31 and following, it is explicitly mentioned that the "-- formation of the liposomes — can be achieved — by simply adding a sufficient amount — of aqueous phase to -- the — substance mixture". The total effect of such considerable dilution is obvious to one skilled in the art and will be commented upon further hereinafter.

Then, when the production of unilamellar liposomes is desired, the addition of the additional amount of aqueous phase must be made suddenly, as pointed out in Column 3, lines 28 and following, as follows: "— that unilamellar liposomes with a minimum size — can be obtained if the equilibrium conditions are changed -- instantaneously, for example, by sudden dilution of the aqueous phase --". As apparent to one skilled in the art and generally speaking, the additional aqueous phase is simply added according to the dilution method which preferably leads to at least a two-fold increase in volume (see Claim 2 of Weder) or even a four-fold increase in volume (see Claim 3 of Weder' and, as also will be clear to one skilled in the art, it is difficult if not impossible to produce unilamellar liposomes having a minimum size when the additional amount of aqueous phase must be added instantaneously or suddenly and, of course, this requirement makes it impossible to conduct such a method on a continuous basis.

In contrast, according to the present invention, an equivalent volume of the filtered out amount of solubilizing- agent containing-aqueous phase is simultaneously or concurrently replaced by or made up with non-solubilizing agent-containing solution so that a constant volume is maintained continuously, and the replacement of the solubi- lizing-agent-containing aqueous phase according to the present invention can be effected or carried out constantly and continuously to maintain a constant volume as the solubilizing agent, i.e., detergent, is filtered out to decrease its concentration in the aqueous phase, in contrast to the dilution of the original volume with an additional amount of aqueous phase according to Weder. Thus, the constant volume tangential filtration (CVTF) method of the present invention, in any case without dilution of the volume of the mixed micelle solution, leads to a constant increase in the molar ratio of bilayer-forming substance to solubilizing agent by filtration off of the solubilizing agent, thereby to decrease its concentration in the aqueous phase as it is replaced by non-solubilizing agent solution, especially with reference to the molar ratio of solubilizing agent to the bilayer-forming substance, e.g., lipid, which is not filtered off but which remains due to the larger structure of the aggregates in the retentate.

According to the flow-through dialysis method, using a semi-permeable membrane which retains the bilayer-forming substance, e.g. , lipid, in the phase to be dialyzed but which is permeable to the solubilizing agent, e.g., detergent, solution, the phase to be dialyzed which contains the lipid- detergent associates (mixed micelles) will be conducted in tubes or channels made of the semipermeable membrane countercurrently along a dialyzing fluid such as water, buffer, and possibly containing the dissolved drug or other medicament. The detergent permeating outwardly through the membrane as a result of the concentration differential is absorbed by the dialyzing fluid and discarded as a result of its destabilizing action on the liposomes thus formed and, because of its skin-irritating potential, the detergent must be removed as completely as possible. Such a method is extremely time-consuming and produces great volumes of detergent-containing dialyzing fluid which must either be discarded or concentrated and recycled for detergent production and such obvious disadvantages are conspicuous to one skilled in the art and result in serious loss of economy of the method.

To repeat, in contrast, the method of the present invention involves the solubilization of an amphiphilic bilayer-forming substance, e.g., phospholipid, with the aid of a solubilizing agent, i.e., detergent, which is subsequently removed in a controlled mode but using tangential filtration under pressure. The phase containing the lipid-detergent associates (mixed micelles) and, if desired, one or several drugs, medicaments, or cosmetics, is filtered against a suitable semipermeable membrane under pressure. The flow direction of the phase is tangential to the membrane surface in contrast to the countercurrent dialysis of Weder USP 4,731,210, with simultaneous replacement of detergent solution, as it is filtered off, by non-solubilizing agent-containing solution so that no dilution occurs and so that the volume of the mixed micelle solution remains essentially constant. The present method is therefore able to prevent the clogging of the membrane pores to a great extent and, in addition, such production of the liposomes by constant volume tangential filtration is a rapid and effective process, involving only a negligible loss in bilayer-forming substance, which can be conducted over extended periods and continuously, and which is accordingly much more commercially acceptable and economic.

* * * * *

Again, in contrast, although tangential filtration has previously been employed for contacting already pre-formed liposomes loaded with osmotic agent with a solution of active agent as in USP 5,049,392, and for separating unencapsulated active ingredient from liposomes after centrifugation and recyclingvia "diafiltration" inatangential-flow filtration device in accord with USP 4,776,991, or for removal of residual solvent after precipitation of liposomes or microparticles in accord with USP 5,100,591, it has never to the best of our knowledge been employed in the formation of the liposomes themselves, much less according to the method of the present invention which involves the maintenance of a constant volume of micelle solution and a constant undiluted concentration of the bilayer-forming substance, e.g., phospholipid, by the introduction of an equal volume of aqueous phase, which may contain additional non-solubilizing materials, simultaneously with the removal of solubilizing agent, i.e., detergent, by tangential filtration under pressure. The retentate thus comprises the liposome-forming micelles and formed liposomes with or without contained drug or other active ingredient in an essentially constant volume of aqueous solution, which may of course be further filtered and concentrated, whereas the off-filtered solubilizing agent, i.e., detergent or surfac¬ tant, can be readily and conveniently recycled in a further solubilizing process, frequently even without further concentration.

In greater detail, in the above-mentioned USP 5,049,392 pre-formed liposomes containing osmotic agent and active agent in diluent are loaded onto the retentate side of a tangential flow filtration unit and large unit volumes of diluent are then passed through the tangential flow unit to cause rupture of the liposomes which then reform and concomitantly incorporate active agent, much like the procedure for incorporating active agent into a liposome which is set forth in USP 4,731,210.

Along the same lines is Brown USP 5,314,695, issued May 24, 1994, who produces prothrombin-liposome compositions and who speaks of removal of detergent by tangential flow diafiltration, among other things, in Column 7 and then proceeds to employ his so-called diafiltration procedure using a tangential flow diafiltration device for removal of detergent in Example 6 in Column 13. Upon inspection, it is found that this Example is no more than the old dilution method of Weder which, prior to the dilution, involves a concentration step, the procedure involving removal of his buffer (CHAPS) by passing 10 volumes of dialysis buffer through the device, followed by adjusting the volume of the dialysate (retentate) back to the original volume. It is therefore clear that the volume of the mixed micelle solution of Brown is first reduced several times by filtration and is then adjusted back to the original volume. The Brown procedure is discussed in greater detail hereinafter in and about comparative Examples D and E, but suffice it to say that, in the mechanism of liposome formation during detergent removal, the starting solution contains mixed lipid/detergent micelles in equilibrium concentration with free detergent molecules. When a certain volume of solution containing exclusively the free detergent is removed, as by tangential filtration, the volume of the retentate of course decreases, but the free detergent concentration remains constant, so that no liposome formation occurs during the filtration process. Fusion of the mixed micelles to finally result in liposomes occurs only if the free detergent concentration is decreased, and this is done according to Brown's Example 6 by diluting the retentate back to its original volume. The liposome preparation of Brown was therefore carried out with several concentration and several dilution steps and not by tangential filtration in accord with the present invention, in which the volume of the retentate is maintained essentially constant, that is, the procedure of the present invention is constant volume tangential filtration (CVTF). The liposome-formation procedure of the present invention thus involves constant volume tangential filtration and not the concentration/dilution procedure of Brown, which has certain inherent disadvantages, including excessive detergent waste and excessively long production times, along the lines of the Weder dilution procedure, as will be pointed out in greater detail hereinafter.

* * *

In general, then, it has been shown that liposomes can be prepared by mechanical procedures or by using organic solvents or detergents, to capsulate:

Mechanical procedures involve hydration of lipids resulting in multilamellar vesicles, which may be disrupted by ultrasonication, homogenization with suitable pressure or extrusion of the MLV through membranes with defined pore size.

