CA1315198C - Liposome continuous size reduction method and apparatus - Google Patents

Abstract

PATENT

LIPOSOME CONTINUOUS SIZE REDUCTION
METHOD AND APPARATUS

ABSTRACT

A method of extruding liposomes from liposomal material comprising extruding the liposomal material through a frit, and apparatus for extrusion.

Description

~31~198 ~ACKGROUND OF THE INVENTION

The filtration and extrusion of liposomes directed to achieving liposome preparations of uniform ~ize are known in the art. However, filtration/extrusion rates have been low being no higher than about 12 ml/minute.

Hunt and Papahad~apolous disclose a "Method for Producing Liposomes in Selected Size RangeQ" in U.S. Patent No. 4,529,561 ("Hunt") by a filtration/extrusion process.

The Hunt process is described either as one of extruding liposomes under pressure through a "uniform-pore-size membrane" or forcing liposomes "through an orifice under pressure" The Hunt filtration-extrusion membrane process is further distinguished in Hunt from the orifice process as being performed at lower pressure. In practice, the membrane process is associated with the retention or buildup of material on the input surface of the filtration-extrusion membrane. This build up reduces flow while requirlng increased driving pressure.

Suzuki et al. (U.S. Patent No. 4,016,100) describes a liposomal preparation with a filtration step utilizing a membrane filter. In Suzuki et al. it i9 also noted that the liposomes are formed in a uniform size.
The filtration process of Suzuki et al. further has a limited through-put capacity due to pressure limitation of membranes and the membrane occlusion that arises from material buildup on the membrane input surface.

It is an ob~ect of this invention to provide a method of extruding liposomes to provide smaller liposome sizes.

It is another ob~ect of this invention to provide a method of producing liposomes of uniform size from liposomal material.

131~98 It i9 an additional ob~ect of this invention to provide a high speed /high volume method of continuous size reduction of liposomes.

SUMMARY ~F THE INVENTION

The instant invention provides a method of extruding liposomes into ~maller sized more uniform populations from liposomal material by extruding the liposomal material through a frit without use of a membrane. Also provided is an extrusion apparatus for such extrusion.
This method is extrusion without filtration in that substantially all of material presented to the extruder is passed through.

Critical to this invention is the discovery that the use of a frit as an extruder element will extrude liposomes into a uniform population of reduced size without frit occlusion while affording substantially total through-put of liposomal material.

As a frit is not a uniform-pore-size element, frit porosity is expressed as a pore size designation. Preferred are frits of pore size designatlons of 500 nanometers as well as frits of smaller pore size designations. These frits are described herein as having pore size designations of 500 nanometers or less. Using a single frit produces the desired result of producing uniform populations of size reduced liposome and has the advantages of structural strength and simplicity. The structural strength of a frit permits use of operating pressures for extrusion far in excess of those that may be used in a membrane filtration/extrusion system. The use of frits of decreasing pore size deqignations is not required.

The invention includes a method of extruding liposomal material by passing the liposomal material through a frit. The invention further includes extrusion of substantially all liposomal material. This method also includes preparing the liposomal material into liposomes prior to extrusion. The invention further includes using a frit with a pore size designation of about 500 nanometers or less. The method additionally lncludes passing the liposomal material through the frit under pressure.
Preferred pressures are in excess of about 100 pounds per square inch.
Pressures of about 1000 psi or greater are more preferred. Extrusion at rates of lOml of liposomal material/minute/cm2 of frit surface area and up to 40ml/minute/cm2are further included in this invention.

The method further includes repeatedly passing liposomal material through the frit. Preferred i9 a method of at least about 3 passes.
More preferred is a method of at least 10 passes through the frit. The method further includes the step of preparing the liposomal material in nonliposomal form (such as by mere dispersal of said materials in a liquid phase) prior to the step of passing the liposomal material through the frit.

The foregoing methods are preferably performed using a metal frit.

The apparatus for extruding liposomes of this invention includes a reservoir ~uitable for containing liposomal material in communication with a pump being in connection with a frit.

