FR2648056A1 - Process for the preparation of lipid microparticles of microcrystalline appearance - Google Patents

Abstract

The subject of the present invention is a process for the preparation of lipid microparticles of microcrystalline appearance of a water-insoluble substance having an affinity for phospholipids and of at least one phospholipid, microparticles stable in suspension in an aqueous solution, characterised in that: a) the said substance and the said phospholipid(s) are dissolved in one or more first organic solvent(s) of the said substance and of the said phospholipid(s), and an extra solvent of the said first solvent(s) and of water; b) the solution obtained of the said substance and of the said phospholipid(s) is mixed with an aqueous solution in an amount less than that beyond which either immiscibility or insolubilisation, especially in the form of a precipitate of the said substance and of the phospholipid(s), is observed, so as to obtain a clear homogeneous phase; c) the said first and extra solvents are evaporated while maintaining the solution in the homogeneous phase in order to recover an aqueous solution containing the microparticles in the form of microsuspensions.

Description

 The present invention relates to a new process for the preparation of lipid microparticles of microcrystalline appearance.

 This type of microparticle, called microcrystals, has been described by the Applicant in particular in the European patent application No. 270 460.

 In the present application, by "crystalline aspect" it is not meant that the crystalline structure is necessarily obtained stricto sensus.

 More specifically, these are microparticles of a water-insoluble substance having an affinity for phospholipids and at least one phospholipid.

 By substance insoluble in water is meant a substance with little or no water-soluble substance and with a phospholipid-affinity substance a chemical compound more particularly capable of interacting with the phospholipids chemically and / or physically. .

 These microparticles offer the advantage of obtaining a stable microsuspension in aqueous solution of a substance which is otherwise insoluble in the aqueous phase, which makes it possible to administer it, in particular when it is a medicinal product in injectable or sprayable form. .

 Furthermore, presumably by virtue of an increased control effect, in particular towards the macrophages on the one hand, and a progressive release effect of the active substance, on the other hand, microparticle preparations give results of therapeutic activity. and of toxicity much more interesting than the lipid lipid vesicles.

 EP 270460 describes a relatively complex preparation process for these microparticles of crystalline appearance, inspired by the processes used for the preparation of liposomes.

The process described in EP 270,460 essentially comprises the following steps: a) The solvent (s) are evaporated from a solution of phospholipid and
said substance, b) The film obtained after evaporation of said solvent (s) is
suspension in an aqueous solution after vigorous stirring.

 To return the film obtained in step b) in suspension, this was done by sonication.

 In the context of an industrial application of the process, the complexity of the process, particularly with the presence of a vigorous stirring step and in particular ultrasonic treatment constitutes a major drawback.

 It has surprisingly been found according to the present invention that this sonication step can be suppressed by means of a new simplified method which is the subject of the invention.

Indeed, the subject of the present invention is a process for the preparation of lipid microparticles with a microcrystalline appearance of a substance insoluble in water having an affinity for phospholipids, and of at least one phospholipid, a stable microparticle suspended in a aqueous solution, characterized in that a) said substance and said phospholipid (s) are dissolved in one or
first organic solvent (s) of said substance and the
said phospholipids, and a third solvent of said first one (s)
solvent (s) and water, b) mixing the resulting solution of said substance and said one or more
phospholipids to an aqueous solution in an amount less than
the one from which one observes either a demixtion or a
insolubilization especially in the form of a precipitate of said substance
and the phospholipid (s) so as to obtain a homogeneous phase
clear, c) The one or more first and third solvents are evaporated while maintaining the
homogeneous phase solution during evaporation, to recover a
aqueous solution containing microparticles in micro form
suspensions.

 In order for the solution to be kept in the homogeneous phase during evaporation, it is ensured that the third solvent does not disappear completely until the evaporation of said first organic solvent (s) has been completed.

 To do this, evaporation will be carried out at a temperature and pressure which take into account the nature of the solvents in question and in particular by using the azeotropic properties of the mixture.

 By third solvent is meant a solvent that mixes water and said first solvents in a homogeneous phase.

 Optionally the third solvent is one of said first solvents.

 In the process, the organic solvents must have a boiling point lower than that of the water at the pressure at which one works.

 In some cases, it may be advantageous in the process according to the invention in step c) to evaporate said solvents at reduced pressure.

 The amounts of said substance and phospholipid (s) dissolved in said solvents should preferably be at least 10 times or even 100 times higher than those which would be dissolved in the amount of water envisaged in the process.

 The simplicity of the process according to the present invention essentially consists in mixing all the constituents and then in removing the organic solvents and thus separating an aqueous phase and an organic phase, the microcrystals being in suspension in the lacquering phase, suggests that as soon as in the presence of the various constituents, a physicochemical reaction of the complexation type occurs, the step of removing the organic solvents and separating the organic phase and the aqueous phase leading to an aggregation in the form of microparticles.

 According to the present invention, microparticles of aspect of structure and of identical size and having the same advantageous pharmacological properties as those obtained by the process previously described in EP 270,460 are obtained.

 Compared with the preparation of liposomes and the preparation of the microcrystals microparticles according to the prior method, the process according to the present invention is much simpler and more economical. In addition, the microparticles obtained are as stable as those obtained by the old process and therefore much more stable than liposomes which suffer from instability which considerably limits their possibilities of use.

 In a particular embodiment of the process according to the invention, the evaporation of the organic solvents and the recovery of the aqueous solution are carried out using a rotary evaporator type installation or by an installation more adapted to large quantities of solvent, such as an evaporator with an upward film (6), the evaporation being carried out under reduced pressure and the aqueous solution being entrained by the vapors of organic solvents before being separated by condensation of the organic phase.

 Advantageously, in this embodiment of the invention, it is possible to subject the recovered aqueous phase containing the microparticles to a second evaporation cycle, or to several evaporation cycles.

 As first organic solvents useful in the process according to the invention, mention may be made of intermediate polarity solvents such as methanol, ethanol, chloroform, dichloromethane or mixtures thereof.

 As third solvents, mention may be made of alcohols such as methanol and ethanol, acetone and THF.

 In particular, an alcohol will be used, in particular methanol or ethanol as a third solvent.

