WO2003039512A1

WO2003039512A1 - Liposomal formulations and related methods

Info

Publication number: WO2003039512A1
Application number: PCT/US2002/019984
Authority: WO
Inventors: Tomas De Paoli; Alfredo Adolfo Hager
Original assignee: Fish, Robert
Priority date: 2001-11-08
Filing date: 2002-06-21
Publication date: 2003-05-15
Also published as: AR031744A1

Abstract

Novel liposomes have relatively high membrane phase-transition temperatures, and little or no residual organic solvent. They can be advantageously dried before use to produce proliposomes. Upon reconstitution and filling with 5-FU or other drugs, the resulting liposomes can carry an unusually high level of active drug, and exhibit especially good storage characteristics. Preferred topical creams and other formulations include at least 20 wt% liposomes, and no more than 0.75 wt%, 0.5 wt%, or even 0.25 wt% 5-FU.

Description

LIPOSOMAL FORMULATIONS AND RELATED METHODS

This application claims priority to Argentina patent application serial no. P010105236, filed on behalf of Tomas De Paoli and Alfredo Adolfo Hager on or about Nov. 3, 2001, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

This invention relates to the field of liposomes.

BACKGROUND

Liposomes

Liposomes are closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may have a single bilayer membrane (unilamellar vesicles), a few bilayers (oligolamellar), or many bilayers (multilamellar). Oligolamellar and multilamellar vesicles are onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer. The bilayers of liposomes are composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. The structure of the membrane bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid monolayers orient toward the center of the bilayer while the hydrophilic "head" orients towards the aqueous phase. (See description in US 4981692 to Popescu, et al., Jan. 1991, the pertinent portions of which, along with those of all other references set forth herein, are incorporated herein by reference in their entirety).

The original liposome preparation of Bangham, et al. (J. Mol. Biol, 1965, 13:238- 252) involved suspending phospholipids in an organic solvent, which was then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase was added, and the mixture was allowed to "swell". The resulting multilamellar vesicles were dispersed by mechanical means. The method provides the basis for the development of the small, sonicated unilamellar vesicles described by Papahadjopoulos et al. (Biochem. Biophys, Acta., 1968, 135:624-638), and large unilamellar vesicles.

Although liposomes as a class have been described for decades, known liposomes suffer from several aspects that render them undesirable for many applications. One aspect is cost. Manufacture of liposomes in commercial quantities requires special facilities and personnel with very specialized training. Even where liposomes can be adequately manufactured, they tend to be relatively inefficient carriers of drugs or other compositions. Although the percentage of an aqueous solution that may be encapsulated depends on several factors, including type of liposomes, their size, osmolarity of media, lipidic composition and manufacturing procedure used, typical lipidic bilayers are at least approximately 4 nanometers thick, so that liposomes of diameter less than about 50 nanometers only encapsulate very small aqueous volumes (0.2 to 1.5 Lt per mol. of phospholipids).

It is also difficult to control the size distribution of liposomes during manufacture. Liposomes vary significantly in size, ranging from about 20 nanometers up to several microns. They are also osmotically active, and therefore can vary the aqueous volume encapsulated as a function of changes in external osmolarity. Liposomes can be separated out as a function of size, but that effort can add substantial additional cost.

Another problem is that liposomes tend to be unstable. Encapsulated small molecules tend to diffuse through the membranes of liposomes until reaching equilibrium with external media. Such behavior is especially problematic in pharmaceutical compositions, where the level of an encapsulated drug is relevant at the time it is used rather than at the time of its preparation. In most cases, and unless special precautions are taken, liposomes that encapsulate materials soluble in water may lose their contents as soon as they have been elaborated. Liposomes also show some instability due to oxidative and microbiologic deterioration. Unilamellar liposomes of diameters, ranging anywhere from 100 to 800 nanometers, and containing aqueous volumes anywhere from 4 to 20 1/mol of phospholipids, tend to be especially unstable. Oligolamellar or multilamellar liposomes that contain intermediate aqueous volumes tend to be more stable than unilamellar liposomes, but are still insufficiently stable for many uses in the pharmaceutical industry.

