CH637825A5 - VEHICLE COMPOSED OF A PHOSPHOLIPID AND A NON-MISCIBLE LIPID, INTENDED TO RELEASE A XENOBIOTIC SUBSTANCE IN THE ORGANISM OF A MAMMAL. - Google Patents

Description

The present invention relates to the delivery to the body of a mammal, and the release into the body of a mammal, of xenobiotic substances. The invention relates more particularly to vehicles for transporting xenobiotic substances in the form of transportable microreservoirs, to a process for forming the microreservoirs.

In many different cases and in many varied circumstances, it is desirable to introduce into the organism of a mammal substances endowed with pharmacological activity which are foreign to the host, these substances being qualified hereinafter as xenobiotic substances. These xenobiotic substances include, without limitation, drugs, diagnostic agents, blood substitutes, biological compounds of endogenous origin, hormones, immunological adjuvants, etc.

In the administration of any xenobiotic substance, a certain degree of specificity must be attained, and the specificity requires that the xenobiotic substance achieves its goal selectively and can be influenced. The lack of specificity linked to the use of numerous xenobiotic substances can therefore deprive these substances of an appreciable part, if not essentially of all, of their potential effectiveness with regard to obtaining the results that are expected. of their use. For example, a chemotherapeutic drug that cannot be retained by the blood plasma for a period sufficient for a significant amount of that drug to reach the target tissue, or an orally administered drug that is intended for blood flow, but which cannot cross the gastrointestinal tract, does not have the degree of specificity which could make it very effective. Thus, in the absence of the desired specificity, a xenobiotic agent cannot essentially, or can only to a limited degree, exercise the pharmacodynamic activities necessary for the exercise of its total potential. Among these 55 pharmacodynamic activities, the kinetics of plasma, the distribution in tissues, the degree of toxicity, the levels of therapeutic substances in vivo, the solubility of xenobiotic agents normally incompatible with other pharmaceutical formulations can be indicated. metabolic activation of xenobiotic agents. “I We have seen in the past that it was desirable to modify and limit, in a beneficial sense, the specificity or pharmacodynamics of many xenobiotic substances whose desirable properties have been recognized. A known approach for limiting the specificity of drugs involves the use of implantation devices established>, normally by surgery, in the organ or near the organ to which the drug is to be delivered. Usually, these implants are formed of a covalent matrix containing the drug to be delivered. The

3

637,825

matrices used can be water-soluble (for example carboxymethylcellulose or polyvinyl alcohol), they can swell with water (for example hydrogels or gelatin), they can be hydrolytic polymers (for example polylactic acids , polyglycolic acids or poly-α-amino acids) or they may be non-hydrolytic polymers (for example a rubber of the organopolysiloxane type). Generally, although these implants can limit the speed at which the drug they contain can be delivered by diffusion or hydrolysis, the influence they can exert on the pharmacodynamics of the drug released is low, or even zero.

One of the most recent approaches that have been suggested to modify and limit the pharmacodynamic activity of xenobiotic agents is the use of a vehicle capable of delivering and releasably adjusting xenobiotic agents at the site of action. in the host organism to which they are administered. The use of liposomes as vehicles has been proposed for this purpose [see G. Gregoriadis, "The Carrier Potential of Liposomes in Biology and Medicines", in "The New England Journal of Medicine", vol. 295, N ° 13, pp. 704-710 (September 23, 1976) and N ° 14, pp. 765-769 (September 30, 1976)]. These known liposomes are formed from phos-pholipids and in particular from phosphatidylcholine in combination with other lipids such as cholesterol, diketyl phosphate, stearylamine and other phospholipids such as phosphatidyl-serine to which phosphatidylcholine is easily miscible. Liposomes are characterized by the miscibility of the components of the vehicle, which means that the phase separation can only be weak or zero between these components. Furthermore, these vehicles lack a high degree of stability, which is mainly due to the oxidation of the phospholipid components and / or to their thermal instability, in particular when they are divided by ultrasound into small particles, a diameter of around 250 Å, for example. In addition, among the liposomes, those which carry a negative charge due to the presence of diketyl phosphate, phosphatidylserine or other negatively charged components, agglomerate in the presence of divalent cations. Finally, when liposomes are injected intravenously, they preferentially concentrate in the liver and spleen, which is at least partly due to their large diameter. Consequently, although the known liposomes used as vehicles are compatible with a certain number of drugs and are in biological compatibility with the organism of a mammal, their natural instability, their tendency to agglomerate notably and their relatively large diameter attenuate their ability to be used as an acceptable means of drug delivery.