 In a particular embodiment of the invention, the organic solvents used in the process will consist of a mixture of chloroform and methanol.

 In this case, the chloroform evaporating normally at 61.2 ° C at atmospheric pressure, that is to say above the boiling point of methanol (64.7 ° C) will operate the evaporation at the temperature of the azeotrope chloroform (81% - methanol (15%) - water (4%)) whose boiling point is only 52.6 ° C at atmospheric pressure.

 As aqueous solution useful in the process according to the present invention, use will advantageously saline solutions such as a solution of NaCl, for example at a concentration of 0.9%.

 Preferably, according to the present invention, the molar ratio phospholipid (s) / substance is between 0.1 and 10.

 If the molar ratio is less than or equal to 2, in particular between 0.8 and 2 and preferably close to 1, a specific interaction is observed with optimum homogeneous production of microparticles whose average size is between 0.1 and 1 p.

 In some cases, it may be advantageous to add one or more sterols to enhance the activity of the active substance, in particular cholesterol or ergosterol.

 In general, according to the invention, phospholipid phosphatidylcholine is preferably used. Phosphatidylcholine may be prepared from egg yolk lecithin, hydrogenated or non-hydrogenated soybean or any other industrial source of lecithin.

 As another phospholipid useful in the process, it is possible to use phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, 3'-O-lysylphosphatidylglycerol, diphsophatidylglycerol, this list being of course not limiting.

 The method according to the invention is particularly advantageous for preparing microparticles of antimycotic drugs, polyene macrolides, such as nistatin and amphotericin B and their aminoacyl derivatives which also exhibit antifungal activity.

 When in the process according to the invention, said substance is amphotericin B and phosphatidylcholine phospholipid, advantageously these two constituents are brought together in equimolar amount to form lipid microparticles of crystalline appearance.

 According to one particular embodiment of the process according to the invention, Amphotericin B and phosphatidylcholine are dissolved in a chloroform / methanol 1: 1 by volume, to which a saline solution in an amount of between 1/20 and 2 is added. The volume of the organic solution of Amphotericin B and phosphatidylcholine.

 Treatment of mycotic infections caused by Candida and Aspergillus is difficult? usually poorly tolerated. Few drugs are active against these two types of microorganisms.

 Polyphenyl macrolide antimycotics such as nystatin and amphotericin B are the most widely used products with activity in both Aspergillus and Candida species.

 The other important class is that of azole derivatives, of which ketoconazole is one, but whose more restricted activity only affects infections caused by Candida.

The clinical use of Amphotericin B is very severely limited by two major disadvantages - first, its high insolubility which requires its administration in
sodium deoxycholate solution; secondly, the intrinsic toxicity of deoxychlotate is added to
the toxic activity of Amphotericin B itself
especially in the kidneys and bone marrow.

 However, whatever its side effects, this antibiotic remains effective in fungal infections that would have a fatal prognosis without this treatment. In this context, it appeared important to reduce the toxicity of Amphotericin B by modifying its intracellular penetration.

 Indeed, a very important factor for the efficacy of antimycotics in general, and for Amphotericin B in particular, is the need to induce an antibiotic action which is synergistic with that of the host cells, which are responsible for the anti-infectious defenses of the patient. 'organization. It has indeed been demonstrated that polymorphonuclear and macrophages can successfully control mycotic infections to the extent that these cells can phagocyte microorganisms and control them in their lysosomal system. Antimycotic drugs must therefore not only reduce the multiplication of infectious agents present in the extracellular milieu but also be able to exert their action inside the phagosomes and lysosomes of polymorphonuclear and macrophages. However, very little is known about the intracellular penetration of polyene macrolides and everything suggests that these substances accumulate at the level of pericellular membranes and reach the intracellular lysosomal compartments only thanks to the membrane flux of the surface. to intracellular spaces.

 The invention has solved these problems by proposing a new galenic form of Amphotericin B based on the interaction of this drug with phosphatidylcholine to form a suspension of microparticles mentioned above.

 This galenic formula allows first to administer Amphotericin B without the use of deoxycholate and it allows to significantly decrease the acute toxicity of products after intravenous injection. These microparticles have the same activity as Amphotericin B solubilized by deoxycholate against extracellular Candida and are more active in vitro in intracellular macrophage infections.

 The properties of the Amphotericin B microparticles have also been compared advantageously with those of the liposomes containing the same macrolide polyene and which are also recommended as a transporter capable of reducing the side effects of amphotericin. These Amphotericin B liposomes have already been the subject of several clinical trials (1,2).

If the formulation in the form of microparticles according to the invention is very advantageous for substances such as amphotericin B, given the sharp drop in toxicity obtained, it should be understood that other active substances can also be used provided that they have an affinity for a phospholipid. Some of them have been mentioned in a non-limiting way in the application EP 270 460
Other features and advantages of the present invention will become apparent in the light of the following description referring to figures.

- Figure 1 shows an ascending film evaporator installation.

- Figure 2: represents the average size of microparticles
Amphotericin B produced by the process according to
the invention.

- Figure 3: represents the frequency in% of the size distribution
microparticles of lot 5 produced by the process
according to the invention.

- Figure 4: represents the cumulative frequency in% of the distribution
microparticle sizes of lot 5.

- Figure S: represents the hemolytic activity of Amphotericin B
as a deoxycholate complex (Fungizone #) and
microparticles produced by the process of
the invention and not lyophilized (lot 3) with respect to blood
human (figure Sa) and murine.

- Figure 6: represents the hemolytic activity of Amphotericin B
in the form of Fungizone and microparticles
freeze-dried products produced according to the process of the invention (batch
5) with respect to human blood after 45 min at 450C.

- Figure 7: represents the weight evolution of the mice after
administration of Amphotericin B in the form of
Fungizone # or microparticles prepared according to
process according to the invention.

FIG. 8 represents the survival of the mice after iv administration
Amphotericin in the form of Fungizone # or
microparticles prepared according to the process according to
the invention (lot 2).

FIG. 9 represents the survival of the mice after iv administration
Amphotericin B in the form of microparticles
prepared according to the process according to the invention (batch 3).