Organic solvents such as ethanol or ether are used to dissolve the membrane lipids. After mixing with aqueous buffer, lipids can form lamellar structure at the arising interfaces of the two solvent systems or when the polarity of the solvent mixture increases. Upon removal or lowering the concentration of the organic solvent, liposomes are formed.

Suitable detergents and membrane lipids form water- soluble aggregates, so-called mixed micelles, which are in equilibrium with detergent monomers. Upon reducing the monomer concentration (by dilution, dialysis, gel chroma- tography, complexation, precipitation, pH jump, temperature jump, or by adsorption to added molecules), the mixed micelles are forced to grow in size and finally to form liposomes.

All of these procedures have at least one important disadvantage. Mechanical procedures lead to degradation of the membrane lipids, to soiling of the products by abrasion of the devices, or to liposomal products with unwanted liposome size or number of membrane lamellae. When using organic solvents, the latter disadvantage is also often true, in addition to the disadvantage of residual solvent molecules. The detergent removal methods disclosed above are time-consuming compared to mechanical procedures and the use of organic solvents. Moreover, when using relatively high lipid concentration to enhance the yield of liposomes and trapping efficiency of drugs or the like, the use of detergents can lead to an undesired increase in the number of membranes in the liposomes and an excessively slow and prolonged procedure. In general, however, detergent removal is a procedure which permits the production of liposomes with a unique membrane, tailored size, and high trapping efficiency for drugs, other medicaments, or cosmetics.

Although excellent liposomes can be prepared by countercurrent dialysis, even having a unilamellar structure with a diameter of 50 nm plus or minus 2 nm or 44 nm plus or minus 2 nm with extreme homogeneity when using low lipid concentrations and high flow rates for the most rapid possible reduction of the concentration of solubilizing agenr in the solution containing the associates being dialyzed, or on the same order using simple dilution by addition of aqueous phase technique, such excellent results can only be obtained at a very low lipid concentration using a very high flow rate and with the generation of a great deal of residual waste filtrate and are accordingly not adapted to continuous, economical, or commercial production of liposomes as is the present method.

A procedure is therefore needed which combines the advantages of the known detergent procedures with an accelerated and efficient removal of detergent, thus strongly reducing preparation time, undesired liposome structures and aggregation at high lipid concentrations, as well as minimization of residual waste-filtrate, and which can be carried out rapidly, efficiently, commercially, economically, and if desired and preferably also continuously. Such is provided by the present invention.

OBJECTS' OF THE INVENTION

It is an object of the present invention to provide a novel method for the production of liposomes and drug or other active-ingredient-containing liposomes directly by constant volume tangential filtration. Another object is the provision of such a method wherein the concentration of solubilizing agent in the mixed micelle solution is reduced by tangential filtration through a membrane which allows passage therethrough of the solubilizing agent solution but excludes the bilayer-forming substance, mixed micelles, and formed liposomes, thereby keeping the formed micelles or liposomes in the retentate. An additional object is the provision of such a method which is carried out without dilution of the volume of the mixed micelle solution containing the bilayer-forming substance and solubilizing agent and any medicament or other active ingredient which may be present therein by the simultaneous or concurrenr introduction of water or aqueous non-solubilizing agent- containing solution in such a quantity to make up for or compensate for the volume of solubilizing agent solution lost by the tangential filtration. Still another object is the provision of such a method which is carried out without dilution of the concentration of the bilayer-forming material in the starting mixed micelle solution of bilayer-forming material and solubilizing agent. A further object is to provide such a method which will permit the employment of high bilayer-forming material concentrations without concur¬ rent production of undesirable multilamellar vesicles or inhomogeneous particle size distribution. A still further object is the provision of such a method which may be carried out continuously and economically and with production of reduced amounts of waste detergent filtrate. A yet addi¬ tional object is the provision of such a method which allows the formation of liposomes, the encapsulation, adsorbtion or incorporation of substances into the liposomes, as well as the removal of non-liposomally attached or incorporated substances and the concentration of the liposome products - all in one procedure and with the employment of a single device. Yet further objects of the invention will become apparent hereinafter and yet additional objects will be apparent to one skilled in the art to which this invention pertains.

* * *

SUMMARY OF THE INVENTION

What we believe and claim to be our invention, there¬ fore, inter alia comprises the following, alone or in combination:

A method of producing liposomes from an aqueous solution comprising bilayer-forming material and detergent, in the form of a solution of mixed micelles and unbound dissolved detergent, comprising the steps of eliminating aqueous detergent-containing solution by tangential filtration of the solution through a membrane adapted to pass the aqueous detergent-containing solution as a filtrate but to retain a mixed micelle- and liposome- containing fraction thereof as a retentate, while simultaneously avoiding dilution of the volume of the mixed micelle solution or of the bilayer-forming material therein, by replacing the detergent-containing solution removed by the tangential filtration by water or a non-solubilizing aqueous solution so as to maintain an essentially constant volume of the mixed micelle solution, and thereby to produce a liposome dispersion as retentate, such a method wherein the bilayer-forming material comprises a phospholipid, sphingolipid, glycolipid, sterol, archebac- terial lipid, or a non-ionic synthetic lipid, such a method wherein the bilayer-forming material comprises a phospholipid and the detergent is selected from the group consisting of sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium ursodeoxycholate, salts of these acids with other cations, n-alkylglycosides, n-alkylmethyl- glycamides, n-alkyloligooxyethylenes, and n-alkylglycosyl- amines, such a method wherein the lipid comprises egg or soy lecithin. such a method wherein the detergent is selected from the group consisting of sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium ursodeoxycholate, salts of these acids with other cations, n-alkylglycosides, n-alkylmethyl- glycamides, n-alkyloligooxyethylenes, and n-alkylglycosyi- amines, such a method wherein the detergent comprises at least one of sodium cholate and octyl glycoside, such a method wherein the non-solubilizing aqueous solution is a buffer-, electrolyte-, and/or active ingredient- containing aqueous solution, such a method wherein the bilayer-forming material comprises a phospholipid, such a method wherein the non-solubilizing aqueous solution is an aqueous buffer solution, such a method wherein the buffer is selected from the group consisting of MOPS, HEPES, TRIS, and phosphate buffers, such a method wherein the volume of the mixed micelle solution and the concentration of the lipid is maintained essentially constant, such a method wherein essentially all of the lipid concentration is maintained in the aqueous mixed micelle solution, such a method which is conducted under a pressure developed in the tangential filtration system, such a method wherein the pressure is up to about one ( 1 ) bar, such a method wherein the pressure is between about 0.2 and about 0.4 bar, such a method including the step of further filtering the retentate to remove residual detergent or liposome-unassoci- ated material, such a method including the step of further filtering the resulting liposome dispersion to concentrate the same into a gel, such a method wherein a constant volume is continuously maintainedby replacementof thedetergent-containing aqueous phase by non-solubilizing-agent-containing solution, such a method wherein the non-solubilizing agent-containing solution is an electrolyte-containing solution, such a method wherein the non-solubilizing agent-containing solution is a buffer-containing solution, such a method wherein the non-solubilizing agent-containing solution is an active ingredient-containing solution, such a method wherein the non-solubilizing agent-containing solution comprises a hydrophilic active ingredient, such a method wherein the non-solubilizing agent containing solution comprises a medicament or cosmetic active ingredient, such a method wherein a lipophilic active ingredient is present in the starting mixture comprising bilayer-forming material, such a method wherein the retentate is recycled until it essentially comprises unilamellar liposomes of desired parti- cle dimensions, such a method wherein the dimensions are between about 30 and 200 nm, such a method which is conducted continuously, such a method wherein the method is conducted until the detergent concentration in the resulting liposome dispersion is reduced to a detergent/bilayer-forming material molar ratio of less than 0.3, and such a method wherein the method is conducted until the detergent concentration in the mixed micelle solution is reduced to a detergent/lipid molar ratio of less than 0.3.