As a result of the ever-increasing role played by xenobiotic agents in the treatment of cancer, diabetes, arthritis, inherited metabolic disorders, diseases related to metal storage, etc., the need for improved vehicles is increasing in the preventive study of diseases such as anaplasmosis and virus infections and in the study of biological mechanisms. The desirability of these sophisticated systems of administration is therefore evident.

Consequently, the main object of the present invention is to offer improved vehicles for the administration of xenobiotic agents in the form of microscopic reservoirs or microreservoirs capable of circulating in the organism of the host. The vehicles in question, intended to deliver xenobiotic agents, must be compatible with a wide variety of xenobiotic agents comprising hydrophobic, hydrophilic or both hydrophobic and hydrophilic compounds, which are also non-toxic and biocompatible with the host organism . These vehicles must be stable for extended periods of storage as well as during their use in the host's organism and they must be favorable to various administration techniques, for example oral, intravenous, intramuscular, intraperitoneal, subcutaneously, topically and by inhalation.

Another object of the present invention is to find vehicles for the administration of xenobiotic substances which are capable of modifying and influencing in a predetermined and beneficial way the pharmacodynamics of the xenobiotic substance delivered and released in the organism. the host. Among the pharmacodynamic activities which undergo this beneficial modification and influence, mention is made of plasma kinetics, distribution in tissues, toxicity, oral absorption, chemotherapeutic ability, metabolism, etc.

Another important objective of the present invention is to find a method of forming vehicles allowing the administration of xenobiotic substances, in the form of microreservoirs capable of circulating in the organism of a mammal to deliver the xenobiotic substance there to a predetermined site. , by effecting a predetermined beneficial change in the pharmacodynamics of the xenobiotic substance.

According to one of its aspects, the invention provides a vehicle for administration biocompatible with a mammal which receives it, for delivering and releasing in the organism of the host a xenobiotic substance whose pharmacodynamics is modified in a beneficial way by the use of the vehicle; this vehicle is characterized by the fact that it is in the form of microreservoirs containing the xenobiotic substance and formed of a phospholipid constituent and of a lipid constituent immiscible with the phospholipid and stable in a physiologically compatible liquid and essentially immiscible with this liquid.

According to another of its aspects, the invention provides a method of forming a vehicle intended to deliver into the organism of a mammal a xenobiotic substance whose pharmacodynamics is modified and limited in a predetermined manner, which method consists in forming microscopic reservoirs of a phospholipid constituent and of a lipid constituent immiscible with the phospholipid which is stable in a physiologically compatible liquid and essentially immiscible with this liquid, and for incorporating into the microreservoirs the xenobiotic substance to be delivered.

The characteristics and advantages of the present invention emerge from the detailed description which follows, made with reference to the appended drawings in which:

fig. 1 is an overall diagram illustrating a process for preparing the vehicles of the invention, intended to deliver a xenobiotic substance;

fig. 2 is a partial diagram illustrating a variant of the method of FIG. 1;

fig. 3 is a schematic representation on a very enlarged scale of a microreservoir of the invention, in the vesicular form;

fig. 4 is a very enlarged schematic representation of a microreservoir of the invention, in a non-vesicular form;

fig. 5 is a very enlarged schematic representation of a vehicle intended to deliver a medicament, of the type described in the prior art;

fig. 6 graphically illustrates the in vitro stability of the microreservoirs in contrast to that of liposomes, the in vitro stability being expressed by the variation as a function of time of the optical density of the microreservoirs and of the liposomes;

fig. 7 graphically illustrates the in vivo stability of the microreservoirs, by the curve of variation of the quantity of cholesteryl oleate (cholesterol ester) remaining in the blood plasma of a rat, as a function of time;

fig. 8 shows an elution profile of microreservoirs containing daunomycin, plotted as a function of the phosphatidylcholine, cholesteryl oleate and daunomycin concentrations in a series of chromatographic fractions;

fig. 9 is a schematic representation illustrating the distribution of a typical drug of the anthracycline class shortly after the injection of this drug in the free form, showing the small amount of the drug which remains in the blood stream "to reach the targeted site;

5

10

15

20

25

30

35

40

45

50

55

60

65

637,825

4

fig. 10 illustrates the degree to which the circulating microreservoirs are able to modify in a beneficial direction the kinetics of daunomycin in the plasma, the graph representing curves of variation of the quantity of daunomycin remaining in the blood stream as a function of the time elapsed after the injection of the drug contained in the microreservoirs and depending on the amount of free daunomycin;

fig. 11 represents a series of curves giving the number of survivors of a group of mice as a function of the time elapsed after the injection at four different doses of microreservoirs, of daunomycin in the microreservoirs and of daunomycin in the free form, the curves illustrating the ability of microreservoirs acting as delivery vehicles to reduce the toxicity of daunomycin;