FIG. 10 shows the mortality at day 14 due to Amphotericin.
B administered iv to mice in the form of Fungizone #,
liposomes and microparticles
EXAMPLE 1 PRODUCTION OF AMPHOTERSCINE MICROPARTICLES
B 1. RAW MATERIALS
Amphotericin B: Amphotericin a is a macrolide antibiotic polyene insoluble in water at neutral pH. It is produced by Streptomyces nodosus and its molecular weight is 924.

 Phosphatidylcholine: L-α; -phosphaticylcholine (or L-α-lecithin) is an amphipatic phospholipid whose polar function of the phosophoric acid ester causes a certain hydrophilicity whereas on the fatty acid side the molecule is hydrophobic. The amphiphilic nature of phosphatidylcholine is used as emulsifier and solubilizer of hydrophobic substances in aqueous media. Its molecular weight is 785.

 L- α -phosphatidylcholine is prepared chromatographically from fresh egg yolk and is 99% pure.

2. METHOD OF PREPARATION
2.1 From one gram of Amphotericin
Amphotericin B microparticle preparations were made using an ascending film evaporator of QUICK FIT (see FIG. 1).

 In a 5 liter flask, 1 gram of Amphotericin B and 0.85 gram of L-42-phosphatidylcholine (pure egg lecithin greater than 99%) are dissolved in 1.5 liters of a mixture of chloroform-methanol (1 1 volume), to which is added, when the solution is clear, 200 ml of NaCl 0.9%.

 The solvent is removed under vacuum in the rising film evaporator. The maximum temperature of the solution at the top of the evaporator is 35.degree.-45.degree. C. After separation of the phases, the aqueous phase containing the microparticles is recovered and subjected to a second evaporation cycle.

 The system is rinsed with distilled water and the final volume is brought to 200 ml using a rotary evaporator, whose heating bath does not exceed 40 C.

 Generally, 82 to 86% of Amphotericin is recovered in the form of a suspension of microparticles at a concentration of between 4 and 5 mg / ml.

2.2 From two Rrams of Amphotericin B
Same as in A, but 2.0 g of Amphotericin + 1.7 g of phosphatidylcholine in 3 liters of chloroform-methanol (1: 1) + 200 ml of NaCl 0.9%.

 83% of Amphotericin is recovered in the form of 260 ml of a suspension of 6.3 mg microparticles in amphotericin / ml.

 The organic phase is free of Amphotericin B.

2.3 Lyophilization
The suspensions of microparticles are lyophylized after addition of the following excipients
2.3.1 Vials containing 5 mg of Amphotericin B in the form of
microparticles
Lactose: 80 mg
Na Citrate: 8 mg
Na phosphate: 7.5 mg
After reconstitution with 1.0 ml of distilled water, each lyophilized vial contains 5.0 mg of Amphotericin B as medium size, about 0.7 μm microparticles formed by equimolar interaction between Amphotericin.
B and Phosphatidylcholine.

Composition after reconstitution
- Amphotericin B: 5.0 mg / ml
- Phosphatidylcholine: 4.25 mg / ml
- Lactose: 80.0 mg / ml
- Na citrate: 8.0 mg / ml
- Phosphate of Na: 7.5 mg / ml
- NaCl: 0.7%
retention
After reconstitution, the solution should be stored at 4 ° C for up to 7 days and should not be frozen.

Aspect
Yellowish suspension of microparticles with an average size of + 0.7 μm.

2.3.2 vials containing 100 mg of Amphotericin B in the form of
microparticles
The suspension of micro particles is centrifuged, the microparticles are resuspended and diluted to 100 mg in 4 ml of distilled water and added
70 mg of lactose. l H2O
40 mg of citrate 3Na.2H2O
30.4 mg Na2HPO4
7.4 mg NaH2PO4 .1H2O
After Iyophylization the flasks contain 100 mg of Amphotericin B as microparticles.

EXAMPLE 2: CONTROL METHOD AND CHARACTERISTICS
PHYSICOCHEMICAL MICROPARTICLES 1. ASSAY
1.1 Determination of amphotericin B by spectrophotometry
a) raw material: the absorbance at 405 nm of a solution
Amphotericin B at approximately 50 g / ml dimethyl sulfoxide is
measured using a spectrophotometer. The concentration is
calculated using the formula: conc. (mg / ml) A405t35.5
(b) in the form of microparticles: to 50 l of the suspension of
about 5 mg / ml, I ml of methanol and 1 ml are added in the order
of 1N HCl. The absorbance at 405 nm of the solution is read and the
The concentration of Amphotericin B is calculated using the
formula: conc. (mg / ml) = A405 / 0.934.

1.2 DOSE OF phosphatidylcholine
The determination of phospholipids whose phosphatidylcholine is carried out by an enzymatic colorimetric test (3) with the ABBOTT analyzer.

The test is based on the release of choline by the action of phospholipase D on phosphatidylcholine and on the action of choline-oxidase on choline which releases betaine and H202.

 In the presence of peroxidase, the produced X2O2 reacts with phenol and amino-4-phenazone to form (monoimino p-benzoquinone) 4-phenazone whose absorbance is read at 500 nm.

2. PHYSICAL CONTROLS
Microparticle preparations are controlled by
- examination of the macroscopic aspect: suspensions of
microparticles (MPs) of Amphotericin EN look
milky yellowish, uniform in color and appearance, without
apparent aggregates. The microparticles do not sediment and
only form a deposit after a few hours; the surrender
suspension is however easy.

- optical microscopy: the suspension of microparticles is
observed under a dark-field microscope (40x objective, eyepiece
20 x). Examination can detect the presence of aggregates
or micro-aggregates. The microparticles observed in
optical microscopy on a black background appear homogeneous
poise of view size. There are few micro-aggregates. The
Microparticles have a non-spherical and irregular shape.

- determination of the dimensions of the microparticles and their
distribution: the average size of the microparticles is
determined using a Coulter Electronics Nano-Sizer,
Ltd. (Harpenden, UK). The size distribution is
determined with a Fritsch Particle Sizer (Fritsch
Laser - Particle Sizer - Analyzer 22; Idar oberstein,
Germany). Microparticles produced at the scale of
Laboratory have a mean size measured at the nanosizer of
+/- 0.7 pm. The average size of the microparticles obtained
according to pilot production is between 0.3 and 0.8 pm
following the preparation. After lyophilization, the size
average of the microparticles is between 0.6 and
1.3 μm (Figure 2).