The apparatus additionally includes a frit, preferably a metal frit, of a pore size designation of about 500 nanometers or less. The appartus further includes a pump which functions at an operating pressure of at least about 100 pounds per square inch.

BRIEF DESCRIPTION OF THE FIGURES

Fig.l i9 a diagramatic side plan of an enlarged section of a frit in cutaway.

Fig.2 is a diagramatic plan of the extrusion appsratus of the present inve~tion.

Fig. 3 is a diagramatic Qide plan of a frit with flow fittings in cutaway.

131~198 DETAILED DESCRIPTION OF THE INVENTION

Hi8h speed/high volume extrusion of liposomes with substantially total through-put of starting liposomal material (lipid, aqueous phase, and optionally organic solvents and/or bioactive agent) is achieved by passage of such starting liposomal material through a frit. As used herein the term liposome referring to extruded product includes both lipid and lipid-drug aggregates as well as true liposo~es.

The term lipid as used herein shall mean any suitable material resulting in a bilayer such that a hydrophobic portion of the lipid material orients toward the bilayer while a hydrophilic portion orients toward the aqueous phase. Lipids include highly hydrophobic compounds such aQ triglycerides, sterols such aq cholesterol and amphipathic lipids.

Of this broad group of lipids, amphipathic lipids are utilized aQ the primary liposomal structural element in the practice of this invention.
The amphipathic lipids have the property of having a polar (hydrophilic) moiety and a hydrophobic moiety. Hydrophilic character could be imparted to the amphipathic lipid molecule through the presence of phosphato, craboxylic, sulphato, amlno, sulfhydryl, nitro, and other llke 8rouPs-Hydrophobicity could be conferred by the inclusion of groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocyclic group. The preferred amphipathic compounds are phosphoglycerides, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid and diphosphatidylglycerol. Synthetic saturated compounds such as dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatldylglycerol, or distearoylphosphatidylcholine or unsaturated species such as dioleoylphosphatidylcholine or dilinoleoylphosphatidylcholine are also usable. Other compounds lacking phosphorous, such as members of the sphingolipid and glycosphingolipid families, are also within the group designated as amphipathic lipid.
.

1315~98 The amphipathic lipids may be utilized admixed with other lipids includlng triglycerides and sterols.

As used herein liposomal material whether in the form of liposomes or in a mlxture of liposome constituents in nonliposomal form comprlses any constituents that form or associate with liposomes. Liposomal material will be understood to include lipids, aqueous media, and optionally organic solvents and bioactive agents.

Liposomal material further includes, under certain circumstances, surfactants and other dispersants that will suspend lipids. Suitable organic solvents are those with a variety of polarities and dielectric properties, which solubilize lipids, include but are not limited to chloroform, methanol, dimethylsulfoxide, methylene chloride, and solvent mixtures ~uch as benzene:methanol. A representative surfactant is an octoxynol such as Triton X-100 (Rohm & Haas). Water, 0.9% saline, as well as citrate or phosphate buffers may also function as lipid dispersants. In some instances, water soluble organic solvents, such as ethanol, will be present in the aqueous phase. Suitable solvents or surfactants or dispersants are those which will distribute lipid throughout a solution and are preferably chosen on the basis of biocompatibility, toxicity, and flammability.

Biologically active a8ents ("bioactive agent") as used herein include but are not limited to antibacterial compounds such as gentamycin, antiviral agents such as rifampacin, antifungal compounds suchas amphotericin B, anti-parasitic compound such as antimony derivatives, tumoricidal compounds such as adriamycin, anti-metabolitès, peptides, proteins such as albumin, toxins such as diptheriatoxin, enzymes such as catalasae, polypeptides such as cyclosporin A, hormones ~uch as estrogen, hormone antagonists, neurotransmitters such as acetylcholine, neutrotransmitter antagonists, glycoproteins such as hyaluronic acid, lipoproteins such as alpha-lipoprotein, immunoglobulins such as IgG,immunomodulators such as interferon or interleuken, * Trade-mark A