Moreover, liposomes whenever produced by the method of tangential filtration as defined in any of the foregoing. GENERAL DESCRIPTION OF THE INVENTION

An aqueous solution of a suitable solubilizing agent, i.e., detergent, and a bilayer-forming substance, e.g., lecithin, and optionally one or more drugs, cosmetics, or medicaments is filtered under pressure through a suitable membrane. The flow direction of the solution is tangential to the membrane surface, which has the advantage that ob¬ struction of the membrane pores is avoided and detergent is effectively and rapidly removed without loss of bilayer- forming substance. Water, buffer, salt solution, or a solution containing a drug or medicament, is simultaneously substituted for the loss of fluid in the retentate without dilution of the volume of the mixed micelle solution or lipid concentration thereof. By such detergent removal liposomes are formed continuously, whereby the drug is incorporated in the membranes, adsorbed on the membranes, or encapsulated in the inner aqueous compartment of the liposomes. The method may be conducted over extended periods or continuously. Without interruption of the procedure, in a second step additional tangential filtration and substitution of wash water or other non-solubilizing agent-containing aqueous solution for the filtrate can be applied to remove residual detergent or liposome-unassociated drug. Without interrup tion, in a third step, the liposome dispersion optionally can be highly concentrated to a jelly consistency by an additional filtration off of the aqueous phase.

Liposomes prepared by this method have a narrow size distribution and are nearly exclusively unilamellar, when suitable detergents are chosen. The mean size of the liposomes can be tailored by the choice of the used deter¬ gent, such as, e.g., sodium cholate, sodium deoxycholate, or n-alkylglycosides, the lipid/detergent ratio, the solvent system employed, i.e., the buffered or unbuffered aqueous solution, the lipid composition, e.g., phospholipids (natural, modified, or synthetic), cholesterol and/or derivatives thereof, or other membrane-forming lipids, and lipid concentration ranging from as low as one might wish up to several hundred millimolars of lipid (jelly-like consistency), and the temperature employed which may range from almost the freezing point of the solvent up to about 65°C.

FURTHER DESCRIPTION OF THE INVENTION THE TANGENTIAL FILTRATION APPARATUS

The tangential-flow filtration apparatus may for example be the Pellicon® (Millipore Corp., Bedford, MA) or the smaller laboratory-scale Minitan®, using a suitably-sized filter or membrane or ceramic "membrane", with a cut off preferably in the range between about 1 and <100, preferably 10-50 kD (kiloDaltons ) . For larger batches, many square feet of filter can be employed in a single system to improve the speed of the method. Alternatively, the tangential flow apparatus may be a FILTRON® device such as an Ultrasette®, a Minisette®, a Centrasette®, or the like, using a tangential flow flat membrane. Particularly advantageous in this respect is the FILTRON® screen channel cassette, having a top and bottom closing membrane and ultrafiltration membranes with screens in between all membranes, thereby providing retentate channels and filtrate channels, all as is well known in the art. Although the micelle solution flows over the membrane surface in a parallel direction in the channels, due to the dense and fine screen which is placed on the various membranes involved, especially when using a screen channel cassette, a wave-like flow of the solution over the membrane surface is effected. Thus, instead of the flow being parallel, a tangential flow is induced inasmuch as, at the bottom point or peak of a wave, the liquid current hits the particular membrane involved in a tangential direction.

The usual tangential filtration (TF) unit consists of a receiver or starting receptacle, a pump, the actual tangen¬ tial filtration instrument ("membrane"), and tubings with integrated rotary slide valve and manometer. The receptacle holding the starting mixed micelle solution is fed with buffer solution (containing active substance as required) to the extent to which filtrate (= detergent-containing, substance-containing buffer) is removed, thereby at any time maintaining a constant volume in the TF unit.

In principle, pressures can be determined in the TF unit at three sites: the feed pressure (P_F) caused by the pump; the filtrate pressure (P_p) which may be controlled by a subsequent valve; and the retentate pressure (P_R) which may also be controlled by a subsequent valve. The working pressure mentioned in the Examples is the retentate pressure

The tangential filtration unit consists of the core piece of the entire process, i.e., one or several ultrafil- tration membranes with the associated hardware, i.e., the so-called filter holder. Dependent on size and material. these filter holders are as stated marketed by Filtron under the trademarks Minisette®, Centrasette®, Maxisette®, Ultrasette®, or by Millipore under the trademarks Pellicon® or Minitan®, among others.

The ultrafiltration membrane is available in different materials and in different molecular weight exclusion limits (pore sizes). As already mentioned, polyether sulfone ultrafiltration membranes of the Omega range with cut-offs between 1 and <100 kD in the Minisette® filter holder were usually employed. In order more rapidly to prepare the liposomes and in larger quantities, no single membrane but one or several membrane cassettes are used (1 cassette = 5 single membranes = 5-fold filter surface), making possible unlimited scale-up.

Membrane cassettes are well understood to be multiple layers of membrane assemblies comprising sheets of ultra¬ filtration membrane placed between waved polymeric screen retentate separators and waved screen filtrate separators. Blocked borders on the filtrate and retentate screens direct the separated fractions to defined collection areas on the bottom cassette hardware manifold.

In such membrane cassettes, the "Feed" mixed micelle solution (after vesicle formation, of course, the detergent- containing liposomal dispersion) flows over the retentate screen, thereby taking on a wave-like motion and flow. At the peak and bottom points of the wave, the "feed" flows over the ultrafiltration membrane in tangential direction. Molecules smaller than the membrane exclusion size (water, detergent, active substance) pass through the membrane, are collected as filtrate, and are discarded or recycled.

Larger particles (mixed micelles, liposomes and active substances) remain between the membranes and are recycled as retentate and refiltered after volume balancing to an essentially constant volume.

Thus, a tangential filtration system as employed according to the invention involves a starting receptacle for the introduction of starting materials or ingredients into the system, a pump for pumping the same onto the filter, and cooperating piping for the conveyance of the fluid starting materials to the pump and thence to the filter. From the filter, cooperating piping takes the retentate in one direction and the filtrate in another direction for recycling. The retentate is recycled back to the starting receptacle until withdrawn from the system when the detergent/lipid ratio reaches a certain predetermined minimum, e.g., less than 0.3.

In operation, the mixed micelle solution enters into the retentate track and moves tangentially to the membrane because of the waves imparted to the dispersion by the undulations in the screen itself, the retentate moving out of the track at the opposite side of the cassette whereas the filtrate escapes through the pore openings in the membrane at the top and bottom of the retentate track and is collected in the filtrate tracks above and below the retentate track, from which it is recovered for recycling. According to the present invention, fresh non-solubilizing solution is introduced into the system at the same rate as the filtrate is removed therefrom, so as to maintain the volume of the mixed micelle solution essentially constant.

DETAILED DESCRIPTION OF THE INVENTION

The invention is further illustrated by the following preparations and examples, which are not to be construed as limiting. Representative Details. Materials. Apparatus, and Manufac¬ turing Conditions employed in the evaluation and validation of the tangential filtration method for producing liposomes: Lipids: Soy lecithin (S.lec)

Egg lecithin

Cholesterol Detergents: Sodium cholate - (cholate)

Sodium deoxycholate n-alkylglycosides

Buffers: Morpholino dipropane sulfonic acid (MOPS)

HEPES (Merck Index ϋ, No. 4573) TRIS (Merck Index ϋ, No. 9684 - Trometh- amine)

Sodiumphosphate; monobasic Sodiumphosphate; dibasic The unit used for tangential filtration was a Filtron Minisette®. Polyethersulfone single membranes (filter surface 0.15 ft²) or membrane cassettes (filter surface 0.75 ft²) of the Omega range were used. Pore sizes (to be more exact: size exclusion limits) varied between 10 kD (kilo- Daltons ) and 100 kD. From the following results the general suitability of membranes <100 kD, preferably 1-50 kD, can be derived.