fig. 12 represents a series of curves of variation of the number of survivors of a group of mice treated by injection of tumor cells as a function of the time elapsed after the injection at two different doses of daunomycin in the microreservoirs and in the free form, these curves illustrating the beneficial modification of the chemotherapy of daunomycin delivered into and released by the microreservoirs;

fig. 13 shows a series of variation curves, as a function of time, of the emission rates of daunomycin from microreservoirs of compositions containing glycerol trioleate as an immiscible constituent of phospholipid and various quantities of cholesterol as an agent influencing the rate of release;

fig. 14 shows a series of curves as a function of time of the emission rates of daunomycin from microreservoirs of compositions containing cholesteryl oleate as an immiscible constituent of phospholipid and various quantities of cholesterol as an agent influencing the rate of release;

the flg. 15 is an elution profile of microreservoirs containing the substance AD 32, as a function of the concentrations of phospholipid, of cholesteryl oleate and of AD 32 in a series of chromatographed fractions;

fig. 16 is a variation curve of the speed of emission of AD 32 from vesicular microreservoirs, showing the effect which results from the use of a small amount of phosphatidic acid in the phospholipid constituent of the composition of the microreservoirs;

fig. 17 illustrates the degree to which the circulating microreservoirs are capable of modifying in a beneficial direction the kinetics in the plasma of the substance AD 32 in microreservoirs and in the free form at a dose of 15 mg / kg;

fig. 18 is a series of graphical representations of the number of survivors of a group of mice having received tumor cells, as a function of the time after the injection at four different doses of the substance AD 32 into the microreservoirs, the curves showing the chemotherapeutic ability of substance AD 32 delivered by microreservoirs;

fig. 19 is an elution profile of microreservoirs containing imidocarb, as a function of the concentrations of phospholipid, of cholesteryl oleate and of imidocarb in a series of chromatographed fractions;

fig. 20 and 21 illustrate the degree to which the circulating microreservoirs are capable of modifying in a beneficial direction the kinetics of imidocarb in plasma at two dose rates, the illustrations being in the form of curves of variation of the quantity of imi- docarb remaining in the bloodstream and ratios of imidocarb to microreservoirs as a function of time after injection of the drug into the microreservoirs and as a function of free imidocarb, at the respective doses of 5 and 4.4 mg / kg ;

fig. 22 is an elution profile of microreservoirs containing estradiol undecanoate, as a function of the phosphatidylcholine and estradiol undecanoate concentrations in a series of chromatographed fractions;

fig. 23 is a histogram showing the concentrations in the blood plasma, at three different times, of estradiol undecanoate administered orally in the form of an ethanolic solution and in microreservoirs;

fig. 24 is an elution profile of vesicular and non-vesicular microreservoirs containing estradiol undecanoate, and

. fig. 25 is a graphic representation of the emission rate of estradiol undecanoate from vesicular and non-vesicular microreservoirs.

The vehicles according to the invention, intended to deliver xenobiotic substances, are formed from a mixture of two or more lipids which are essentially immiscible. More particularly, the delivery systems are formed from compositions comprising a phospholipid component and a lipid component immiscible with the phospholipid, which may be a cholesterol ester or a triglyceride or a mixture of cholesterol esters or triglycerides or of these two types of constituents. The phospholipid component may contain more than one phospholipid; the composition of the microreservoir can also contain a constituent modifying the bonds and / or a constituent influencing the rate of release. Examples of phospholipids which are suitable for the implementation of the invention are phosphatidylcholine, phosphatidylseine, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid and sphingomyelin. It is also possible to use mixtures of two or more of these phospholipids. Among these compounds, phosphatidylcholine is appreciated, alone or as a mixture with other phospholipids.

Phosphatidylcholine is a term applied to compounds that are esters of fatty acids with glycerophosphoric acid and choline. Thus, phosphatidylcholine can be represented by the following formula:

FA — CH2 O

I II

FA '- CHCH2 - O - P - O - (CH2) 2 - N - (CH3) 3 I

o-

in which FA and FA 'are fatty acid residues. The fatty acids used to form these esters can be saturated or unsaturated and can contain about 12 to 20 carbon atoms. These fatty acids are, without limitation, palmitic, stearic, oleic acids, etc. The phosphatidylcholine used in vehicles delivering an active substance according to the invention can be isolated from egg yolk by the method described by Litman [“Biochemistry”, 12: 2545 (1973)] or other natural sources such as beans or can be synthesized by a suitable process such as that described by Robles and Van der Berg in "Biochim. Biophys. Acta ”, 187: 520 (1969).