The inherent inefficiency of liposomes, their size distribution and instability are less relevant in the cosmetic industry, which markets a number of products in liposomes that encapsulate hydrosoluble and liposoluble active principles. (Korting HC, et al.; Eur. Clin. Pharmacol. 39: 349-351, 1990; EgbariaK and Weiner N. Cosmetic / 'Toiletries 106.79- 93,1991; Jungingef HE, et al., Cosmetic & Toletries 106: 45-50, 1991 and Mezei M. En O. Braun-Falco HC. Korting and Maibach, eds, Griesbach Conference on Liposome Dermatic Heidelberg: Springer- Verlag, Berlin, 206-214, 1992). The use of liposomes in the food industry is also infrequent, with liposomes used to supplement milk products and soy beverages being some of the few commercially available items, (see US Patent No. 5534268 to De Paoli et al., July 1996).

Proliposomes

Proliposomes are dried liposomes that can be reconstituted to form liposomes on an as needed basis. One of the earliest descriptions is set forth in European Patent 0158441 B2 to Leigh, (June 1992). Proliposomes are advantageous because, among other things, they can be used to encapsulate both hydrosoluble and liposoluble compounds with high efficiency. The patent disclosed a version of proliposomes containing phospholipids, water, and a water miscible organic solvent. The basic principles of proliposomes production are also described in "Liposomes, The Pro-Liposome Approach", Arnaud, Jean-Pierre (Sept. 1993), published by Lucas Meyer Company.

A major potential application of proliposomes is in the preparation of topical pharmaceutical formulations for treating dermatological diseases. Such proliposomes would ideally be devoid of organic solvents, available in powder form, have high stability during long periods of time, be reconstituted by an aqueous solution that contains an active ingredient, and use lipids that have phase-transition temperatures above 37 °C. Against those standards the previously known proliposomes are problematic. In the Leigh proliposomes, for example, at least some organic solvent remains in the final product, which makes them undesirable for use in foods and the many pharmaceutical industries. In addition, up to 40% of the weight of Dr. Leigh's proliposomes is water, which decreases their medium life and makes them more susceptible to oxidation and microbial degradation. Other proliposomes described in the literature are also inadequate. US Pat. No. 6045828 to Bystrom et al. (April 2000), for example, teaches proliposomes that use phase-transition temperatures below body temperature (37 °C).

Topical Pharmaceuticals

A particularly attractive use of liposomes and proliposomes in topical pharmaceutical formulations is treatment of actinic keratoses. Actinic keratoses are epithelial precancerous lesions thought to be induced by solar radiation. They have been treated for many years with topical preparations, but unfortunately with mixed results. Peeling agents such as carboxylic acids, particularly hydroxy acids and keto acids, and especially glycolic acid and lactic acids (as well as their esters, salts and lactones) produce cosmetic improvement, but they are considered to be relatively ineffective in removing actinic keratoses (see US Pat. No. 4234599 to Van Scott et al, Nov. 1980).

Fluorouracil (5-fluoro-2-2,4(lH,3H)-pyrimidinedione, 5-fluorouracil, or simply 5- FU) has been found to be clinically effective in treating actinic or solar keratoses, and superficial basal cell carcinomas. 5-FU is believed to create a thymine deficiency that causes unbalanced growth and cell death. The effects are most marked on cells that grow quickly and rapidly take up 5-FU. Unfortunately, the compound is associated with significant side effects, including erythema, photosensitivity, hyperpigmentation, burning, pain, itching, skin inflammation, and ulceration.

Researchers have addressed the issue of side effects in many different ways. US 5627187 to Katz (1997) teaches a combination of 5-FU with a superficial dermal peel agent selected from the group of hydroxy carboxylic acids, keto acids, halogenated carboxylic acids, and salicylic acid. Other literature teaches combinations of 5-FU with coriticosteroids. Various treatment schedules have been implemented in an effort to reduce the side effects of 5-FU. US 4849426 to Pearlman (1989) teaches pulsing of 5-FU (with an appropriate penetrating solvent) in intervals from once every 3 to 30 days, to once every 4 to 14 days. At the other extreme, researchers have suggested applying 5-FU 4 times a day for between 7 and 21 days. See Unis, N.E. "Short Term Intensive 5-Fluorouracil Treatment of Actinic Keratoses", Dermatol. Surg., vol. 21, no. 2, pp 162-163, Feb. 1995. More recently it has been discovered that low doses of 5-FU (less than 2%) can be effective, even if given for short durations and relatively low frequency. WO 00/00159 to Tobey et al. (publ. 2000) teaches that 0.5% 5-FU can effectively treat actinic keratoses when applied daily for 7 to 14 days.