The flow rate was between 75 and 250 ml/min. As this parameter depends on the pump used and the diameter of the tube it is not critical and, as can be taken from the following, has no influence on the resulting quality of the liposomes.

Dependent on flow rate and membrane used, there is a specific system pressure in each tangential filtration system which, for kinetic reasons (conversion rate of mixed micelles into liposomes) will not be too high. In the following experiments a suitable retentate pressure (P„) was up to about one (1) bar, preferably between about 0.2 and about 0.4 bar.

The liposomes by tangential filtration are usually produced at room temperature. However, production was also successful at 4°C and 65°C. The latter higher working temperature ensures the suitability of the tangential filtration process for the production of liposomes from hydrated lecithin and synthetic phospholipids such as dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phos- phatidylcholine (DPPC), and distearoyl phosphatidylcholine (DSPC).

The aforementioned lipids are used in concentrations between 1 and 200 mM/100 ml (corresponding to 0.78 g/1 to 156 g/1).

In the case of Na cholate ("cholate" in the Tables), the (molar) lipid/detergent ratio varied from 1:1.4 to 1:8.5. Higher detergent amounts do not lead to a further reduction of the resulting vesicle size but considerably prolong the duration of filtration until the detergent is completely removed, thus increasing the amount of waste material. Only at a detergent lipid ratio of 5:1 or higher can lecithin be dissolved by octylglycoside (- octylglucopyranoside).

Mixed Micelle Solution I: The mixed micelle solution is usually prepared according to the classic film method: Lecithin was dissolved in 30 ml of ethanol. Cholesterol was dissolved in 10 ml of chloroform. Both solutions were mixed in a round-bottom flask, the organic solvent being removed by rotary evaporation under vacuum at 40°C. The detergent was dissolved in 100 ml 10 mM MOPS buffer (pH 7.3) and the lipid film was resuspended in this solution until a clear micelle solution occurred. (Used for Examples 8-13). Mixed Micelle Solution II: Much more elegant and economic is the direct dissolution of all components in 100 ml of the MOPS buffer. The solution is stirred until the micelle solution is clear (used for Examples 1-7, 14, 15). By means of tangential filtration, the detergent concentrations of the individual mixed micelle solutions were reduced until a molar ratio of detergent/lipid of < 0.3 was achieved. The volume of filtered out detergent-containing filtrate was continuously substituted or replaced by an equal volume of MOPS buffer (volumetric control).

Example A: Detailed Description of the General Manufacturing Process:

1.270 g of egg lecithin and 0.999 g of sodium cholate was introduced into a 100-ml measuring flask. The pH was adjusted to 7.3 with 1 mM of phosphate buffer. The ion intensity was adjusted with sodium chloride to 0.16 and the solution made up to the mark. This mixed micelle solution was stirred until it became clear. According to the lipid and solvent amounts used, the molar lipid/detergent ratio was 0.72. Subsequently, the mixed micelle solution was subjected to tangential filtration. The Minisette® was equipped with a 10 kD membrane cassette of the Omega range (polyether sulfone membrane). The flow rate was adjusted to 450 ml/min, the resulting working pressure being 0.5 bar. The experiment was carried out at room temperature. Tangential filtration was continued until a detergent concentration of 1 mg/ml was obtained. The time required was 11 min. The amount of detergent-containing filtrate was 314 ml, which volume was continually replaced by introduction of an equal volume of phosphate buffer. The mean particle size of the resulting liposomal dispersion was determined by laser light scattering. Comparative Example B: Comparison with Weder USP 4.731.210 Dilution Method (Weder's Example 1):

Starting from a micelle solution containing 1.27% (w/w) of lecithin, Weder, after a dilution of 1:10, obtains a 0.127% liposomal dispersion of a mean particle size of 30 ± 2 nm. After this dilution, the cholate concentration is 1 mg/ml. In order to obtain a suitable lipid concentration for dermatologic application, the liposomal dispersion would have to be concentrated to at most 1/10 of its volume in a second step. This means that 900 ml of detergent-containing aqueous phase ( = waste) would have to be removed to obtain 100 ml of a 1.27% final product.

Comparative Example C: The Present Constant Volume

Tangential Filtration:

Using the same materials and concentrations, by means of the present tangential filtration process, there is obtained in a single step without any dilution 100 ml of a 1.27% liposomal dispersion having a mean particle size of 42.1 ± 9.3 nm from 100 ml of mixed micelle solution (1.27% of lecithin). During the production time of only 11 min, a total of 314 ml of detergent-containing aqueous phase is filtered off. The same volume is continuously replaced with bu fer solution during the entire process to keep the volume of the mixed micelle solution constant. After this 11-minute period, the residual sodium cholate concentration has also decreased to 1 mg/ml.

Summary:

In summary, it can be stated: the same liposome quality can be obtained by the present tangential filtration metho , using the same materials and concentrations. However, in comparison with the Weder dilution method, the most essential advantages are:

1. No 2nd (concentration) step required

2. Reduced manufacturing time

3. Decrease of 900 ml waste volume to 314 ml.

* * *

Now, considering the Brown USP 5,314,695 disclosure at Column 7, line 31 and Example 6, Column 13:

Brown Procedure - Example 6. Column 13 Brown's Pyrosart® or Ultrasart® filter unit from

Sartorius Corp. is a tangential filtration system which is comparable to Minisette®. Brown prepares a mixed micelle solution from a phospholipid mixture which generally contains phosphatidylserine PS and phosphatidylethanolamine PE (cf. Brown column 5, lines 8, 9: not less than 2.5 mol percent PE/PS) and a detergent. This mixed micelle solution is filtered through the TF unit, unbound dissolved detergent being thereby separated together with water (column 13, lines 30-32). The resulting concentrated retentate (incorrectly described by Brown as dialysate) exclusively contains mixed micelles and is then made up to the original volume with a detergent-free dialysis buffer (lines 33-34; TBS containing 150 nM trehalose as dialysis buffer). This process is repeated 10 times (lines 35, 36). In total, the 10-fold amount of the initial volume of dialysis buffer passed through Brown's TF unit. Also, after the 10th cycle, a concentrated retentate results which can be made up to the initial volume as required (lines 37-39).

By reducing the mixed micelle solution and making it up to the initial volume with detergent-free buffer. Brown only applies Weder's dilution method. The liposomes are formed instantly, mainly in the second concentration and dilution cycle, through the addition of a "dialysis buffer". Generally speaking, the addition of a detergent-free buffer to a lipid-detergent mixed micelle solution effects a reduction of the amount of detergent in the mixed micelles as a result of the saturation concentration of the detergenr in the buffer. Due to this loss in detergent, the mixed micelles are converted into liposomes.

In Brown's method (and also in the case of Weder), this addition of the buffer and the formation of the liposomes happens at once. Brown has to reduce the mixed micelle solution (and later the liposomal dispersion) to at most 1/10 of the initial volume to achieve a 95% removal of the detergent after 10 cycles. Similarly to Weder, Brown therefore dilutes his mixed micelle retentate in a ratio of 1:10.

At least after the second dilution step, all mixed micelles have been converted into liposomes (see also the accompanying figure) . The following eight (8) concentration and dilution cycles serve only to remove the detergent from the liposomal dispersion. In the present process. as much detergent-free buffer is continuously added as detergent-containing buffer is filtered off. Thus the volume remains constant, and there is no dilution. As the detergent is continuously removed from the mixed micelles, there is also a continuous formation of liposomes.