In the detailed description of the invention given below, the phospholipid constituent of the vehicles delivering a substance is illustrated, for reasons of convenience, by phosphatidylcholine, term by which we mean the esters which correspond to the general formula given below -above. In some of the microreservoir formulations, small amounts of phosphatidic acid are incorporated into the phospholipid constituents to improve the binding of the xenobiotic substance to the microreservoirs. It should also be noted that other phospholipids, such as those which have been mentioned, which satisfy the conditions indicated below with regard to chemical and physical properties, can be used in place of phosphatidylcholine.

The cholesterol or triglyceride ester component used as a component of the vehicle must be essentially immiscible with the phospholipid component, just as it must be essentially insoluble in a physiologically compatible liquid such as a saline solution in physiological equilibrium, containing NaCl or KCl. The cholesterol or triglyceride ester component must also be apolar or nonpolar to the point that it does not form a monolayer, and it must be present at a concentration chosen so that it is essentially immiscible with the double phospholipid layer. .

5

10

15

20

25

30

35

40

45

50

55

60

65

5

637,825

The cholesterol of formula C27H15OH is a secondary monounsaturated alcohol, quick to form esters with saturated and unsaturated fatty acids such as oleic acid, stearic acid,

palmitic acid, etc. In general, fatty acids having 10 to 18 carbon atoms are preferable. Suitable methods of forming these cholesterol esters include condensing a fatty acid chloride with cholesterol and isolating these esters from natural sources.

In choosing the fatty acid for the formation of the cholesterol ester, it is preferred that it is an acid having a low degree of unsaturation (for example no more than about two double bonds per molecule fatty acid). In general, the esters formed from fatty acids having higher degrees of unsaturation give complexes intended to distribute xenobiotic substances which have greater stability than those which are formed from fatty acids with higher unsaturation.

The triglycerides suitable for use in accordance with the invention are fatty acid esters of glycerol which correspond to the general formula: q

II

HjC-O-C-RJ

I

O

II

hc-o-c-r2

o

11

h2c-o-c-r3

in which the variables R !, R2 and R3 of the fatty acids forming the esters can have 10 to 18 carbon atoms. Examples of these fatty acids include palmitic, stearic, myristic, oleic and linoleic acids. These triglycerides are advantageously prepared by condensation of the fatty acid chloride with the glycerol. They can also be isolated from natural sources.

In the general description which follows, it is assumed for convenience that the ester used is a cholesterol ester. Of course, one could also use a triglyceride, a mixture of cholesterol esters, a mixture of triglycerides or a mixture of one or more cholesterol esters with one or more triglycerides in the formation of microreservoirs used as delivery vehicles. The examples are reproduced in which a cholesterol ester or a triglyceride is used as a component immiscible with the phospholipid.

The vehicles of the invention used to deliver the xenobiotic substances are in the form of what are called microreservoirs in the present specification. These microreservoirs can take one of two forms, namely a vesicular form which has a small partitioned wall containing the medium used to form the microreservoir, or a non-vesicular form in which the cholesterol ester and / or the triglyceride are contained. in the phospholipid monolayer. As will be shown later, the ratio of phosphatidylcholine to the cholesterol ester determines which of these forms predominates in any synthetic process. The choice between these forms depends mainly on the nature of the xenobiotic substance to be delivered and released, on the pharmacodynamics of the desired xenobiotic substance and on the mode of administration.

Two different synthetic routes for the formation of microreservoirs as well as for the incorporation of the xenobiotic substance in these microreservoirs are illustrated schematically in FIGS. 1 and 2. The preparation method illustrated in fig. 1 is based on the use of ultrasound to form the microreservoirs constituting a popular process. In this process, the phosphatidylcholine and the cholesterol ester are mixed in an inert organic liquid which is a solvent for these two constituents at the same time, for example chloroform; and, if necessary, the xenobiotic substance is added to this solution as indicated by an arrow in dashed lines in FIG. 1. The solvent is then removed by evaporation under vacuum and a dry residual mixture remains which is then hydrated by the addition of a physiologically compatible liquid, for example an aqueous solution of NaCl or KCl of suitable concentration and a buffer. If the xenobiotic substance is not added to the initial mixture, it is added to the resulting liquid suspension before the formation of the microreservoirs.

In the formation of the liquid suspension, it is preferable to use amounts of the residual mixture equivalent to about 1-5% by weight of the physiologically compatible liquid. Although the amount of xenobiotic substance added to the composition containing the phospholipid and the cholesterol ester (or the triglyceride) may vary, it is preferable to incorporate up to about 2%, relative to the weight of residue, of the xenobiotic substance in microreservoirs.