1 3 ~
vasodilators, dyes such as Arsenazo III, radiolables sucha 9 14C, radio-opaque compounds such as 90Te, fluorescent compoundQ such as carboxy fluorscein, receptor binding molecules such as estrogen receptor protein, anti-inflammatories such as indomethacin, antigalucoma agents such as pilocarpine, mydriatic compounds, local anesthetics such a~
lidocaine, narcotics such as codeine, vitamins such as alpha-tocopherol, nucleic acids such as thymine, polynucleotides such as RNA polymers, psychoactive or anxiolytic a8ents such as diazepam, mono- di- and polysaccharides, etc. A few of the many specific compounds that can be entrapped are pilocarpine, a polypeptide growth hormone such as human growth hormone, bovine growth hormone and porcine growth hormone, indomethacin, diazepam, alpha-tocopherol itself and tylosin. Antifungal compounds include miconazole, terconazole, econazole, isoconazole, tioconazole, bifonazole, clotrimazole, ketoconazole, butaconazole, itraconazole, oxiconazole, fenticonazole, nystatin, naftifine, amphotericin B, zinoconazole and ciclopirox alamine, preferably miconazole or terconazole. The entrapment of two or more compound simultaneously may be especially desirable where such compounds produce complementary or synergistic effects. The amounts of drugs administered in liposomes will generally be the same as with the free drug; however, the frequency of dosing may be reduced.

High speed/high volume extrusion as a function of the surface of the extruding element ~here a frit) as used herein refers to extrusion rates over about lOml/minute/cm2 frit surface area and preferably over about 20ml/minute/cm2 and most preferably over about 40ml/minute/cm2 or greater. With a frit of 2.5cm surface area these rates of extrusion are 25, 50, and lOOml/minute respectively. High speed/high volume is also expressed as high rate extrusion.

A frit element as used herein is a structural member comprised of generally bead-like material partially con~oined, usually by compression and/or sintering. Among the many materials useful in frit preparation are ceramic, glass metal and metal/carbide bead-like materials which are con~oined into a frit. ~ue to the low affinity of lipid for metal, metal 131~8 frits are preferred for facilitating substantially total through-put of material. The preferred metal frit is of stainless steel.

Frits are available from a number of sources such as Ace Scientific Company, (East Brunswick, N.J.), Scientific Systems, Inc.(State College, E'enn.) and Ranin Instrument Company (Woburn, Mass.). Frits are characterized in having nonuniform pore size over a pore size range.
Frit pore size ran8e is experimentally determined on the basis of material that passes through or i9 excluded by the frit. This defines a range of particle sizes a portion of which lie above and below,the stated pore size. As used herein frit pore size of a frit will be referred to as a pore size "designation" based upon such an empirical tests well known in the art. Paths through the frit are not straight paths and path walls are irregular. Frits may be prepared in a number of shapes including rod or disc shapes.

The detailed section of a frit 1 used in this invention is depicted in detailed cut away side view in Figure 1 and comprises a matrix of essentially solid subunits 2 par~ially sintered together leaving interstitial spaces 3. Sintered subunits form a partially continuous surface 4 yielding nonstraight path trans-frit openings 5. The partial continuity of surface i9 sufficiently continuous such that the frit strength is a function of the length of its cross sectional axis 6 Frits are conveniently about 2 cm in diameter and about 0.1 cm thlck, but these dimensions are not crltical. Frits as large as 20 cm in diameter or larger, may be utilized. Frits in rod shape are useful including those of about 10 inches in length and about 1 inch in diameter. Preferred frits have a pore size designation of about 500 nm down to those of about 200 nm. Frits of a pore size designation of about 100 nm or less tend to be of prohibitively hi8h resistance to flow and are not preferred for this application.