These different approaches to liposome formation and the different product times required according to Brown and the present process are shown in the graph of the accompanying Figure, which presents a comparison of liposome formation using the constant volume tangential filtration procedure of the present invention and the concentration- dilution procedure of Brown. REFERENCE TO THE DRAWING Reference is now made to the accompanying Figure which shows the results of two (2) different approaches to liposome formation and the different production times required according to the present constant volume tangential filtration (CVTF) method and the concentration-dilution method of Brown, in which the percentage of liposomes formed is plotted against the time in minutes, the time required to form 100% of the liposomes according to the CVTF method of the present invention requiring 20 minutes as compared with 60 minutes according to the concentration-dilution procedure of Brown, comparative examples from which the data has been plotted being presented hereinafter in this Specification, all of which will be more fully understood from the preceding and following discussion and from the comparative examples.

In the following comparatives Examples, the processes are compared in detail: Comparative Example D: The Present Constant Volume Tanσen- tial Filtration Method (CVTF)

780 mg soy lecithin and 860 mg of Na cholate were introduced into a round-bottom flask, made up to 100.0 g with phosphate buffer pH 7 and stirred with a magnetic stirrer. Subsequently, the clear mixed micelle solution was subjected to tangential filtration for about 20 min. at room temperature. Filtration was carried out using a Mini- sette®TF system equipped with a polyethersulfone cassette of the Omega type having a cut-off of 10 KD. The flow rate was about 240 ml/min. The inlet pressure was about 0.2 bar. During the entire manufacturing process the detergent- containing buffer solution filtered off was continuously substituted by detergent-free phosphate buffer pH 7 (volumetric control). The final detergent content was 5.6%, the waste volume being 385 ml.

Comparative Example E: Brown USP 5.314.695 Concentration- Dilution Method

According to Brown: The liposomes are also produced using a Minisette®TF system equipped with a polyethersulfone membrane of the Omega type having a cut-off of 10 KD. Also in this case 780 mg soy lecithin and 860 mg Na cholate were introduced into a round-bottom flask, made up to 100.0 g with phosphate buffer pH 7 and stirred until a clear mixed micelle solution had formed. This mixed micelle solution was concentrated to a residual volume of about 8 ml per filtration cycle, and was subsequently made up to the initial volume using phosphate buffer pH 7. This process was repeated 10 times. At a flow rate of also 240 ml/min. , this process took 60 min. and produced a waste volume of 920 ml.

The final detergent content was 4.8%.

* * *

The continuous production of liposomes according to the present CVTF process clearly shows advantages as to the quality of the resulting liposomes, the duration of the process, and the waste produced:

Parameters Brown Constant-Volume TF

Medium particle size 63.1 nm 35.5 nm

Distribution range 38.8 nm 16.8 nm

Polydispersity 0.319 0.099

Duration of production 60 min. 20 min.

Waste volume 920 ml 385 ml

By the present process much smaller liposomes can be produced which are in a much narrower distribution range and considerably more homogeneous (polydispersity as a measure for the homogeneity of a vesicular population: PD * 0 - completely homogeneous, all vesicles have the same size; PD = 1 - completely heterogeneous, each vesicle has a different diameter) . Despite the higher quality of the liposomes, this process takes only 1/3 of the time required by Brown. In addition, only 40% of the waste described by Brown is produced by the present CVTF process.

The reduction of the initial volume to about 1/10 to 1/20 according to Brown leads to a concentration of the mixed micelle solution or the liposomal dispersion. At the same time the viscosity gradually increases to an extent that, in the end, the product is almost gel-like and can no longer be passed through a TF unit. At lipid concentrations higher than those described in Brown's patent, the viscosity of the initial mixed micelle solution is clearly higher. Thus the formation of a non-pumpable consistency occurs much earlier. Therefore, a reduction to 1/10 or even 1/20 of the initial volume becomes impossible. As a result of this, the subsequent dilution by Brown can only be 1:1 to 1:5. This means that more than 10 cycles described are required to separate the detergent. Thus waste volume and duration of manufacture increase considerably. In contrast to the CVTF process in which both lipid concentration (and hence the viscosity of the dispersion) remain constant during the entire process, Brown's method is clearly limited with regard to the maximum processable lipid concentration.

Further, in considering the relative advantage of the present process as compared to Brown, the following must be considered: The lecithin concentration in liposome-containing pharmaceuticals or cosmetics common in the market usually ranges between 10 and 20 mMolar. Two approaches for producing such formulations are possible: a) preparation of the liposome dispersion with the desired lecithin concentration, or b) preparation of a liposome concentrate with a lecithin concentration much higher than the final concentration in the formulation. This concen¬ trate is then incorporated into the formulation and thereby diluted to the desired lecithin concentration. If, for example, the liposome concentrate is 100 mMolar, one (1) part of this concentrate and nine (9) parts of an aqueous gel are mixed together to give a liposomal gel with a lecithin concentration of 10 mMolar. Thus, the concentrate becomes diluted 1:10. To produce one (1) kg of this 10 mMolar liposomal gel, one can prepare according to approach a) one (1 ) kg of a 10 mMolar liposome dispersion (and jellify it), or, according to b), one can dilute 100 g of a 100 mMolar liposome concentrate with 900 g of an aqueous gel.

Provided that the production method/device is suitably adapted, it takes the same time and effort to produce one ( 1 ) kg of a 10 mMolar liposome dispersion or a 100 mMolar liposome concentrate. But in case b) one is able to produce not only one (1) kg, but rather 10 kg of a liposomal gel, because of the 1:10 dilution!

So approach b) is much more economic, because one can produce the 10-fold amount of the final formulation in the same time. Producing 100 mMolar liposome concentrates. Brown's method is very ineffective. Because Brown does not keep the volume constant by buffer substitution, he concentrates the mixed micelle solution (or liposomal dispersion) to a gel- like consistency, which can no longer be passed through the TF unit. Starting with a 10 mMolar mixed micelle solution, he can concentrate this solution to about 1/10 of the starting volume before obtaining a gel. 9/10 of detergent- containing buffer are thereby filtered off. After a 10-fold repeat of this process (_^filtering off 10x9/10 of detergent containing buffer) he has removed at least 95% of the detergent content.

Starting with a mixed micelle solution containing 100 mMol or more lipid, Brown can concentrate these solutions only to about 1/5 or even 1/2 of the starting volume before obtaining a gel-like consistency. Only 4/5 or even 1/2 of the detergent-containing buffer is filtered off during one concentration step. His concentration-dilution process therefore has to be repeated more than 10 times (in the aforementioned examples as many as 2 to even 20 cycles) to remove 95% of the detergent content.

Increasing lipid concentrations therefore result in further increasing production times (=number of concentra- tion-dilution cycles) . In contrast to the present process, in which consistency remains constant during the entire process because of keeping the volume constant, and which in any case requires only 1/3 of the time required by Brown, Brown's method is clearly limited to low lipid concentra¬ tions.