As shown in fig. 1, the microreservoirs are then formed by ultrasonic treatment of the saline solution in a non-oxidizing atmosphere, for example in a nitrogen atmosphere. It has been noted that the formation of microreservoirs is more complete when the ultrasound treatment is carried out at a temperature which is equivalent to the melting point of the cholesterol ester or of the triglyceride used as a component immiscible with phospholipid or which slightly exceeds this temperature. . The ultrasonic treatment is carried out at a suitable level of energy and for a period sufficient for the microreservoirs to have the desired diameter. For example, it has been found sufficient to use a supply power of 120 W for 20 min. Alternatively, the ultrasonic treatment of the saline suspension can be replaced by the forced passage of this suspension through a small diameter orifice to form the microreservoirs.

As shown in fig. 2, a variant of the formation of microreservoirs in an aqueous medium, for example in saline solution, resides in the formation of a solution of the phospholipid, the cholesterol ester and the xenobiotic substance using an organic solvent miscible with l water and the subsequent introduction of this solution into a physiological aqueous salt solution. Alternatively, the xenobiotic substance can be added to the saline solution before the injection step. To illustrate this procedure, mention is made of the injection of an ethanolic solution of the lipid mixture into an aqueous solution with stirring; or else the slow injection of an ether solution of the lipid mixture into an aqueous compartment.

The cloudy liquid resulting from the formation of the microreservoirs in the aqueous suspension medium is then centrifuged to produce a clear phase and a cloudy upper phase, the latter containing larger non-vesicular microreservoirs, for example with a diameter between approximately 300 and around 1000 Â. The clear phase is chromatographed to produce a fraction of small non-vesicular microreservoirs (for example of diameter equal to approximately 250 Å) and of vesicular microreservoirs whose diameters range from approximately 190 to approximately 300 Å.

The distribution of the microreservoirs between the vesicular and non-vesicular forms is determined by the molar ratio of the phospholipid component to the immiscible component of the phospholipid. By using phosphatidylcholine and cholesterol oleate as examples illustrating a composition of microreservoirs, it can be demonstrated that the use of a proportion of approximately 67 to 97 mol% of phosphatidylcholine gives microreservoirs mainly vesicular, while the use more than about 50 mol% of cholesterol ester gives mainly non-vesicular microreservoirs.

The microreservoirs of the invention have remarkable structural properties due to the use of a mixture of the phospoholipid component and of a lipid component (cholesterol ester, triglyceride or their mixtures) which is essentially immiscible with phospholipid. This structure, in its vesicular and non-vesicular forms, is illustrated schematically in FIGS. 3 and 4. The insolubility of both phospholipids (represented by phosphatidylcho-

5

10

15

20

25

30

35

40

45

50

55

60

65

637,825

6

line in fig. 3 and 4) that of the immiscible lipid constituent (represented by a cholesterol ester) generates an organized structure of microreservoir according to which contact between the immiscible lipids and the aqueous medium is avoided or reduced to a minimum. This organization gives much more stability to the microreservoirs than one would expect from a vehicle formed in accordance with the indications given by Gregoriadis (loc. Cit.) In the prior art. This can be seen by comparing figs. 3 and 4 with fig. 5, which schematically represents the structure of a reservoir delivering a medicament according to Gregoriadis. It appears from fig. 5 that, when miscible lipids are used to form the liposome, the resulting structure is lamellar, which gives rise to an unstable realization probably due to the oxidation of phospholipids which destabilizes the lamellar organization, or to the crystallization of phospholipids if the temperature drops below their transition temperature.

The stable structure of the microreservoirs of the invention shown in FIGS. 3 and 4 can be attributed, at least in part, to a thermodynamic driving force which prevents exposure of the cholesterol ester or the triglyceride to the aqueous medium. As a result, the structural completeness of the microreservoirs of the invention is greatly improved. This in turn means that the ability of microreservoirs to influence and modify the pharmacodynamics of the xenobiotic substances incorporated therein is likewise favored compared to other distribution reservoirs. In addition, the lack of polarity of the microreservoirs, which is the consequence of the use of cholesterol esters or triglycerides in place of cholesterol and a similar substance, essentially prevents the aggregation of the microreservoirs. This is naturally a very advantageous characteristic, since such an aggregation would otherwise release the cholesterol esters or the triglycerides in an aqueous medium, and that this would result in a strong reduction in the free energy of the system. Consequently, the individual microreservoirs, which are similar in all respects to the circulating natural serum lipoproteins, are particularly well adapted to the circulation in the blood plasma of a mammal in the organism of which they are incorporated and to the retention in the plasma where they exert their maximum efficiency.

The great improvement in the stability in vitro of the microreservoirs of the invention, compared to that of the liposomes whose use has been recommended in the prior art, is illustrated in Example 1 and in FIG. 6.