A continuing problem, however, is that 5-FU does not penetrate skin very well. FLUOROPLEX® (1% topical solution of 5-FU) and EFUDEX® (2% or 5% solution of 5- FU) both utilize propylene glycol as a penetrating agent. AZONE® (1- dodecylazacycolheptan-2-one), DMSO (dimethyl sulfoxide), DMA (dimethylacetamide), DMF (dimethyl formamide), and l-methyl-2-pyrrolidone have all been suggested for enhancing percutaneous absorption. These and other penetrating enhancing agents are described in US 4849426 to Pearlman (1989). Low dose 5-FU could be delivered in liposomes, but as discussed above, satisfactory liposomes are not yet publicly known.

Thus, a need remains to provide improved liposomes and proliposomes. There is also a continuing need to utilize such technology in the field of topical pharmaceutical agents, especially in the use of 5-FU.

SUMMARY OF INVENTION

The present invention provides systems and methods for producing and using hovel liposomes having relatively high membrane phase-transition temperatures, and little or no residual organic solvent.

One aspect of novelty provides formulations for topical application of an active drug, comprising a base in which are dispersed liposomes or other carriers having an average size of less than 12 microns, and that contain on average at least 2 wt% of the drug. By adjusting the lipid composition, either the kind of phospholipids used and the fatty acids saturation grade of the same the carriers can contain larger percentages of the drug, including on average at least 4 wt% or even 5 wt% of the drug.

Where the carriers comprise liposomes, it is preferred that at least 80% of the carriers are unilamellar, although higher percentages are preferred, including at least 90% and even at least 99% of the carriers being unilamellar. Liposomal carriers are preferably manufactured using little or no organic solvent, such that they contain on average no more than 5 wt%, 3 wt%, or even 1 wt% organic solvent.

The lipids of the liposomes are selected to have a membrane with overall phase transition temperature of at least 37 °C, and more preferably between 41 °C and 62°C. At least partially as a result, the liposomes lose less than 20%, 10%, or even 5% of their drug contents at 3 months of storage. Preferred liposomes are relatively small, having a mean size of between 5 and 10 microns, and more preferably between 6 and 8 microns.

Especially preferred liposomes have membranes with an average transition temperature of at least 37°C, and contain no more than 2% or even 1% organic solvent. In terms of active drug content, preferred formulations have a total concentration of the drug of no more than 2.5 wt%, and the ratio of average concentration of drug in the carriers to average concentration of drug in the formulation is between 2 and 8 inclusive.

Contemplated drugs include nucleoside analogs, and especially 5 fluorouracil (5- FU). Preferred formulations include at least 20 wt% liposomes, and no more than 0.75 wt%, 0.5 wt%, or even 0.25 wt% 5-FU.

A typical method of manufacture includes the steps of providing a lipid reagent having a phase transition temperature of at least 37°C; combining the lipid reagent with an aqueous reagent; heating and agitating the combination of lipid and aqueous reagents; and cooling and drying the combination. The drying step can be facilitated by applying a pressure of no more than 1 Torr at a temperature of 20-40°C. Salt can be added to alter osmolarity.

Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the invention, along with the accompanying drawings, in which like items are represented by like numerals.

BRIEF DESCRIPTION OF THE DRAWINGS5

Fig. 1 is a flowchart depicting steps in the production of the liposomes according to an aspect of the present invention.

Fig. 2 is a lateral elevation with partial cutaway of the device developed to carry out the drying of proliposomes.

Fig. 3 is a diagram of a vacuum control circuit of the device shown on Fig. 3.

Fig. 4 is a distribution curve of the sizes of the liposomes, prior to the dehydration stage.

Fig. 5 is a distribution curve of the sizes of the liposomes of Fig. 4 after rehydratation. Fig. 6 is a distribution curve of the sizes of the liposomes after rehydratation with a tampon-solution (boric/borate 1,2 M. sodium chloride 0,9%, pH 8) that contained 5- fluorouracil at 5% p/v.