Processing the same lipid concentrations, both methods yield about the same number of liposomes. However, an important difference is that, higher lipid concentrations (resulting in greater numbers of liposome) can be produced according to the present process without extending the process time and without excessive waste detergent filtrate. Examples 1-15 - Constant Volume Tangential Filtration Production of Liposomes:

The following Tables show the experiments and the relevant manufacturing conditions which have been applied in validating the present constant volume tangential filtration technique. In this context, both the influence of the parameters "flow rate" and "membrane pore size", as well as the effects of formula variations as to lipid concentration, lipid/detergent ratio, type of detergent, and cholesterol added, were investigated. Tables

composition (g) lipid— molar ratio membrane flow mean cone. lip/det (ml/min) diameter

(nm)

1. S-lec 0.078 1 mM 1:1.6 10 KD 250 40.2(8.9) cholate 0.068

2. S-lβc 0.78 10 mM 1:1.6 10 KD 250 44.8(8.2) cholate 0.685

3. S-lec 1.32 17 mM 1 :1.6 10 KD 250 48.6(9.8) cholate 1.16

4. S-lec 3.9 50 mM 1 :1.6 10 KD 250 53.3(10.6) cholate 3.427

5. S-lec 7.8 100 mM 1 :1.6 10 KD 250 89.1 (20.4) cholate 6.854 composition (g) lipid— molar ratio membrane flow mean dia¬ cone. lip/det (ml/min) meter (nm)

2. S-lec 0.78 10 mM 1 :1.6 10 KD 250 44.8(8.2) cholate 0.685

6. S-lec 0.78 10 mM 1 :1.6 10 KD Z5 47.6(9.4) cholate 0.685

7. S-lec 0.78 10 mM 1 :1.6 50 KD 250 55.0(11.6) cholate 0.685 composition (g) lipid— molar ratio membrane flow mean dia¬ cone. lip/det (ml min) meter (nm)

8. S-iec 0.669 11.5 mM 1:1.6 10 KD 250 42.9(8.5) cholest 0.111 cholate 0.780

9. S-lec 0.669 1 1.5 mM 1:1.4 10 KD 250 58.0(20.7) cholest 0.111 cholate 0.690

composition (g) lipid— molar ratio membrane flow mean dia¬ cone. lip/det (ml/min) meter (nm)

10. S-lec 0.235 4.1 mM 1 :8.5 10 KD 250 35.1 (9.0) cholest 0.046 cholate 1.523

11. S-lec 0.2305 4.1 mM 1:8.5 10 KD 75 34.5(6.8) cholest 0.046 cholate 1.523

12. S-lec 1.0241 18.4 mM 1 :8.5 10 KD 250 34.7(4.2) cholest 0.205 cholate 6.7703

13. S-lec 1.0241 18.4 mM 1 :8.5 10 KD 75 35.7(10.8)

composition (g) lipid— molar ratio membrane flow mean dia¬ cone. lip/det (ml/min) meter (nm)

14. E-lec 1.00 12.8 mM 1:5 50 KD 250 144.9(33.3) OG 1.89

15. E-lec 1.00 12.8 mM 1:2.9 50 KD 250 60.3(10.8) OG 0.46 cholate 0.945

Comments:

Both the present constant volume tangential filtration method and vesicle quality are independent of the flow rate and hence of the extent of the membrane flow (cf. Examples 2 and 6). On the other hand, even though not very pronounced, there is a direct correlation between vesicle diameter and membrane pore size (cf. Examples 6 and 7). However, this correlation may be utilized for the production of larger liposomes only up to a membrane cut-off of 50 kD. In the case of greater pore diameters, pronounced lipid losses are to be expected as liposomes are also filtered out. The process is distinguished by excellent reproducibility. By selecting the suitable membrane, a targeted vesicle quality can be constantly produced, independently of the other equipment parameters and the peripheral equipment (pump rate, tube diameter, etc.).

Both vesicle size and quality can be controlled not only via the membrane but also above all via the qualitative and quantitative composition of the formulation. For example, with constant conditions, particle size increases in direct proportion with the lipid concentration used (cf. Examples 1-5). It is even possible to double the liposomal diameter (40.2nm -> 89.1 nm). A much stronger influence on vesicle size can be exerted through the selection of the detergent used for solubilization/mixed micelle formation. By replacing sodium cholate by octylglucoside (cf. Example 14), particle size may even be almost tripled (cf. Example 7: 55.0 nm -> 144.3 nm). In contrast, when using an admixture of octylglucoside and cholate (No. 15), only slight insignificant changes occur.

The vesicle size can also be controlled via the lipid/detergent ratio, i.e., via the structure/composition of the mixed micelles. The more detergent is used to solub- ilize the lipid, the smaller are the resulting liposomes. The effect (cf. Examples 8, 9, 12) is less pronounced as compared to the aforementioned control procedures. In contrast to the vesicle-increasing property of cholesterol, which has often been described in the literature and which was also observed when preparing liposomes by high-pressure homogenization, such influence could not be observed in the case of the present constant volume tangential filtration (cf. Examples 2, 8). Examples 2. 5. and 8 - Constant Volume Tangential Filtration - Detailed Description

The benefits of the present technology are even more evident in the case of Examples 2, 5, and 8. (See Tables)

Example 2:

For a 100-ml batch, 780 mg of soy lecithin and 685 mg of Na cholate were dissolved in 10 mM MOPS buffer (pH 7.3) under stirring until the mixed micelle solution was clear. Subsequently this solution, having a lipid concentration of 10 mMol and a (molar) lipid/detergent ratio of 1:1.6, was subjected to constant volume tangential filtration. The Minisette® was equipped with a 10-kD membrane cassette of the Omega range (polyethersulfone membrane). The flow rate was adjusted to 250 ml/min. and the resulting working pressure was 0.2 bar. The experiment was carried out at room temperature. Tangential filtration was continued until a detergent/lipid ratio of < 0.3 was obtained. The time required was 12 min; the amount of detergent-containing filtrate was 300 ml which was continually substituted by MOPS buffer during the filtration procedure. The resulting liposomal dispersion was filtered through a 0.22 μm cellulose acetate membrane, the mean particle diameter being determined by laser light scattering (44.8 ± 8.2 nm).

Example 5:

Also in this case the batch size was 100 ml. Soy lecithin was added in a quantity of 7.8 g and Na cholate in a quantity of 6.854 g. The lipid concentration was 100 mM, the lipid/detergent ratio being 1:1.6. 10 mM MOPS buffer (pH 7.3) was also used in this case. The liposomes were prepared and determined under the conditions described in Example 2. As a result of the higher lipid concentration, a working pressure of 0.4 bar was achieved. The constant volume tangential filtration process lasted 75 min and 1.5 liters of filtrate were produced. The filtrate was continually substituted by MOPS buffer, thereby keeping a constant volume. The mean particle size determined was 89.1 ± 20.4 nm.

Example 8:

669 mg of soy lecithin was dissolved in 30 ml of ethanol. Ill mg of cholesterol was dissolved in 10 ml of chloroform. Both lipid solutions were combined in a round- bottom flask and converted into a lipid film at 40°C under vacuum. Resuspension was made with 100 ml of 10 mM MOPS buffer (pH 7.3) containing 780 mg sodium cholate until a clear mixed micelle solution was obtained. The total lipid concentration was 11.5 mM, the lipid/detergent ratio being 1:1.6. All other working conditions were the same as described in Example 2. The working pressure was 0.2 bar. The constant volume tangential filtration process lasted 12 min., producing 300 ml of filtrate as waste material. The filtrate was continually substituted by MOPS buffer. The mean particle size was 42.9 ± 8.5 nm.

The data are summarized in the following Table.

Example Compo¬ Lipid Batch Mean particle Deter¬ Dura¬ Ftitrate no. sition concen¬ size size gent tion of volume tration lipid filtra¬ ratio tion

2 Lecithin 10 mM 100 ml 44.8±8.2nm 0.289 12 min. 300 ml

5 Lecithin 100 mM 100 ml 89.1±20.4nm 0.285 75 min. 1500ml cholate

8 Lecithin 11.5 mM 100 ml 42.9±8.5nm 0.276 12 min. 300 ml choles¬ terol cholate

Comments Regarding Examples 2 and 5:

The resulting vesicles produced in Examples 2 and 5 were extremely homogenous. They were characterized by only a single bilayer (that is, they were unilamellar) as seen from electron microscope photographs.

As is well known, the occurrence of multilamellar liposomes cannot be prevented using detergent dialysis when starting from a lipid concentration of 30 mMol or above, corresponding to only one-third of the lecithin employed in the foregoing Example 5. As is also well known, the physical stability of a liposomal dispersion which contains multilamellar vesicles of inhomogeneous particle size distribution is considerably reduced and generally unsatisfactory. In contrast to the constant volume tangential filtration method of Examples 2 and 5, the countercurrent dialysis method is also markedly restricted with regard to lipid concentrations, essentially only to those below about 30 mMol, which can be processed without the production of undesirable multilamellar liposomes.