Example 1:

Various mixtures of 60 µmol of egg yolk phosphatidylcholine, various quantities of cholesteryl oleate and 4 mg of daunamycin are evaporated to dryness under vacuum, starting from a chloroform solution of these constituents of the microreservoirs. 5 ml of 0.1 M KCl solution and 10 mmol of trihy-droxymethylamine are added to the residue obtained (buffer of pH equal to 8.0); the resulting suspension is then treated with ultrasound for 15 min at 51 ° C. under a nitrogen atmosphere with an input power of 120 W. The suspensions are then centrifuged at 100,000 x g for 1 h to remove any non-dispersed lipid.

Thin layer chromatograms of centrifuged liquors show no signs of degradation of phosphatidylcholine or daunomycin. 4 ml aliquots of each sample are then placed in stoppered cuvettes and the samples are incubated at 37 C. At various times, the optical densities of the two solutions are determined at 400 nm and 600 nm. The results thus obtained are reproduced graphically in FIG. 6 by the variation as a function of time of the ratio of these optical densities, namely A400 / A600. Since the two structures initially have the same molecular size, the A400 / A600 ratio gives an indication of any increase in particle diameter due to structural disorganization of the liposome.

Fig. 6 illustrates the considerable difference in stability between the microreservoirs of the invention and liposomes of similar size. While the absorbance of microreservoirs containing 33% cholesteryl oleate, as well as lower amounts of the cholesterol ester, is relatively stable, that of the liposome has increased rapidly since the start of the evaluation period and at the end by 10 d, the liposomes have degraded beyond any practical limit. As already assumed, the pronounced degradation of the liposomes is probably due to the interaction with the aqueous medium and the aggregation into larger particles, these two factors not existing in the case of microreservoirs because of their stability. structural and their lack of polarity.

In addition to their stability in vitro, the microreservoirs of the invention also have a very high stability in vivo compared to liposomes. This superiority is illustrated in Example 2 and in FIG. 7.

Example 2:

100 jimol of egg yolk phosphatidylcholine, 10 nmol of 14C-labeled cholesteryl oleate and 10 jimol of egg yolk phosphatidic acid are dissolved in chloroform and the solution is evaporated to dryness under vacuum. 5 ml of 0.154 M NaCl and 10 mmol of trihydroxymethylamine (pH 8.0) are added to the dry residue of the lipid mixture. The resulting suspension is treated with ultrasound for 15 min at 51 ° C. under a nitrogen atmosphere. The ultrasonically treated mixture is then chromatographed on a 2.5 x 40 cm Se-pharose 4B column. The individual fractions which coincide in the elution profile of the phospholipids and of the cholesteryl oleate are combined and then concentrated by ultrafiltration. 1.1 ml of these concentrated microreservoirs are injected into the tail vein of a rat. Plasma samples are taken at various intervals and the radioactivity associated with the microreservoirs contained in the plasma sample is measured. The results thus obtained are reproduced graphically in FIG. 7 as a function of the time elapsed after the injection.

Although the kinetics are complex, we can see from fig. 7 that, at equilibrium, the clearance of the microreservoirs has a period of 5.5 h. In contrast, ultrasound-treated liposomes with a similar negative charge have a plasma period of only 8 min [see R.L. Juliano and D. Stamp, “Biochem. Biophys. Res. Comm. " 63: 651 (1975)]. It seems logical to admit that the multiplication of the period by a factor close to 40 that is obtained in the case of microreservoirs can be attributed to an improvement in structural stability.

The association of a xenobiotic substance incorporated into the microreservoirs is illustrated in Example 3 and in FIG. 8.

Example 3:

2180 jimol of egg yolk phosphatidylcholine, 242 jimol of 14C carbon-labeled cholesteryl oleate (specific activity = 1.6 × 10 4 disintegrations / min (dpm / nmol)) and 36 mg of daunomycin are mixed together in chloroform. and the solution is evaporated to dryness under vacuum. The dry mixed residue is rehydrated by adding 110 ml of 0.1M KCl solution and 10 (tmol of trihydroxymethylamine (pH 8.0). This suspension is then treated with ultrasound using a Branson W- 185 for 15 min at 51 ° C. under a nitrogen atmosphere.

The liquid treated with ultrasound is then chromatographed on a Sepharose 4B column of 2.5 x 40 cm. The individual fractions resulting from the chromatography are then analyzed by the method of Gomori ["J. Lab. Clin. Med. ”, 27: 955 (1949)] to assay phosphatidylcholine, by radioactivity to assay for ceryl oleate and by fluorescence measurements to assay for daunomycin. The results of these analyzes are reproduced in FIG. 8, on which the analytical results of cholesteryl oleate, phosphatidylcholine and daunomycin were superimposed.

For the diameter of the vesicular microreservoirs, values of about 200 to 300 Å have been found, based on their elution profile on the

5

10

15

20

25

30

35

40

45

50

55

60

65

7

637,825

gel column which had been previously calibrated using ultrasound treated egg yolk phosphatidylcholine liposomes.