DETAILED DESCRIPTION

General Procedures

Figure 1 lists production of proliposomes 10 according to the following general steps: step 12 - providing a lipid reagent having a transition temperature of at least 37°C; step 14 - combining the lipid reagent with an aqueous reagent; step 16 - heating and agitating the combination of lipid and aqueous reagents; and step 18 - cooling and drying the combination. In subsequent steps the proliposomes are "filled" with one or more active components. The active components can be incorporated into liposomes either by encapsulation in hydrophilic compartments of the liposome (when the active component is water-soluble), or by encapsulation into the lipid bilayers, when the active component is lipophilic. A preferred method is set forth in greater detail as follows:

a) Provide a mixture of lipids containing phospholipids and at least two hydrogenated lecithins as described below;

b) At room temperature moisturize a mixture of the phospholipids with a tampon solution comprising between 0 and 1.8 p/v of sodium chloride, having pH between 3.5 and 8.5 inclusive, and more preferably between 5.0 and 8.0 inclusive (all ranges herein are deemed to be inclusive unless expressly defined otherwise), until obtaining a suspension comprising between 1 - 10% p/v of the phospholipids mixture;

c) Heat the suspension at a temperature sufficient to reach the phase-transition temperature of the lecithins. Preferred temperatures are between of 55 °C and 75 °C;

d) Submit the heated suspension to homogenization using (1) 80 mm rotors turning from 1000 to 3000 rpm and (2) ultrasound for 60 to 180 minutes at a frequency between 24 and 40 kilocycles per second, using between 30 and 60 watts per liter of suspension; e) After homogenization any bacteriostatic agent may be incorporated into the suspension of liposomes, such as methyl-paraben, propyl-paraben or imidazolidinilurea, each of them in a proportion of 0.1 to 0.5% wt% of suspension, and more preferably between 0.2 to 0.4% wt% of suspension;

f) Dehydrate the liposomes to form proliposomes by vacuum drying at a temperature of 20-50° C. Note that if the liposomes are not dehydrated immediately, they must be preserved at a temperature of 2 - 8° C;

g) The proliposomes can be rehydrated through combination with substantially any aqueous solution containing substantially any suitable active principle, and agitating at a temperature of 55-75 °C for between 60 and 80 minutes. Although both liposoluble and hydrosoluble principles can be encapsulated in this manner, the expected yield differs. Encapsulation of liposoluble active principles usually results in yield ranges between 70 and 100%, while encapsulation of hydrosoluble principles usually results in yield ranges of at least 50%; and

h) Optionally, liposomes can be coated, for example as set forth in US Pat. No. 5552156 to Burke, (Sept. 1996).

Dehydration Equipment

Figure 2 depicts a device designed by the inventors to properly dehydrate liposomes into proliposomes. Prior to the development of this device it was extremely difficult to dehydrate liposomes that had been prepared in large volumes in an aqueous solution, especially where such dehydration process needed to be carried out in a sterile manner.

The device 30 generally comprises a hermetically closed dehydration chamber 32, which contains an agitator 33 with two sets of removable steering paddles 35 that move about a rotating axis 34. Each of the paddles 35 extends radially from the axis 34 up to within a few millimeters or less of an interior wall 36 of chamber 32. The agitators 33 are mounted on the axis 34 by means of cylinders 34'. Agitator 33 is easily mounted and dismounted so that it may be cleaned, enabling the use of various different sets of paddles 35 as needed. Chamber 32 is preferably selected to have a relatively large capacity, such as 7 liters. Chamber 32 and its contents are also preferably selected to be constructed of a strong, readily cleaned material such as AISI 316 steel. The only exception is the sealing material to door 37, which is preferably made of acrylic material to provide a hermetic seal, and to allow visualization of the dehydration procedure being carried out in the interior of the chamber 32. A "type L" joint 39 is placed transversally at an extreme 38 of chamber 32. Door 37 is mounted by hinges and wedges that regulate its perpendicular position relative to the longitudinal geometric axis of chamber 32 in such a way that a uniform and convenient pressure is applied to the profile of the front side of joint 39. In this manner, the door 37 provides a hermetic seal, while also allowing a convenient access to the interior of chamber 32 to extract the powder obtained, and for cleaning.

To facilitate drying, chamber 32 has an outlet 40 for connection to a vacuum source, and some sort of heater, which in this instance is represented by heating bands 41 & 42 formed by electric resistances that surround the perimeter of the chamber 32. The heating bands are advantageously controlled by a programmable temperature controller (not shown in the diagram) connected to a temperature sensor having a "J" type thermocouple 43, resting on the external phase 44 of the chamber 32 and between the said heating bands 41 and 42.