- molecular sieving - search for said substance
article: The presence of free Amphotericin B may be
highlighted by molecular sieving of an aliquot from the
suspension of microparticles on a column filled with
sepharose gel 68 (PHARMACIA Fine Chemicals, Upsala,
Sweden), eluted with NaCl 0.9%. The microparticles come out
in a volume corresponding to the dead volume of the
column and Amphotericin B free as well as L4-
phosphatidylcholine go out in a corresponding volume
to the total volume of the column. The limit of detection of the
percentage of Amphotericin and free PC is 0.1%.

The dosage of Amphotericin is measured by
the absorbance at 405 nm as previously described.
frequency (%) of the size distribution is illustrated in
Figure 3 and the cumulative frequency, in%, is represented in
Figure 4. The median size is 0.99 pm and the size
average of 1.69 pm. 50% of the microparticles have a
size between 0.6 and 1.7 μm and 90% of the micro
particles have a size between 0.25 and 5.2 μm.

No particle has a size greater than 16 μm, and
only 0.1% of microparticles have a size
greater than 12 μm.

3. STRUCTURE
The Amphotericin B microparticles can be chemically characterized as being composed of Amphotericin B and L -phosphatidylcholine in a molar ratio of 1, and can therefore be represented as follows, n being unknown

Example 3 Pharmacological Studies
The toxicology and pharmacology of Amphotericin B as microparticles were compared with those of Amphotericin B as a complex with deoxycholate (DOC), which is the commercial form of Amphotericin B (Fungizone). than that of multilamellar liposomes as described by SCULIER; ot al. (2). These liposomes are composed of phosphatidylcholine, cholesterol and stearylamine in a molar ratio of 4: 3: 1, have an average diameter of 0.1 pm and a lipid-amphotericin molar ratio of about 70.

1. TOXICOLOGY
1.1 In Vitro Toxicity
1.1.1 Toxicity to etythrocvests
The haemolytic effect of Amphotericin B in the form of deoxycholate complex (Fungizone or in the form of microparticles was tested with respect to human or murine red blood cells, following the protocol described by METHA et al (METHA et al. Biochim Biophys Acta 770 (1984), 230-234).

Protocol
Red blood cells from aliquots of human blood as well as murine blood (female OFI mice 25-30 grams) collected on
EDTA are washed 3 times and then diluted to 2% in NaCl 0.9% and distributed in test tubes. After addition of increasing amounts of Fungizone # (0 to 30 μg final / ml) or Amphotericin B microparticles (0 to 1000 μg Amphotericin / ml), the erythrocytes are incubated at 37 ° C. for 45 minutes.

 After centrifuging the samples at 3000 RPM for 10 minutes, the haemolytic effect is determined by measuring the absorbance at 550 nm of supernatant, which reflects the amount of hemoglobin released.

Results
Non-freeze-dried microparticles
The hemolysis of human erythrocytes (FIG. 5a) and murine (FIG. 5b) by Fungizone # is complete after 45 minutes at a concentration of 30 μg / ml. The hemolyzing doses 50% (DH 50) and 10% (DH 10) of the erythocytes are calculated by interpolation of the data and are shown in the table below.

Table 1: Haemolytic effect of Amphotericin B in the form of
Fungizones and microparticles against erythrocytes
human and murine

<tb><SEP> DH10 * <SEP> DH50 **
<tb><SEP> (g / ml) <SEP> (g / ml)
<tb> Human erythrocytes <SEP>
<tb> Complex <SEP> DOC <SEP> (Fungizone #) <SEP><SEP> 1.4 <SEP> 6.8
<tb> Microparticles <SEP> 150 <SEP>><SEP> 150
<tb> Murine erythrocytes <SEP>
<tb> Complex <SEP> DOC <SEP> (Fungizone #) <SEP><SEP> 1.8 <SEP> 6, 9
<tb> Microparticles <SEP> 97 <SEP>><SEP> 150 <SEP>
<tb> 4 DH10 = hemolyzing dose 96% erythrocytes after 45 min at 35 ° C * + DH50 = hemolyzing dose 50 96 erythrocytes after 45 min at 37 ° C
Freeze-dried microparticles
Haemolysis of erythrocytes by the Amphotericindeoxycholate complex (DOC or Fungizone complex # and by the microparticles of Ampho B freeze-dried and then released

1.1.2 Toxicity to macrophages
The in vitro cytotoxicity of Amphotericin B in Fungizon form of microparticles and liposomes was determined on the continuous line of cultured murine macrophages 3774-G8.
RPMI enriched with 10% fetal calf serum. These macrophages are infected at 104 Candida trorncalis for 105 macrophages. The cytotoxicity is determined by the reduction of the macrophage-reducing enzyme activity towards the tetrazolium salts (4). The cytotoxicity is expressed in the form of the Amphotericin B concentration which reduces by 50% the enzymatic activity (CT50). The results shown in
Table 2 indicate that in the form of liposomes, Amphotericin B is 4 times less toxic in vitro than as a deoxycholate complex.

In the form of microparticles, Amphotericin B is even less toxic in vitro: 33 times less cytotoxic than Fungizone and 7.7 times less toxic than in the form of liposomes.

<Tb>

<SEP> Formulation <SEP> CT50 (M) <SEP> CT50rel.a
<tb> Complex <SEP> DOC <SEP> (Fungizone <SEP> 4.0 <SEP>
<tb> Li <SEP> prosomes <SEP> 17 <SEP> 4.2
<tb> Microparticles <SEP> 132 <SEP> 33
<Tb>
aCT50rel: CT501CT50 report of the complex Doc 1.2 In vivo toxicity
1.2.1 Acute toxicity
1.2.1.1 Preliminary results in mice (LD50)
Amphotericin B in the form of a deoxycholate complex (Fungizon, liposomes and microparticles was injected intravenously to female OF1 mice of 25 to 31 grams, at a rate of 5 to 6 mice per dose. covered a 14-day post-dosing period (mortality and body weight).