Example 16 - Production of a Liposomal Dispersion Containing An Active Substance and its Further Processing into a Gel Preparation: 1.0 g of soy lecithin and 0.878 g of Na cholate were introduced into a beaker of appropriate size. The lipid concentration was 12.8 mM, the lipid/detergent ratio being 1:1.6. The beaker was made up to 100 g using a phosphate buffer (pH 6.5) containing an active substance of the following formulation: Sodium phosphate monobasic (x2 H₂0) 8.42 g/1, sodium phosphate dibasic (x2 H₂0) 6.26 g/1, dexpanthenol 66.7 g/1 in purified water. Constant volume tangential filtration was carried out with the aid of a 10 kD membrane cassette (Omega range) at a flow rate of 250 ml/min and a working pressure of 0.2 bar. Filtration was continued until a detergent/lipid ratio of < 0.3 was obtained. During this time (15 min) 340 ml of filtrate was obtained which was continually substituted with active substance-containing phosphate buffer of the above composition but containing 50 g/1 dexpanthenol. The resulting 100 ml of liposomal dispersion (particle size 84.2 ± 15.9 nm) was reduced by further filtration without buffer substitution until a volume of 75.7 g was obtained. Under stirring, the liposomal concentrate was diluted with a mixture of 7.0 g isopropanol and 7.8 g glycerol and jellified by adding 2 g of Na polyacrylate. Finally, 7.5 g of octyldodecyl myristate was admixed with this liposomal preparation. The finished emulsion gel contained liposomes having a size of 93.2 ± 18.3 nm; the dexpanthenol content was 5%.

Example 17 - Production of a Liposomal Gel Containing a Lipophilic Active Ingredient:

0.780 g of soy lecithin and 0.156 g of dl-α-tocopherol nicotinate was introduced into a round-bottom flask of a suitable size and dissolved in ethanol. Subsequently, the solvent was removed with the aid of a rotary evaporator at 45°C under vacuum. The resulting lipid film was redispersed with 97.564 g of the following buffer solution: 8.42 g/1 of sodium phosphate monobasic (x 2 H₂0), 6.26 g/1 of sodium phosphate dibasic (x 2 H₂0), 13.87 g/1 of sodium cholate, 10 g/1 of phenoxyethanol in purified water. The resulting clear mixed micelle solution was subjected to constant volume tangential filtration. The Minisette® was equipped with a 10 kD membrane cassette of the Omega range (polyether sulfone membrane). The flow rate was adjusted to 450 ml/min, the resulting working pressure being 0.5 bar. The experiment was carried out at room temperature. Constant volume tangential filtration was continued until a detergent/lipid ratio of < 0.3 was obtained. Production took 45 min., the resultant waste material being 1200 ml of filtrate. From the outset, the filtrate was continually replaced by an equal volume of the above-mentioned phosphate buffer without sodium cholate. The mean liposome particle size was 32.5 ± 7.7 nm.

Finally, 1.5 g of preneutralized polyacrylic acid was admixed therewith to form a liposomal hydrogel.

* * *

Suitable bilayer-forming substances for use according to the method of the present invention may include sphingolipids, glycolipids, sterols (as for example cholesterol), archebacterial lipids, non-ionic synthetic lipids, phospholipids and in particular lecithins and the like, as is well known in the art, representatively from the disclosure of USP 4,731,210 and especially from line 35 of Column 8 through line 3 of Column 9 thereof. The best-suited building elements for liposome preparations are soy or egg lecithin. (USP XXII; NFXVII; published 1989 and official from January 1, 1990). On pages 1942 and 1943 thereof, the official monograph of lecithin is clearly set forth. In addition to these two natural lecithins, other synthetic or natural phosphatidyl cholines with saturated or unsaturated fatty acid chains of 16-20 carbon atoms can also be used. In addition to soy and egg lecithins, also rape and safflower lecithins are of natural origin. The definition of fatty acid chain lengths of 16-20 carbon atoms representatively comprises palmitic acid, margaric acid, stearic acid, nonadecanoic acid, arachic acid, palmitoleic acid, oleic acid, linoleic acid, elaeomargaric acid, elaeostearic acid, gadoleic acid, and arachidonic acid. As will be understood by one skilled in the art, since phosphatidyl-choline (PC) should be esterified twice, the resulting PC esters, which are in fact the phosphatidylcholines, are for example: dioleoyl-PC, dipalmitoyl-PC, but also mixed esters such as margaroyl-stearoyl-PC, palmitoyl-oleoyl-PC, and the like. These well-defined phosphatidylcholines are not produced by extraction or otherwise from natural lecithins, but are synthesized de novo and are consequently referred to as synthetic lecithins. In brief, the bilayer-forming material may comprise a phospholipid, sphingolipid, glycolipid, sterol (as for example cholesterol), archebacterial lipid, or a non- ionic synthetic lipid.

As solubilizing agents, usual detergents or surface active agents may be employed, such as for example bile salts such as sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium ursodeoxycholate, and also salts of these with other cations such as potassium, sugar derivatives such as n-alkylglycosides, n-alkylmethylglyc- amides, n-alkylglycosylamines, ionogenic substances, nonionic solubilizing agents such as n-alkyloligooxyethylenes, and mixtures thereof, as is well known in the art, and repre¬ sentatively from USP 4,731,210, Column 9 thereof, lines 3-37, all hereinafter referred to as "detergent", and especially those selected from the group consisting of bile salts such as sodium cholate, sodium deoxycholate, sodium chenodeoxy¬ cholate, sodium ursodeoxycholate, and also salts of these acids with other cations, such as potassium; detergents selected from the group of n-alkylglycosides, n-alkylmethyl- glycamides, n-alkyloligooxyethylenes, and n-alkylglycosyl¬ amines.

As far as medicaments, drugs, and cosmetics which may be included within, in, or on the liposomes according to the present invention, as well as additional materials which may be included in the tertiary systems of bilayer-forming substances and solubilizing agent, i.e., detergent, with water, which form the lipid-detergent associates or mixed micelles, these are also well known to one skilled in the art and representatively from USP 4,731,210, at Column 9, lines 38 through Column 10, line 22 thereof. Lipophilic agents suitable for bilayer-binding are, for example, acne therapeutics such as hexachlorophene, tretinoin, or minocy- cline. Other topical agents such as α-tocopherol nicotinate, tromantadine, croconazole, minocycline, sodium heparin, dexpanthenol, meclocycline, cyproterone, cyproterone acetate, 2-tert.-butyl-4-cyclohexylphenyl nicotinate-N-oxide, plant extracts, corticosteroids, androgens, ethinyl estradiol, non- steroid antiphlogistics, dihydropyridines, spironolactone, erythromycin esters, local anaesthetics, estradiol esters, or antihistaminics can also be incorporated. The amount of active substance can be varied in dependence upon the therapeutic requirements. For example, 10 mg to 50 g of active ingredient can be used per 100 g of lecithin.

As to use and mode of administration of liposomal medicaments or cosmetics and suitable forms into which they may be converted for administration, these are likewise well known in the art and such forms may be topical, parenteral, solutions, aerosols, emulsions, hydrogels, lyophilizates, and the like, all as is already known to one skilled in the art and representatively from USP 4,731,210, Column 10, lines 23-65 thereof. Liposome preparations for topical application are usually mixed with polymer jellifying agents to increase viscosity and improve application. For this purpose, especially polyacrylic acid and derivative (0.2 to 2.5%) gels, gel-forming cellulose and cellulose derivative (0.2 to 3%) gels, and sodium salts of acrylic acid/ acrylamide copolymerisate - 1 to 4% gels; commercially available as Hostacerin PN73®, in the concentrations indicated may be used. The gel is prepared according to commonly-known procedure. Especially preferred are mixtures consisting essentially of egg or soy lecithins and the described jellifying agents and active substances.