The rotating axis 34 is connected externally to some form of motion inducer, such as electric motor 45. Suitable motors have adjustable speed (preferably between 0 and 58 rpm) and are capable of bi-directional rotation. The drive train rotating axis 34 is coupled through a motor reducer 46, a coupling 47, and a rotating latch 48 for high vacuum. The electric motor 45 is connected to an electronic aviator (not shown in the diagram). The rotating latch 48 is mounted on spherical bearings and sealed by a high-vacuum stopper, and is designed to be mounted in such a way that a very precise concentricity may be kept between axis 34 of agitator 33 and the geometric axis of chamber 32, thus obtaining a substantially uniform distance between paddles 35 and the internal wall 36 of chamber 32.

The vacuum generator preferably comprises a PIRANI high vacuum pump 49, with two heads. The first head 50 is connected directly to the aspirator of the pump, and the second head 51 is connected to the outlet 40 of chamber 32. Also connected to the chamber are a BOURDON type vacuum gauge 52, a valve 53 to allow air to break the vacuum, and a BENERT or McLEOD vacuum gauge 54. This vacuum-generating device also includes an isolation valve 55. All connections are preferably fast couplings rated for high vacuum.

A first test was carried out with chamber 32 empty to determine stagnancy. After 4 hours of degasification a pressure at the chamber was reached of 0.002 Torr (millimeters of mercury). Afterwards, insulation valve 55 was closed and pressure in the chamber 32 increased at a rate of 0.0004 Torr per second per each liter in the chamber, and this value is deemed most acceptable for this type of equipment.

Lipids

A wide range of lipids can be used in preparing liposomes according to the present invention. Examples listed in US Pat. No. 6045828 to Bystrom et al. (April 2000) include natural and synthetic phosphoglycerollipids, sphingolipids, and digalactosylglycerolipids. Amongst natural lipids the patent mentions sphingolipids (SL) such as sphingomyelin (SM), ceramide and cerebroside; galactosylglycerolipids such as digalactosyldiacylglycerol (DGalDG); phosphoglycerolipids such as egg-yolk phosphatidylcholin (e-PC) and soya- bean phosphatidylcholin (s-PC); and lecithins such as egg-yolk lecithin (e-lecithin) and soya-bean lecithin (s-lecithin). Amongst synthetic lipids are mentioned dimyristoyl phosphatidylcholin (DMPC), dipalmitoyl phosphatidylcholin (DPPC), distearoyl phosphatidylcholin (DSPC), dilauryl phosphatidylcholin (DLPC), l-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), l-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), and dioleoyl phosphatidycholin (DOPC). Amongst mixtures of lipids are mentioned the following: SM/PC, SM/Cholesterol, ePC/Cholesterol, sPC/Cholesterol, PC/PS/Cholesterol, DMPC/DPPC, DMPC/DPPC/CH, DMPC/CH, DPPC/DOPC, DPPC/DOPC/CH, DLPC/DPPC, DLPC/DPPC/CH, DLPC/DMPC, DLPC/DMPC/CH, DOPC/DSPC, DPSM/DMSM, e-lecithin/Cholesterol and s-lecithin/Cholesterol. The '828 patent also suggests the use of charged lipid such as dimyristoyl phosphatidylglycerol (DMPG), diphospalmitoyl phosphatidylglycerol (DPPG), dimyristoyl phosphatidic acid (DMPA), dipalmitoyl phosphatidic acid (DPP A) or stearylamine (SA).

An important feature in the production of liposomes according to the steps of Fig. 1, however, is that the lipids or lipid combinations employed have phase-transition temperatures greater than 37° C. For that purpose mixtures containing hydrogenated lecithins are preferred because they are readily available, and have a lower cost than that of the pure phospholipids. Altering the degree of hydrogenation can readily control the phase transition temperature. Preferred compositions contain a mixture of at least two phospholipids, preferably hydrogenated present in amounts between 60 and 80% wt% relative to composition of proliposomes. Especially preferred compositions comprise two or more hydrogenated lecithins in the amounts described below:

a) Between 4 - 20% in weight of dipalmitoyl phosphatidyl choline, preferably 12,5%

b) Between 20 - 80% in weight of distearoyl phosphatidyl choline, preferably 50%;

c) Up to 0,6% in weight of palmitoyl phosphatidyl choline, preferably less than 0,3%;

d) Up to 2,4% stearoyl phosphatidyl choline, preferably less than 1,2%;

e) Between 0 and 3% in weight of dipalmitoil phosphatidyl ethanolamine, preferably 1,5%;

f) Between 0 and 12% in weight of distearoyl phosphatidyl ethanolamine, preferably 6%;

g) Between 0 and 3% in weight of dipalmitoil phosphatidyl inositol, preferably 1,5%; and;

h) Between 0 and 12% in weight of distearoyl phosphatidyl inositol, preferably 6%.