Microparticles prepared according to the process according to the invention
MP Lot 2
In this experiment at the dose of 4 mg / kg, the induced average weight loss of 20% in 2 days and no mouse survives more than 2 days (Figures 7 and 8).

 At 10 and 15 mg doses of Amphotericin / kg, the microparticles of the lot do not induce weight loss, despite the death of 2 out of 6 mice at a dose of 13 mg / kg. At 20 mg / kg microparticles induce 100% mortality within 24 hours.

MP Lot 3
In this experiment, at a dose of 2 mg / kg, no weight loss or mortality was observed after iv administration of Fungizon. At doses of 3 and 4 mg / kg, weight loss (2% with 1 nadir at days 4-5) is reversible, despite a high mortality (80%) at the dose of 4 mg Amphotericin / kg. No mice survived more than 48 hours iv administration of 4 mg / kg Fungizone 2) (Figure 9).

The microparticles at 16 mg in Amphotericin / kg induce 40% mortality and an average weight loss of 20% at day 4, weight loss which is recovered at day 15. At higher doses, mortality is total at day 6 ( dose of 22 mg amphotericin / kg) at day 3 (dose of 28 mg
Amphotericin / kg) and day 2 (34 mg Amphotericin / kg).

Determination of LD50
The results of the above experiments make it possible to calculate a provisional value of LDSo for Amphotericin B in the form of deoxycholate complex (FungizoneS3, liposomes and microparticles.

 The percentages of mortality at day 14 are plotted against the logarithm of the dose (Figure 10).

 The LDSo are calculated by linear regression of probit of the% mortality as a function of the logarithm of the dose and the values obtained are shown in Table 3.

Table 3: Acute Toxicity of Amphotericin B as a Complex
with deoxycholate (Fungizone #), liposomes or micro
particles

<tb><SEP> LD50 <SEP><SEP> Interval of
<tb><SEP> Formulation <SEP> mBE <SEP> conf <SEP> iance <SEP>
<tb><SEP> (p <0.05)
<tb><SEP> DOC <SEP> Complex (Fungizone #) <SEP><SEP> 2.5 <SEP> 1.8 <SEP> - <SEP> 3.5 <SEP>
<tb><SEP> 3.4 <SEP> 3.1 <SEP> - <SEP> 3.9
<tb> Liposomes <SEP> 33, S <SEP><SEP> 24.9 <SEP> - <SEP> 4S, 2
<tb> Microparticles
<tb><SEP> preparation <SEP> lot <SEP> 2 <SEP> 15.7 <SEP><SEP> 15.0 <SEP> - <SEP> 16.4
<tb><SEP> preparation <SEP> lot <SEP> 3 <SEP> 15.4 <SEP> 14.3 <SEP> - <SEP> 16.6
<Tb>
The results indicate that the death-inducing dose of 50% of the mice 14 days after single iv administration of the products (LD50) is 2.5 mg / kg for amphotericin B as liposomes, 33.5 mg / kg. kg as liposomes and 15.4 to 18.3 mg / kg as microparticles.

 The microparticles prepared according to the process according to the invention do not differ from each other in terms of LD50.

 In the form of microparticles Amphotericin is therefore about 4.5 to 7.4 times less toxic than in the form of deoxycholate complex, from the point of view of acute toxicity.

 In the form of liposomes, Amphotericin B is 10 to 13 times less toxic than in the form of a DOC complex.

2. PHARMACOLOGY
2.1 In vitro antifungal activity
2.1.1. Models of extra cellular infections
The direct antifungal activity of Amphotericin B as a DOC complex (Fungizone, Jiposomes and microparticles) was determined on Candida tropicalis using the microparticles prepared by the laboratory method (EP 270,460).

The direct antifungal activity of Amphotericin B in the form of microparticles prepared according to the previous pilot and freeze-dried was compared to that of Fungizont on various yeasts and fungi in the laboratories of the Institute of Tropical Medicine in
Antwerp.

2.1.1.1 Direct activity against Candida tropicalis Protocol
The direct antifungal activity of Amphotericin B in the form of Fungizonei) of microparticles or liposomes is measured on the growth of Candida tropicalis in RPMI medium containing 20% fetal vecal serum. Inhibition of growth after 16 h is observed at increasing concentrations of Amphotericin B (14 concentrations ranging from 0.012 to 100 nM> .The minimum inhibitory concentration (MIC) is the lowest concentration that is not observed at all. growth of
Candida.

Results
Amphotericin B MIC in the form of microparticles or in the form of Fungizone is identical (Table 4). In contrast, Amphotheratin B encapsulated in liposomes composed of phosphatidylcholine, cholesterol and stearylamine in a molar ratio of 4: 3: 1 is 8-fold less active than free amphotericin.

Table 4: MIC Amphotericin B in Fungizone Form of
liposomes or microparticles, with respect to Candida
tropicalis

<tb><SEP> Formulation <SEP> MIC <SEP> (pM) <SEP>
<tb> Complex <SEP> DOC <SEP> (Fungizone #) <SEP><SEP> 0.2
<tb> Liposomes <SEP><SEP> 1.56 <SEP>
<tb> Microparticles <SEP> 0.2
<Tb>
2.1.1.2 Direct activity with respect to various yeasts and
mushrooms
2.1.1.2.1 Protocol
Yeast Material
Candida albicans (10)
Candida lusitaniae (9)
Candida tropicalis (10)
Torulopsis glabrata (10)
Candida krusei (10)
Candida parapsilosis 110)
Candida guilliermondii (10)
Candida (pseudotropicalis) kefyr (8)
Cryptococcus neoformans (45) Sporothrix schenckii:
20 strains
17 patients from Peru
1 South African (generalized sporotrichosis)
2 from Brazil
Culture centre
Yeast morphology Agar (Difco) pH 7.3 (phosphate buffer
Mother solution of antifungals in distilled water.

Final concentrations in
0.016; 0.032; 0.063; 0.125; 0.25; 0.5; 1; 2; 5; 10
inoculum
Yeast suspension or yeast phase of S. schenckii
About 107CFU in distilled water (= standard N 2 Mc Farland)
seeding
The Petri dishes are seeded using a multipoint inoculator (Denley - Tech Limited).