Figure 2 depicts the preferred extrusion apparatus 10 with a feed reservoir 9 for liposomal material with a capped access port 12. The input reservoir is connected through line 14 to a pump 16 leading through 131~198 line 18 to frit 1, positioned in-line. The liposome material pumped through the frit passes through line 21 and through 3-way valve 23 and is received into collection vessel 22 collecting extruded material. Line 24 entering into input reservoir 20 may be used to degas the liposomal material if connected to a vacuum source, pressurize the liposomal material if connected to a pressure source, or direct a particular gas such as nitrogen to the liposomal material. Return line 25 from three-way valve 23 into the feed reservoir permits the recycling of extruded material.

In using this apparatus the lipid material may be introduced to the pump 16 by any pump means known to the art. When piston pumps are employed, liposomal material may be drawn into the pump head itself.
External pumping of feed stock from the feed reservoir 9 by an ex~ernal pumping device 27 and a first two-way valve 26 and a second two-way valve 28 deliver feed stock to pump 16. The pump head then provides energy for circulation of the liposomal material through the frit. The invention is not limited to piston pumps and any suitable pumping means may be used including dlaphragm pumps. In embodiments with a sufficient hydrostatic head and thus a sufficient trans-frit pressure differentail the pump is not necessary.

Fig. 3 is a diagramatic side plan of a frit with flow fittings 30.
With such fittings a frit is placed in-line in the apparatus of this invention. Liposomal material is pumped through line 18 fixed in pressure tight connection to vessel 38 by primary entry fitting 34 and secondary entry fitting 36. Liposomal material enters chamber 40 and is extruded through frit 1 into space 42 and is removed through line 21.
Line 21 is fixed in pressure ti8ht connection to frit 1 in vessel 38 by exit fitting assembly 44 comprised of frit seat 46, primary exit fitting 48 secondary exit fittin8 50 and tertiary exit fitting 52.

The hi8h rate of extrusion is enabled by the strength of the frit, which does not suffer occlusion and is not deformed by the pressure of the extrusion step. Membrane type extrusion is rate limited by the _g_ 13~198 minimal resistance to deformation of membrane material. The frit extrusion rate i9 primarily limited by the pumping pregsure. PreAsures of over 200 psi are preferred with about lO00 psi or more most preferred. A preferred pump is the Ranin Rabbit HPX, a piston type pump. Pumps may be utilized at preAsureA of from about 50 p9i and higher.

While the trans-frit pressure will be the primary determinant of the extrusion rate, other factors such as fluid viscosity will affect the extrusion rate. Those skilled in the art will understand that at smaller frit pore size designations liposomal materials of lesser viscosity are preferred. Further if the frit material is lipophilic, lipid adhesion to the frit may compromise frit pore size.

It is an advantage of this invention that a single frit may be utilized to accomplish the extrusion of this invention.

The invention will be better understood by reference to the following examples, while not being limited thereby:

Pre~aration of Li~osomal Material and Extrusion Liposomal material was prepared by stirring 100 gm of egg phosphatidylcholine into 1000 gm of methylene chloride at ambient temperature and pre~sure. Next 1000 gm of O.9X saline containing 100 mg/ml streptomycin was added with stirring. The methylene chloride was removed by evaporation and the resulting liposomal material was comprised of liposomes with a mean diameter of 0.54 micron.

50 ml of the resulting liposomal material was placed in a feed reservoir. This feed reservoir was connected to a piston pump (Ranin Rabbit HPX) equipped with a 25 ml/minute preparative pump head. This pump was in fluid connection with a stainless steel frit ( Scientific Systems, Inc.) with a pore size designation of 500 nanometer. The frit was 5mm in diameter and 0.7 mm thick.

~3151~8 The 50ml of the resultin8 liposomal material was extruded at a rate of 25 ml/minute with the contents extruded 3 times through the frit.
l'otal recovery of the material was not less than 99.99X. After each pass t:hrough the frit the mean dlameter of the extruded liposomes was determined by quaqi-elastic light scattering methodology. The results are shown in Table 1. A mean liposome diameter of 0.27 micron was achieved. The extrusion of the liposomes yielded a reduction in liposome ~ize from a mean diameter 0.54 micron size to an mean diameter of 0.27 micron. Thus the extrusion method yielded a 50% reduction in liposome size.