Of course, the proportion present in each one of the lecithins in the proliposomes depends on the characteristics required in the final liposomes. Those skilled in the art will understand how to adapt the proportions of the listed lipids to achieve desired size and other characteristics in the final liposomes.

Active components

Although some of the disclosure set forth herein focuses on 5-FU as a pharmaceutically active ingredient for treatment of actinic keratoses, other drugs can be substituted for 5-FU, or added to 5-FU to achieve an additive or synergistic effect to treat the same disease. A 5-FU derivative is described in GB 2 168 350 to Hong (publ. 1986). Contemplated co-drug formulations include at least two drug compounds ionically linked, or covalently linked to one another via a physiologically labile bond. US Pat. No. 6051576 to Ashton et al. (April 2000) teach numerous such formulations in which 5-FU is one of two co-drugs.

Still other active ingredients can be used to treat other diseases. Numerous examples are listed in the 6045828 patent. Non-liposomal components of a treatment program are also contemplated, including for example sensitizing skin cells to 5-FU treatment as taught in French patent no. 2235094 to Jolivet, 1999.

Administration

Liposomal preparations are contemplated to be administered in any suitable manner, including for example administering topically in the form of a gel, ointment or cream. To that end patches, adhesive strips, and/or pads may be used to maintain contact between the preparation and the skin being treated.

Liposomal preparations contemplated herein can include any suitable bases, carriers, excipients, and so forth. Preferred combinations include the conventional cosmetic compositions that contain materials such as one or more of the following: sunscreens, penetration enhancers, moisturizers, surfactants, emollients, colorants, conditioners, antimicrobials, astringents, detergents, bulking agents, and so forth, as long as such components do not significantly interfere in the operation of the liposomes.

Contemplated formulations of 5-FU are from about 0.1% to 2% or more, and even up to 5%. The composition may be administered from between once every other week to 5 or more times a day, and the treatment regiment may last from a single application to 8 weeks or more. At present the most preferred treatment regiment is 0.5% cream administered once per day for 14 days.

Depending on formulation and treatment regimen, at least 1%, 2%, 3% or more of the available applied drug may penetrate to the dermis.

Indications

A number of pathological conditions which occur in man, animals and plants may be treated more effectively by encapsulating the appropriate compound or compounds in liposomes. These pathologic conditions include but are not limited to infections (intracellular and extracellular), cysts, tumors and tumor cells, allergies, etc. Formulations contemplated herein can therefore be used to treat a very wide range of dermatological disorders. For 5-FU containing liposomes, it may be reasonable to treat actinic damage of all sorts, non-malignant lesions including warts, psoriasis, and so forth, as well as some pre- malignant or malignant conditions, depending on the active drug, and the treatment schedule.

Major Advantages

The methods and compositions disclosed herein can produce liposomes having desirable size distributions appropriate for a range of therapeutic needs. For example, sub- micronic liposomes can be produced that are useful for topical pharmaceutical preparations because they are relatively more able to penetrate the deep layers of the skin than liposomes of larger sizes.

In general, the size distribution of the liposomes obtained from proliposomes can be selected as needed for the final formulation with the active ingredient by adjusting the lipid composition, either the kind of phospholipids used and the fatty acids saturation grade of the same. As an example, if we increase the phosphatidyl choline content of the proliposome, the medium size will be lower. For the same phospholipids composition the medium size can be increased with a higher content of saturated fatty acids. For a given phospholipids composition of the proliposomes, we can increase the medium size of the liposomes, by increasing the proliposomes concentration in the liposomal formulation. As an example, for a concentration of 7% of proliposomes, the mean size is 3.5 to 4.5 micron and for a concentration of 9% proliposomes, the mean size is increased to 4.5 to 5.5 micron. For a given proliposomes composition and concentration, the mean size of the liposomal formulation can be increased by increasing the sodium chloride concentration."