 The inoculum being about 104CFU.

Incubation
For yeasts: 25 C
S. schenckii: 370C
Reading
After 48 hours, the growth is compared to those of control boxes without antifungals.

 The minimum inhibitory concentration (MIC) in μg / ml is that at which no growth is visible to the naked eye for 48 hours.

2.1.1.2.2 Results: the results are included in the
table 5
Table 5: Activity of Amphotericin B as a DOC Complex
(Fungizone R) and wicroparticles with respect to various
yeasts and mushrooms
MIC in g / ml after 48h
CANDIDA ALBICANS (CA) F * L4 F L4 '1 66259 stonatis Rwanda AIDS 0.25 0.25 0.25 0.5 2 66197 vaginitis B 0.25 0.25 0.5 0.5 3 66010 vaginitis Spanje sero A 0.5 0.25 0.5 1 4 66011 vaginitis Spanje sero B 0.5 0.25 0.5 1 5 66063 worldspoellag B 0.25 0.25 0.25 1 6 66172 mondspoeling B 0.25 0.25 0.25 1 7 66185 aondspoeling B? >0.5> 0.5 2 5 8 64251? B 0.125 0.25 0.125 0.5 9 39971 baby - Iumbaal vocht B 0.25 0.25 0.25 0.5 10 66188 vaginitis 0.25 0.25 0.25 0.5
CANDIDA LUSITANLAE (CL) 1 57996 nagel B 0.5 1 0.5 1 2 61700 leukemia B>2>2>10> 10 3 64180 nagel B>2>2>10> 10 4 65996 world B 0.5 1 5 65911 world B 0.5 1 6 59819 world B 0.5 1 0.5 1 7 60758 hemocultuur B>2>2>10> 10 9 66274 nagel B 0.5 1 10 66297 risicopatient B 0.5 1
* = FungizoneR R L4, L4 'and L5 = batches of Amphotericin B microparticles
lyophilized prepared according to the invention under different conditions.

Table 5: (continued 1)
MIC in g / ml after 48h
CANDIDA TROPICALIS (CT) F L4 F L4 '1 66013 Spanje urine 0.25 0.25 0.25 0.5 2 66070 Prond B 0.5 0.25 0.5 1 3 66003 FIV + 0.5 world 0.25 0.5 1 4 66189 vaginitis B 0.25 0.25 0.5 1 5 66222 hemocultuur>0.5> 0.5 2 5 6 66263 stomatitis Rwanda 0.5 0.5 0.5 2 7 66234 vaginits B 0.5 0.5 0.5 2 8 64485 lumbaal vocht Kinshasa 0.5 0.5 0.5 2 9 65940 vaginitis Spanje 0.5 0.5 0.5 2 10 65444 tongveger B 0.5> 0.5 0.5 2
TORULOPSIS GLABRATA (TG) i 61764 sputum vrow pediatrics B
(Leuven) 0.5> 0.5 0.5 1 2 61770 long B tLeuven) 0.25 0.5 0.25 0.25 3 63748 vaginitis Spanje (Martine2) 0.5 0.5 4 63865 Infection na verwijderen
van beupprothese NL 0.5 0.5 5 64109 vaginitis B 0.5 0.5 0.5 1 6 64542 hewocultuur B 0.5> 0.5 0.5 1 7 66224 mondveger B 0.5 0.5 0.5 1 8 66305 longiecrety B 0.5 0.5 9 63228 mondspoeling B? 0.5 0.5 10 49124 vaginitis (Dr. Zaouk
<Dr Swinne) 0.5 0.5
Table 5: (continued 2)
MIC in g / ml after 48 hours
CANDIDA KRUSEI (CK) F L4 F L4 '1 66175 (ayeloide leukemia) 0.5> 0.5 0.5 1
hemocultuur B 2 65993 hemocultuur B>0.5> 0.5 1 2 3 65934 vaginitis Spanje 0.5> 0.5 0.5 1 4 65935 vaginitis Spanje 0.5> 0.5 0.5 1 5 63045 expectoration B 0.5> 0.5 0.5 1 6 64714 hemocultuur B 0.5> 0.5 1 2 7 62928 urine B>0.5> 0.5 0.5 1 8 57994 urine B 0.5> 0.5 1 1 9 58080 keelveger B 0.5> 0.5 1 1 10 60699 urine B>0.5> 0.5 1 2
CANDIDA PARAPSILOSIS (CP) 1 66326 etter gehoorgang B 0.125 0.5 2 65995 hemocultuur 0.5 1 3 64708 sputum 0.5 1 4 63447 nagel B 0.5 1 5 63554 osteitis Rwanda 0.5 1 6 63267 ooretter B 0.5 1 7 63210 nagel 0.5 1 8 62746 hemocultuur Rwanda 0.5 1 9 62675 nagel B 0.5 1 10 63263 teen B 0.5 1
Table 5: (continued 3)
MIC in g / ml after 48h
CANDIDA GUILLIERMONDII (Cc) F L4 1 65994 nagel 8 0.5 0.5 2 66215 mondspoeling BIV + 0.5 0.5 3 66193 hemocultuur B 0.5 0.5 4 66176 hemocultuur myeloide
leukemia B 0.25 0.5 5 62001 nagel B 0.5 0.5 6 60664 huid B 0.5 0.5 7 66071 teen schilfers B 1 2 8 64763 huidschilfers B 0.5 0.5 9 11013 hand B 0.25 0.5 10 62652 nekwonde B 0.5 0.5
CANDIDA (PSEUDOTROPICALIS) KEFYR (CP) 1 66058 vaginitis 0.5 1 2 63145 vaginitis 0.5 1 3 58114 sputum B 0.5 1 4 58130 zwarte tong, post AB B 0.5 1 5 34441 dierenziektenbestrijding
Torhout B 0.5 2 6 57676 koe B 0.5 1 7 39219 urine B 0.5 1 8 66199 vaginitis B 0.5 1
Table 5: (continued 4)
MIC in g / ml after 48h
SPOROTHRIX SCHENCKII F L4 L4 'IV 63582>10> 10
RV 63583 5> 10
RV 63584 0.5 1
RV 63585 0.5 1
RV 63586>10> 10
RV 63587 0.5 2
RV 63588 0.5 1
RV 63590>10> 10
RV 63592>10> 10
RV 63593 0.5 1
RV 63594 0.5 5
RV 63595 0.5 5
RV 63596 1 0.5
RV 63597 5> 10
RV 63603 0.5 1
RV 63604 0.5 1
RV 63605 2 5
RV 66551 2 5
RV 66553 0.5 2
RV 66557 0.5 2
RV 14980>10> 10
RV 66552 10> 10 RV 66558 0.25 1
RV 66559 0.25 2
RV 66600 <0.125 0.25
RV 66601 0.25 1
RV 66603>10> 10
RV 66604 10> 10
RV 66606 0.25 1
RV 66607 <0.125 1 Table 5: (Continued 5)
MIC in g / ml after 48h
F L4 L4 '
RV 66609 0.25 5
RV 66610 1> 10 IV 66611>10> 10
RV 66612 c 0.125 1
RV 66618 0.5 2
RV 66621 0.25 2
RV 66627 5> 10
RV 66629 1 5
RV 66631 2 0.5
RV 66632 2> 10
RV 66636 10> 10
RV 66637 2 2
RV 66639 <0.125 <0.125
RV 66643 <0.125 5
RV 66646 2 5
RV 66651>10> 10
RV 14970 10> 10
RV 14955>10> 10
RV 52146 10> 10
RV 63602
RV 52147
Table 5: (continued 6)
MIC in g / ml after 48h
CRYPTOCOCCUS NEOFORMANS F L4 F L5 1 45979 8 - 0.0063 0.5 2 45977 C - a 0.0063 0.5 3 45978 C - K 0.0063 0.5 4 45980 D - &alpha; 0.0063 0.5 5 45981 D - a 0.0063 0.5 6 66216 for Europa AIDS 0.25 0.5 7 66513 var neoformans Hexico
AIDS 0.25 0.5 8 66534 "0.25 0.5 9 66440 var grattii Mexico
AIDS 0.25 0.5 10 66536 "0.'5 Il 55446 A omgeving Afrika 0.25 0.25 12 55447 A" 0.25 0.25 13 55448 A "0.5 0.5 14 55449 A" 0.25 0.25 15 55450 A "0.25 0.25 16 5637 @ A omgeving Maleisië 0.5 0.5 17 56375 A "0.5 0.5 18 56376 A" 0.5 0.5 19 56377 A "0.25 0.25 20 56378 A" 0.5 0.5 21 53960 A for Afrika 0.5 0.5 22 54034 A "0.5 0.5 23 54138 A" 0.25 0.25 24 54424 A "0.5 0.5 25 54427 A "0.25 0.25 26 56056 A for Peru 0.25 0.25 27 53806 A for Europa 0.25 0.25 28 54830 A" 0.5 0.5 29 55032 B var. gattii Maleisie
para 0.125 0.1'5
Table 5: (continued 7)
MIC in g / ml after 48 h
F L5 30 55033 B n 0.125 0.125 31 55034 B "0.125 0.125 32 55035 B l 0.125 0.125 33 3463 B" Zaire para 0.125 0.125 34 20185 B 2 "0.125 0.125 35 48370 B" China for 0.125 0.125 36 52644 C 2t USA for 0.125 0.125 37 52645 C "" 0.125 0.125 38 57477 D para Europa 0.125 0.125 39 51276 D "0.125 0.125 40 36214 D" 0.125 0.125 41 34802 D t 0.125 0.125 42 62692 Prim. huid 0.125 0.125 43 32908 D sapro>1> 1 44 32910 D sapro>1> 1 45 33370 A / D for Europa 0.125 0.125
The results indicate that the MICs obtained with MP lot 4 and lot 4 'are in general 1 to 4 times higher than those obtained with the
Fungizone 1 on Candida Albicans, C. Lusitano, C. Tropicalis,
Torulopsis Clabrata. C. Krusei, C.Parapsilcsis, C.Guilliermondii and
C.Kefyr as well as on Sporothrix Schenckii. On Cryptococcus neoformans, the MPs (lot 4 and lot 5) are as active as Pungizone R on 35 of the 45 strains, 2 times less active on 5 of the 45 strains and much less active on the strains BX, C- &alpha;, D -&alpha; Ca and Ba.