Extrusion of LiDosomal Material Lipo~omal material was prepared by the method of Example 1 but utilizing amikacin instead of streptomycin. Next 300 ml of the resulting liposomal material was pumped through a frit with 500 nanometer pore size designation as in Example 1. Four passes through the frit were made with the results shown in Table 2. The starting liposomal material had a mean diameter of 0.41 micron and after extrusion a mean diameter of 0.18 microns. This extrusion represents a reduction in liposome size of over 50%.

PREP~RATIO~ AND EXTRUSION : PILOCARPINE - ALPHA TOCOPHEROL HEMISUCCINATE

Five gram-q of pilocarpine base were added to a weighed 500ml round bottom flask and stopper, and the total mass in grams recorded.
D~alpha-tocopherol acid succinate (12.75 g, corresponding to a 1:1 M
ratio of pilocarpine:D-alpha- tocopherol) was added to the flask and the contents again weighed. Methylene chloride (50 ml) was added and the flask agitated to dissolve the solids and the flask again weighed. The fla~k was placed on a rotary evaporator in a water bath at 55C and 1315~98 rotated for 30 minutes (no vacuum applied). After 30 minutes, the flasX
and contents were again weighed, then rotary evaporated with vacuum at 55C. The weight of the flask was recorded every 30 minutes thereafter until two successive weighings were within O.lg. The preparation wa~
then cooled to room temperature (25C), stoppered, and stored at 4C.

The 500 ml round bottom flask containing the pilocarpine - D-alpha tocopherol was placed on a rotary evaporator; with the water bath temperature set at 55 C. The material was warmed for 30 minutes, then the water bath temperature reduced to 35 C. An aqueous solution of 0.1% (w/v) sorbic acid 0.1% (w/v) sodium EDTA dihydrate (92 ml) was added and the suspension vortically mixed. The final volume was ad~usted to 125 ml with additional aqueous phase. The resulting liposomes were extruded 10 times as in Example 1 producing size-reduced liposomes having a uniform average mean diameter.

PREPARATION A~D EX~RUSION : PILOCARPINE - ALPHA-TOCOPHEROL HEMISUCCINATE

The materials ant procedures of Example 3 were followed using a 5.0 liter round bottom flask and stopper, 30 g of pilocarpine base, 76.5 g of D-alpha-tocopherol acid succinate (corresponding to a 1:1 M ratio of pilocarpine:D-alpha- tocopherol), and 300 ml of methylene chloride.

The 5 liter round bottom flask containing the pilocarpine- D-alpha tocopherol was placed on a rotary evaporator; with the water bath temperature set at 55C. The mixture was warmed for 30 minutes, then the water bath temperature was reduced to 35C. An aqueous solution of O.OlX (w~v) ~orbic acid with 0.01% (w/v) disodium EDTA dihydrate (550 ml) was added and the suspension mixed for 1-1.5 hours using an agitator b~ade. The final volume was ad~usted to 750 ml with addltional aqueous phase. The resulting liposomes were processed by method of Example 1 passing the liposomes 10 times through a stainless steel frit having a nominal pore size desi8nation of 500 nm.

V 131~ 98 TABLE l Mean Diameter PassaQe No. (microns) % Size Reduction 0 0.54 __ l 0.38 30 2 0.35 36 3 0.27 50 Mean Diameter Passa~e No. (mlcrons) X Slze_Reductlon 0.41 __ 1 0.34 17 2 0.29 29 3 0.26 35 4 0.18 57 ~315198 LIPOSOME CONTINUOUS SIZ~ REDUCTION
METHOD A~D APPARATUS

FIELD OF THE INVE~TION

Liposomes comprise a class of bilayer lipid structures with a host of applications, particularly in the pharmaceutical arts. A number of distinct liposomal structures have been identified such as stable plurilamellar vesicles (U.S. Patent No. 4, 522,803) multilamellar vesicles (Bangham et al., 1960 J. Mol. Biol. 13:238-252), and reverse-phase evaporation vesicles (U.S. PaSent No. 4,235,871).
lS Particularly of interest are unilamellar vesicles as described by Papahad~apolous and Miller in 8iochim. B~o~hYs. Acta. 135: 624-638 (1967).