We can see from fig. 8 that the elution profiles of phosphatidylcholine (according to the phosphate analysis method), cholesteryl oleate (according to the radioactive analysis method) and daunomycin (d 'after the fluorescence analysis method) coincide, which indicates that the xenobiotic substance (daunomycin) has been associated with vesicular microreservoirs. Since daunomycin is a relatively hydrophobic drug, with certain hydrophilic characteristics, it is reasonable to assume that the site of drug localization resides in the lipid organization of the microreservoirs.

Among the pharmacodynamic activities of a xenobiotic substance which can be modified in order to make this substance more effective, mention is made of the kinetics in plasma, the degree of toxicity and the therapeutic efficacy. Daunomycin is currently being tested clinically as a chemotherapy drug against cancer; but it is a relatively hydrophobic substance and very difficult to maintain in the plasma stream, as evidenced by the fact that, less than 2 min after administration, approximately 95% of this substance has left blood flow and are passed through various organs, leaving only about 5% to reach the target tissue, in this case a tumor. This is illustrated in fig. 9 which reproduces the distribution of a typical drug of the anthracycline class, administered intravenously, 2 min after the injection. We finally know that daunomycin is a toxic substance, in particular because it tends to concentrate in the cardiac tissues, which requires not only to carefully control its administration, but also to administer it at very low doses.

The following examples 4 to 6 and FIGS. 10 to 12 illustrate the ability of the microreservoirs of the invention circulating in the blood stream to modify the kinetics in the plasma, the toxicity and the chemotherapeutic efficacy of daunomycin.

Example 4:

200 (imol of egg yolk phosphatidylcholine, 20 nmol of cholesteryl oleate and 4 mg of daunomycin are dissolved in chloroform, then the solution is evaporated to dryness under vacuum. Then 5 ml of NaCl are added to the resulting dry residue 0.154M and 10 (trihydroxymethylamine imol (pH 7.4). This aqueous suspension is then treated with ultrasound for 15 min at 51 ° C. under a nitrogen atmosphere. The daunomycin which is not contained in the vesicular microreservoirs produced is separated from the latter by passage over a gel column of Sephadex G-50 2.5 × 20 cm The volume of the voids which contained daunomycin incorporated in the microreservoirs is concentrated to 3 mg of daunomycin / ml.

A control sample of free daunomycin is also prepared at the same concentration and in the same buffer.

Three rats are injected intravenously with microreservoirs containing daunomycin; and six other rats are injected intravenously with free daunomycin. The dose of daunomycin contained in the microreservoirs is 4 mg / kg; the dose of free daunomycin is 4 mg / kg for three rats and 10 mg / kg for the three remaining rats. 0.5 ml of blood is taken from each rat at various times. The plasma is separated from the blood samples and the daunomycin is assayed by the fluorescence fusion method. The results thus obtained are reproduced graphically in FIG. 10 as a function of the time after injection.

It appears from fig. 10 that the use of circulating microreservoirs as a vehicle for the distribution of daunomycin changes in a beneficial way and improves the kinetics of this drug in plasma. From a comparison of curves A and B representing the same dose rate, it can be seen that, after approximately 15 min, the concentration of daunomycin in the microreservoirs circulating in the plasma is approximately 4 μg / ml; while the concentration of free daunomycin in the plasma is equal to

about 0.4 Hg / ml. Consequently, at this stage, the concentration in the blood stream of daunomycin transported by the circulating microreservoirs is some ten times greater than when this active substance is introduced directly into the blood stream. Furthermore, as shown in FIG. 10, free daunomycin is practically absent from the blood stream after 33/4 h, while its concentration after this time interval reaches approximately 0.4 (ig / ml in the circulating microreservoirs. Thus, the concentration in the current active substance transported in circulating microreservoirs after 33 / th is equivalent to the concentration which exists after 15 min only when the active substance is introduced in the free form.

Another comparison of curve A with curve C (free daunomycin at a dose of 10 mg / kg) shows that, after 1 h, the concentration of the active substance in the plasma is approximately 1.3 | ig / ml when this substance is transported by the circulating microreservoirs whereas it is approximately 0.5 μg / ml when the active substance is in the free form; after 3 h, these values are, respectively, equal to approximately 0.5 μg / ml and approximately 0.3 ng / ml; after 6 h, they are, respectively, equal to approximately 0.21 ng / ml and approximately 0.14 μg / ml. Consequently, the use of the circulating microreservoirs according to the invention can circulate in the blood stream concentrations of this active substance which are considerably greater than those which are obtained when the same active substance is administered in the free form. at doses two and a half times larger than when administered in the form of microreservoirs.