2.1.2 Intracellular infection model
The antifungal activity of Amphotericin B with respect to intracellular infections is determined in vitro using the J-774-G8 macrophage line infected with Candida Tropicalis as a model.

 The activity of Amphotericin B in the form of microparticles is compared with that of Amphotericin B in the form of DOC complex (Fungizone #) as well as that of Amphotericin entrapped in multilamellar liposomes.

2.1.2.1 Protocol
J-77448 macrophages cultured in RPMI medium containing 10 mM MES pH7.2 supplemented with 10% delipidated human plasma are infected at the rate of 1 Candida tropicalis by 10 macrophages.

The action of drugs is measured in boxes
A 24-well LIMBRO containing 105 infected macrophages adhering to glass slides, and the range of concentrations tested ranged from 0.048 μM to 100 μM.

The percentage of infected cells is determined after 16 hours of coculture. On microscope slides, the total number of macrophages (NT) and the number of marcrophages infected with Candida (Ni) are counted after MAY-GRUNA'ALD-GIEMSA staining. The percentage of infection is the ratio Ni / NT (expressed in%)
The concentration, expressed in pM, which reduces by 50%
Intracellular infection (IC50) is then determined.

2.1.2.2 Results
The results, shown in Table 6, show that the microparticles are twice as active as Fungizone (E) and 8 times more active than liposomes with respect to intracellular infection.

Table 6: Activity of Amphotericin B in the form of Fungizone,
Jiposomes or microparticles with respect to infections
intracellular (J-774G8 macrophages infected with Candida
tropicalis)

<tb><SEP> Formulation <SEP> IC50 <SEP> (M)
<tb> Complex <SEP> DOC <SEP> (Fungizone <SEP> 0.2
<tb> Liposomes <SEP> 0.8
<tb> Microparticles <SEP> 0.1
<Tb>
These studies of the activity of Amphotericin B microparticles are very important to ensure the ability of these microparticles to penetrate phagocytic cells such as polymorphonuclear and macrophages.