By using the methods and apparatus disclosed herein, the size distribution of liposomes can be substantially conserved across dehydration and eventual rehydration. In one test, for example, we demonstrated that the median (average) size of liposomes before dehydration was of 10.02 microns, where 90% of the particles were smaller than 17.02 microns and 10% were of less than 3 microns (Fig.4). Subsequently this same group of liposomes was dehydrated using the methods set forth herein, and were rehydrated to evaluate the distribution of sizes of the particles. The results showed that median size was of 9.89 microns, with 90% of the particles having a size smaller than 22 microns, and 10% having a size smaller than 1.92 microns (Fig.2). These results indicate that the distribution of sizes of rehydrated liposomes corresponds to that of the original liposomes.

In another test, a sample of the same group of the original dehydrated liposomes was rehydrated with a tampon solution (buffer boric/borate 1.2 M, sodium chloride 0.9%, pH 8) containing 5-fluorouracil at 5% p/v. Median size of the rehydrated liposomes was of 6.90, microns with 90% of the particles having a size of less than 13.16 microns, and 10% having a size of less than 2.00 microns (Fig.3) (The components of Figure 3 have the standard representations). These liposomes with 5-fluorouracil at 5% p/v were used to prepare a creamy composition by mixing the same with a compatible cream base, obtaining a cream containing 5-fluorouracil at 0.5% p/v.

The composition of the present invention also allows the substantially instantaneous encapsulation of active principles into the proliposomes to form liposomes, which can take place immediately before their use. This makes the proliposomes especially useful when the active drug or other compound being carried would rapidly diffuse into the surrounding media.

Another major advantage is that contemplated proliposomes can be used to prepare liposomes with different osmolarities, which affects permeability and thereby modulates release of active principles. Different osmolarities can be achieved by modifying the amount of sodium chloride or other salt present in the liposomes. Experimental osmolarities reached a range from 0 to 1% p/p, with preferred osmolarities ranging from 0.85 to 0.95%.

Examples

The inventive subject matter can be illustrated by the following examples, which should not be interpreted as a limitation in the scope of same. On the contrary, it should be clearly understood that other modifications, applications and equivalents of them will be suggested to those who are well acquainted with this subject, without deviating from the spirit and essence of the present invention and/or scope of the attached examples. Example 1: Production Of Proliposomes In Powder

60 grams of a 1 :4 mixture of DPPC and DEPC were moisturized, with 940 grams of a tampon solution boric/borate 0.2M, sodium chloride 0.9%, pH 7.2. The sodium chloride was ACS quality. The suspension was submitted to ultrasound of 24 kcycles/second for 120 minutes at a temperature of 65 °C, with continued agitation. Once that treatment was completed, 4 g methylparaben and 4 g propyl-paraben were added to the suspension. Liposomes obtained were fractionated in containers of 100 gr. and kept in the freezer at 2 to 8 °C until dehydrated. Size distribution of liposomes was analyzed using a Coulter light scattering laser having a measuring range from 0.375 microns to 2000 microns.

The dehydration stage of liposomes was carried out by placing 800 grams of liposomes in the 7 liter high vacuum chamber 32. Dehydration was initiated at 25 °C and a final vacuum of 0.1 millimeters of mercury. That process produced 57.5 grams of a uniform powder of proliposomes.

7 grams of the proliposomes obtained from the previous stage were rehydrated with 93 grams of water at 65 °C for 60 minutes, using a magnetic agitator. Liposomes obtained from the rehydrated proliposomes were analyzed by the light scattering Coulter laser described above.

Example 2: Preparation Of Liposomes With 5-Fluorouracil At 5% From Proliposomes

12.5 grams of 5-Fluorouracil were dissolved in 230 grams of a tampon solution of boric/borate 1.2 M, sodium chloride 0.9%, adjusting pH to 8.00 with NaOH 4N. The weight was adjusted to 250 grams. The solution of 5-Fluorouracil was heated at 65°C, and then slowly added to 17.5 grams of proliposomes, agitating continuously for 60 minutes and maintaining temperature af 65 °C. 250 grams of a suspension of liposomes were obtained. The same procedure used on Example 1 for the size-distribution analysis was used.

To study the percentage of microencapsulated 5-Fluorouracil, liposomes were separated using high-speed centrifugation. Concentration of 5-Fluorouracil was determined in the pellet and in the supernatant material. The amount of 5-Fluorouracil of the pellet of liposomes and supernatant was calculated by dissolving aliquot amounts of both in ethanol and measuring absorbance in a spectrophotometer at 265 nm.