Frozen and thawed multilamellar vesicles (FATMLV) are described in "Solute Distributions and Trapping Efficiences Obscrved in Freeze-Thawed Multilamellar Vesicles, " Mayer et al., Bioch~a et Bio~hvsica Acta. 817:
193-196 (1985).

A problem encountered in the liposome art ls size variablllty of the liposomes. Size unifonmity is a particular consideration in the pharmaceutical use of liposomes for ln~ection. In~ected liposomes of over about 5 microns (5000 nanometers) in diameter may block capillarie~. Liposomes of uniform 3ize will have both predictable and ~electable distribution characteristics. This invention concerns an improved method and apparatus for achieving uniform liposome size.

A

Claims (25)

1. A method of extruding liposomes from liposomal material in the form of liposomes the step of passing the liposomal material through a frit under pressure.

2. The method of claim 1 wherein substantially all of the liposomal material is extruded.

3. The method of claim 2 wherein the frit is comprised of metal.

4. The method of claim 1 wherein the frit is of a pore size desig-nation of about 500 nanometers or less.

5. The method of claim 4 comprising passing the liposomal material through the frit at a rate of at least about 10 ml/minute/cm2 frit surface area.

6. The method of claim 5 wherein the rate is at least about 40 ml/minute/cm2 frit surface area.

7. The method of claim 1 wherein the pressure is at least about 200 pounds per square inch.

8. The method of claim 7 comprising the steps of passing the liposomal material through the frit at least about 3 times.

9. The method of claim 8 comprising the steps of passing the lipo-somal material through the frit at least about 10 times.

10. The method of claim 8 wherein the liposomal material comprises egg phosphatidylcholine.

11. The method of claim 4 comprising the step of preparing the liposomal material into liposomes prior to passing the liposomal material through the frit.

12. The method of claim 1 comprising the step of passing the lipo-somal material through the frit at a pressure of at least about 100 pounds per square inch.

13. The method of claim 12 comprising the step of preparing the liposomal material into liposomes prior to passing the liposomal material through the frit.

14. The method of claim 1 comprising the step of preparing the lipo-somal material in a nonliposomal form prior to the step of passing the liposomal material through the frit.

15. An apparatus for extruding liposomes comprising:
a) a feed reservoir suitable for containing liposomal material;
b) a pump in connection with said feed reservoir; and c) a frit through which said pump extrudes said liposomal material.

16. The apparatus of claim 15 wherein the frit is comprised of metal.

17. The apparatus of claim 16 wherein the frit is of a pore size designation of about 500 nanometers or less.

18. The apparatus of claim 15 wherein the pump functions at an operating pressure of at least about 200 pounds per square inch.

19. The apparatus of claim 15 wherein the frit is of a pore size designation of about 200 nanometers or less.

20. The apparatus of claim 15 wherein said pump pumps liposomal material through the frit at a rate of 10 ml/minute/cm2 frit surface area.

21. A method of extruding liposomes comprising:
a) placing a liposomal material into a feed reservoir;

b) pumping said liposomal material by a pumping means from the feed reservoir to a frit; and c) passing the liposomal mixture through the frit.

22. The method of claim 21 wherein the placing of a liposomal mixture into a feed reservoir comprises placing said liposomal material into the feed reservoir in the form of liposomes.

23. The method of claim 21 wherein the passing of the liposomal material through the frit comprises passing the liposomal material through a metal frit.

24. The method of claim 22 wherein passing the liposomal material through a metal frit comprises passing the liposomal material through a metal frit having a pore size designation of less than about 500 nanometers.

25. The method of claim 21 wherein the passing of the liposomal material through the frit is at a rate of 10 ml/minute/cm2 frit surface area.