 The results in Table 6 illustrate that Fungizone is as active on intracellular forms as on extracellular forms whereas microparticles such as liposamates are more active on intracellular forms than on extracellular forms.

The intracellular penetration of the microparticles inside the macrophages could also be confirmed by microscopic observations.

3. CONCLUSIONS
The toxicity of Amphotericin B can be significantly reduced by its incorporation into liposomes. These liposomes retain their activity on Candida in vitro and in vivo in mice.

Several dozen patients have already been treated with these amphotericin B liposomes in the United States in the LOPEZ-BERENSTEIN team and
Belgium at the Bordet Institute (1,2,5).

 These clinical trials have confirmed the best tolerance and toxicity of this dosage form and have provided significant evidence of increased activity.

 The ER-CELLTARG research group has developed microparticles consisting of Amphotericin B and phosphatidylcholine.

These microparticles are distinguished from liposomes by their lipid / amphotericin molar ratio close to 1, unlike liposomes where this ratio varies from 90 to 70. These microparticles also do not have the microscopic structure of liposomes.

 The haemolysing effect of the microparticles is reduced at least 50-fold compared to Amphotericin in the form of Fungizone and the cellular toxicity studied in vitro on macrophages showed a reduced toxicity of 33 times compared with Fungizone and 8 times compared to liposomes.

 The acute toxicity of these microparticles after intravenous administration expressed as LD50 is 5 to 7 times lower than Amphotericin B and 2 times higher than that of liposomes.

 The antifungal activity of Amphotericin B in the form of microparticles has been determined in vitro and is generally similar to that of Fungizone and probably higher than that of Amphotericin B in the form of liposomes.

 In addition, in a study carried out on cultured macrophages infected with Candida tropicalis, the ratios of the concentrations of the cytotoxic activities / intracellular antifungal activities for the three forms of amphotericin B were 20 for free Amphotericin B (Fungizone). , 21 for liposomes and 1320 for microparticles. The latter form therefore appears in this case much less cytotoxic while remaining more active than the reference products.

 The results recorded in vitro show a very active capture of microparticles by macrophages, which could explain the high activity of these microparticles loaded with Amphotericin B are found concentrated in the same intracellular compartment probably lysosomal.

 BIBLIOGRAPHIES 1. LOPEZ-BERENSTEIN G. and JULIANO R.L.

Applications of liposomes to the delivery of antifungal agents
Liposomes. From Biophysics to therapeutics.

2. SCULIER J.P., COUNE A., BRASSINNE C., LADURON C., HOLLAERT C.

First trials of intravenous use of ultrasonic liposome
human therapeutics
Medicine / Science 3 (1987) 41-44 3. TAKAYAMA et al.

 Clin. chem. Acta 79 (1977) 93-98 4. MOSMANN C.T.

J. Immunol. methods 65 (1983) 55-63 5. SHIRKODA A., LOPEZ-BERENSTEIN G., HOLBERT JM, LUNA MA,
Hepatosplenic fungal infection: CT and pathologic evaluation after
treatment with liposomal AMB.

Radiology 159 (1986) 349-353 6. Analytical Chemistry Vol. 21 n0 4 April 1949 page 527-528, William
Bartholomey

Claims (14)

 1. Process for the preparation of microcrystalline lipid microparticles of a water-insoluble substance having an affinity for phospholipids, and of at least one phospholipid, stable microparticles suspended in an aqueous solution, characterized in that a) dissolves said substance and said phospholipid (s) in one or  first organic solvent (s) of said substance and said one or more  phospholipids, and a third solvent of said first solvent (s) and  water, (b) mixing the resulting solution of the substance and the at least one  phospholipids to an aqueous solution in an amount less than  the one from which one observes either a demixtion or a  insolubilization especially in the form of a precipitate of said substance  and phospholipids, to obtain a homogeneous phase  clear, c) The said first and third solvents are evaporated while maintaining the solution  in a homogeneous phase to recover an aqueous solution containing the  microparticles in the form of microsuspensions.  2. Method according to claim 1, characterized in that in step c) the evaporation of said first solvent is before the total evaporation of the third solvent.  3. Process according to claim 1 or 2, characterized in that the evaporation of the organic solvents and the recovery of the aqueous solution are carried out using an ascending film evaporator type installation, the evaporation being carried out under reduced pressure and the aqueous solution being entrained by the vapors of organic solvents before being separated by condensation of the organic phase  4. Process according to claim 3, characterized in that the recovered aqueous solution containing the microparticles is subjected to a second evaporation cycle.  5. Method according to one of the preceding claims, characterized in that the molar ratio between the phospholipid or phospholipids and said substance is between 0.1 and 10.  6. Method according to claim 5, characterized in that the molar ratio is between 0.8 and 2, preferably close to 1.  7. Method according to one of the preceding claims, characterized in that the phospholipid is phosphatidylcholine.  8. Method according to one of the preceding claims, characterized in that said (s) first (s) organic solvent (s) of the phospholipid solution and said substance are selected from organic solvents common intermediate polarity such as chloroform, dichloromethane, methanol and ethanol.  9. Method according to one of the preceding claims, characterized in that the third solvent is selected from methanol, ethanol, acetone and THF.  10. Method according to one of the preceding claims, characterized in that the organic solvents consist of a mixture of chloroform and methanol, the evaporation of chloroform by evaporation of the azeotrope chloroform-methanol-water before evaporation. remaining methanol.  He. Method according to one of the preceding claims, characterized in that the solution of said substance and phospholipids also comprises one or more sterols such as cholesterol or ergosterol.  12. Method according to one of the preceding claims, characterized in that the aqueous solution is a saline solution.  13. Process according to the preceding claim, characterized in that the aqueous solution is a 0.9% NaCl solution.  14. Method according to one of the preceding claims, characterized in that said substance is selected from anti-mycotic agents, macrolides, polyene.  15. Method according to the preceding claim, characterized in that said substance is chosen from nistatin, amphotericin B and their antifungal derivatives.  16. microparticle microparticles stable microcrystalline suspension in an aqueous solution comprising a substance insoluble in water and at least one phospholipid, obtained by a method according to one of the preceding claims.