Example 3: Preparation Of A Cream With 0.5% Of 5-Fluorouracil.

Liposomes with 5-Fluorouracil at 5% p/v obtained in example 2 were used to prepare a cream of pharmaceutical use, mixing 90% of a compatible cream base with 10% liposomes. The 5-FU cream was used to carry out a small clinical trial in patients with actinic keratoses. The cream showed similar therapeutic effect as compared with commercial preparations that contain 5% of the active ingredient, but the time of treatment was shorter. In addition by using lower concentrations of 5-fluorouracil the adverse effects of the drug were significantly reduced.

CONCLUSION

Thus, numerous systems, methods, and apparatus for producing and using liposomes have been described. While specific embodiments and applications of this invention have been shown and described, it would be apparent to those skilled in the art that many more modifications are possible without departing from the inventive concepts herein. The invention, therefore, is not to be restricted except in the spirit of the appended claims.

Claims

What is claimed is:

1. A formulation for topical application of an active drug, comprising a base in which are dispersed carriers having an average size of less than 12 microns, and that contain on average at least 2 wt% of the drug.

2. The formulation of claim 1 wherein the carriers contain on average at least 4 wt% of the drug.

3. The formulation of claim 1 wherein the carriers contain on average at least 5 wt% of the drug.

4. The formulation of claim 1 wherein the drug comprises a nucleoside analog.

5. The formulation of claim 1 wherein the drug comprises 5 fluorouracil.

6. The formulation of claim 1 wherein at least 80% of the carriers are unilamellar.

7. The formulation of claim 1 wherein at least 90% of the carriers are unilamellar.

8. The formulation of claim 1 wherein at least 99% of the carriers are unilamellar.

9. The formulation of claim 1 wherein the liposomes contain on average no more than 5 wt% organic solvent.

10. The formulation of claim 1 wherein the liposomes contain on average no more than 3 wt% organic solvent.

11. The formulation of claim 1 wherein the liposomes contain on average no more than 1 wt% organic solvent.

12. The formulation of claim 1 wherein on average the liposomes lose less than 20% of their drug contents at 3 months of storage.

13. The formulation of claim 1 wherein on average the liposomes lose less than 10% of their drug contents at 6 months of storage.

14. The formulation of claim 1 wherein on average the liposomes lose less than 5% of their drug contents at 12 months of storage.

15. The formulation of claim 1 wherein the liposomes have a mean size of between 5 and 10 microns, inclusive.

16. The formulation of claim 1 wherein the liposomes have a mean size of between 6 and 8 microns, inclusive.

17. The formulation of claim 1 having a total concentration of the drug of no more than 2.5 wt%.

18. The formulation of claim 1 in which the ratio of average concentration of drug in the carriers to average concentration of drug in the formulation is between 2 and 8 inclusive.

21. The formulation of claim 1 in which the carriers have a membrane with an average transition temperature of at least 37°C.

22. The formulation of claim 1 in which the carriers have a membrane with an average transition temperature of between 41 °C and 62°C inclusive.

23. The formulation of claim 1 in which the carriers have a membrane with an average transition temperature of at least 37°C and contain no more than 2% organic solvent.

24. The formulation of claim 1 in which the carriers have a membrane with an average transition temperature of at least 37°C and contain no more than 1% organic solvent.

25. A method of manufacturing a composition: providing a lipid reagent having a phase transition temperature of at least 37°C; combining the lipid reagent with an aqueous reagent; heating and agitating the combination of lipid and aqueous reagents; and cooling and drying the combination.

26. The method of claim 25 further comprising adding a drug containing no more than 1 wt% organic solvent.

27. The method of claim 26 wherein the drug comprises a nucleoside analog.

28. The method of claim 26 wherein the drug comprises 5 fluorouracil.

29. The method of claim 25 further comprising adding a quantity of a salt.

28. The method of claim 25 wherein the step of agitating includes sonication.

29. The method of claim 25 wherein the aqueous reagent includes at least 99.99 wt% H2O.

30. The method of claim 25 wherein the step of drying comprises subjecting the combination to 20-40°C, at a pressure of no more than 1 Torr.

31. A topical formulation of no more than 0.75 wt% 5 fluorouracil having at least 20 wt% liposomes.

32. The formulation of claim 31 having no more than 0.5% 5-FU.

33. The formulation of claim 31 having no more than 0.25% 5-FU.