Alternatively and optionally, common adjuvants such as, for example, an antioxidant, e.g. , vitamin E or butyl-hydroxy toluene (BHT), alcohols, and preservatives such as phenoxe- thol, sorbic acid, Kathon CG^tB (Merck Index 11, No. 6677), or parabens can be added in usual amounts.

* * * It is accordingly seen from the foregoing that the method of the present invention provides a novel method for the production of liposomes by constant volume tangential filtration under pressure which permits the employment of higher lipid concentrations for the production of liposomes having extremely small particle size and a single bilayer and without the presence of multilamellar liposomes or inhomogeneous particle size distribution, which is remarkably reduced in comparison with prior art practice and results. The method involves the removal of solubilizing agent solution as the filtrate and retention of the bilayer-forming material - solubilizing agent associates in the form of mixed micelles and forming and formed liposomes in the retentate, which is not diluted by the addition of aqueous phase as in prior art methods, but wherein the volume of the mixed micelle solution is maintained by simultaneous replacement with an aqueous solution of buffer, electrolyte, or drug or other active ingredient to the extent necessary to make up for the loss in volume due to filtration off of the solubi¬ lizing agent solution, which thereby incrementally increases the molar ratio of bilayer-forming material to solubilizing agent and incrementally and continuously induces liposome formation in the retentate. The method may be practiced rapidly and conveniently and with higher lipid concentrations than according to prior art procedures and provides a novel, rapid, and convenient method for the production of liposomes and drug-, medicament-, or cosmetic-containing liposomes which may be conducted commercially.

In addition, the procedure of the present invention can advantageously be conducted on a continuous basis, with an unlimited scale up and minimal waste detergent filtrate, and with unprecedented uniformity of product and rapidity of production of liposomes of desired particle sizes, so that the constant volume tangential filtration method of the present invention now stands alone as the paramount process for the production of liposomes of any desired small particle size and content, including medicinal, drug, or cosmetic con¬ tent. Moreover, the process of the present invention is highly advantageous, not only from the standpoint of rapidity and convenience of large scale production, but also economics, when compared to dilution, concentration-dilution, or the usual high-pressure homogenization, ultrasound, and extrusion methods of liposome production, as will be immediately apparent to one skilled in the art.

It is to be understood that the present invention is not to be limited to the exact details of operation, or to the exact compounds, compositions, methods, procedures, or embodiments shown and described, as various modifications and equivalents will be apparent to one skilled in the art, wherefore the present invention is to be limited only by the full scope which can be legally accorded to the appended claims.

Claims

We Claim:

- 1 -

A method of producing liposomes from an aqueous solution comprising bilayer-forming material and detergent, in the form of a solution of mixed micelles and unbound dissolved detergent, comprising the steps of eliminating aqueous detergent-containing solution by tangential filtration of the solution through a membrane adapted to pass the aqueous detergent-containing solution as a filtrate but to retain a mixed micelle- and liposome- containing fraction thereof as a retentate, while simultaneously avoiding dilution of the volume of the mixed micelle solution or of the bilayer-forming material therein, by replacing the detergent-containing solution removed by the tangential filtration by water or a non-solubilizing aqueous solution so as to maintain an essentially constant volume of the mixed micelle solution, and thereby to produce a liposome dispersion as retentate.

- 2 -

A method of Claim 1 wherein the bilayer-forming material comprises a sphingolipid, glycolipid, sterol, archebacterial lipid, non-ionic synthetic lipid, or a phospholipid.

- 3 -

A method of Claim 2 wherein the bilayer-forming material comprises a phospholipid and the detergent is selected from the group consisting of sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium ursodeoxycholate, salts of these acids with other cations, n-alkylglycosides, n- alkylmethylglycamides, n-alkyloligooxyethylenes, and n- alkylglycosylamines.

- 4 -

A method of Claim 2 wherein the lipid comprises egg or soy lecithin.

- 5 -

A method of Claim 4 wherein the detergent is selected from the group consisting of sodium cholate, sodium deoxycholate, sodiumchenodeoxycholate, sodiumursodeoxycho¬ late, salts of these acids with other cations, n-alkylglyco¬ sides, n-alkylmethylglycamides, n-alkyloligooxyethylenes, and n-alkylglycosylamines.

- 6 -

A method of Claim 5 wherein the detergent comprises at least one of sodium cholate and octyl glycoside.

- 7 -

A method of Claim 1 wherein the non-solubilizing aqueous solution is a buffer-, electrolyte-, and/or active ingredient-containing aqueous solution.

- 8 -

A method of Claim 7 wherein the bilayer-forming material comprises a phospholipid.

- 9 -

A method of Claim 7 wherein the non-solubilizing aqueous solution is an aqueous buffer solution.

- 10 -

A method of Claim 9 wherein the buffer is selected from the group consisting of MOPS, HEPES, TRIS, and phosphate buffers. - 11 -

A method of Claim 2 wherein the volume of the mixed micelle solution and the concentration of the lipid is maintained essentially constant.

- 12 -

A method of Claim 2 wherein essentially all of the lipid concentration is maintained in the aqueous mixed micelle solution.

- 13 -

A method of Claim 1 which is conducted under a pressure developed in the tangential filtration system.

- 14 -

A method of Claim 13 wherein the pressure is up to about one (1) bar.

- 15 -

A method of Claim 14 wherein the pressure is between about 0.2 and about 0.4 bar.

- 16 -

A method of Claim 1 including the step of further filtering the retentate to remove residual detergent or liposome-unassociated material.

- 17 -

A method of Claim 16 including the step of further filtering the resulting liposome dispersion to concentrate the same into a gel.

- 18 -

A method of Claim 11 wherein a constant volume is continuously maintained by replacement of the detergent- containing aqueous phase by non-solubilizing-agent-containing solution.

- 19 -

A method of Claim 18 wherein the non-solubilizing agent- containing solution is an electrolyte-containing solution. - 20 -

A method of Claim 18 wherein the non-solubilizing agent- containing solution is a buffer-containing solution.

- 21 -

A method of Claim 18 wherein the non-solubilizing agent- containing solution is an active ingredient-containing solution.

- 22 -

A method of Claim 21 wherein the non-solubilizing agent- containing solution comprises a hydrophilic active ingredi¬ ent.

- 23 -

A method of Claim 21 wherein the non-solubilizing agent containing solution comprises a medicament or cosmetic active ingredient.

- 24 -

A method of Claim 1 wherein a lipophilic active ingredient is present in the starting mixture comprising bilayer-forming material.

- 25 -

A method of Claim 1 wherein the retentate is recycled until it essentially comprises unilamellar liposomes of desired particle dimensions.

- 26 -

A method of Claim 25 wherein the dimensions are between about 30 and 200 nm.

- 27 -

A method of Claim 25 which is conducted continuously.

- 28 -

A method of Claim 2 wherein the method is conducted until the detergent concentration in the resulting liposome dispersion is reduced to a detergent/lipid molar ratio of less than 0.3. - 29 -

A method of Claim 25 wherein the method is conducted until the detergent concentration in the resulting liposome dispersion is reduced to a detergent/bilayer-forming material molar ratio of less than 0.3.

- 30 -

Liposomes whenever produced by the method of tangential filtration as defined in Claim 1.

- 31 -

Liposomes whenever produced by the method of tangential filtration as defined in Claim 2.

- 32 -

Liposomes whenever produced by the method of tangential filtration as defined in Claim 8.

- 33 -

Liposomes whenever produced by the method of tangential filtration as defined in Claim 23.

- 34 -

Liposomes whenever produced by the method of tangential filtration as defined in Claim 25.

- 35 -

Liposomes whenever produced by the method of tangential filtration as defined in Claim 28.