The change in the plasma kinetics of a drug such as daunomycin (as shown in Fig. 10) means that this substance exerts a less toxic effect, since it is maintained longer in the blood stream and it therefore cannot concentrate in organs such as the liver, kidney and heart. This also means that any administered dose is more effective when transported by circulating microreservoirs than when it circulates freely, since the targeted tissue is all the more exposed to the dose of daunomycin as this substance remains larger amount in the blood stream. Since the efficiency with which this substance destroys tumor cells is based on the ability of these cells to fix said substance, maximum contact between the active substance and the cells is essential for obtaining maximum efficiency.

Finally, the modification of the kinetics in the plasma of a xenobiotic substance as illustrated in FIG. 10 offers the possibility of reducing the doses; indeed, if the behavior, for example, of the dose of 10 mg / kg corresponding to curve C could be considered satisfactory for chemotherapeutic purposes, it is obvious that the dose rate could be reduced below the value 4 mg / kg if the drug was delivered and released by the circulating microreservoirs. Such a reduction in dose would also significantly reduce toxicity.

The fact that a marked reduction in the toxicity of daunomycin results from the use of circulating microreservoirs is illustrated in Example 5 and in FIG. 11.

Example 5:

1000 jimol of egg yolk phosphatidylcholine, 100 (imol of cholesteryl oleate and 20 mg of daunomycin) are dissolved in chloroform and the solution is evaporated to dryness under vacuum. 20 ml of KCl 0 are added to the dry residual mixture , 1 M and 10 pmol of trihydroxymethylamine (pH 8.0) The aqueous suspension is treated with ultrasound for 15 min at 51 ° C. under a nitrogen atmosphere. The liquid treated with ultrasound is chromatographed on a column of gel of Sepharose 4B 2.5 x 40 cm. Only the fractions which present a concordance between the elution profiles of phosphatidylcholine, cholesteryl oleate and daunomycin are collected together. The combined fractions are then concentrated by ultrafiltration. using an XM-50 membrane, at a final concentration of daunomycin of 3 mg / ml.

5

10

15

20

25

30

35

40

45

50

55

60

65

637,825

A similar sample of microreservoirs not containing daunomycin is also prepared and concentrated to the same concentration of microreservoirs. A control solution of free daunomycin is also prepared at a concentration of 3 mg / ml in 0.1 M KCl and 10 mol of trihydroxymethylamine (pH 8.0) intended to be used as free daunomycin.

The microreservoirs containing daunomycin, the microreservoirs not containing the active substance and the free active substance are injected at different doses into mice, in a single intraperitoneal injection. Ten BDF mice are used in each experimental protocol; the results obtained are shown graphically in FIG. 11. These graphs illustrate the survival rate of mice as a function of time. It should be noted that the microreservoirs themselves, in the absence of daunomycin, are devoid of toxicity. At all doses, the use of circulating microreservoirs to transport daunomycin reduces the toxic effects of the active substance compared to the effects produced by free daunomycin. At reduced doses, the ability of dispensing vehicles to reduce toxicity increases compared to the free active substance. It is assumed that this reduction in toxicity can be explained at least in part by the ability of the circulating microreservoirs to keep daunomycin in a blood stream and away from tissues and organs, especially the heart.

The chemotherapeutic effect of daunomycin delivered by means of the vehicles according to the invention is illustrated in Example 6 and in FIG. 12.

Example 6:

The procedure of Example 5 is followed to prepare vesicular microreservoirs with and without daunomycin and to prepare daunomycin intended to be used as free active substance. Several mice are injected intraperitoneally

1 x 106 P388 tumor cells. 24 h later, single daily intraperitoneal doses of the vesicular microreservoirs are injected with and without daunomycin and free daunomycin for a period of 5 d after tumor implantation. A group of mice used as a control group does not receive any active substance in any form. Five mice are used for each experimental protocol and two different dose rates, namely 4 mg / kg and 2 mg / kg. The results obtained in these tests are represented graphically in FIG. 12 by the variation in the number of survivors as a function of time.

The mice to which the microreservoirs without daunomycin are injected have a survival rate (not represented graphically) similar to that of the control mice (curves C on the graphs). At a dose of 4 mg / kg. daunomycin delivered by circulating microreservoirs exerts a chemotherapeutic effect, while the free active substance at the same dose rate deploys a significantly greater toxicity than tumor cells. At the low dose of

2 mg / kg, daunomycin delivered by circulating microreservoirs exerts a greater chemotherapeutic response than does the free active substance.

In the formulation of the microreservoirs of the invention, it may be desirable to include one or more agents modifying the binding of the xenobiotic substance, capable of raising or reducing the relative amount of xenobiotic substance retained by the microreservoirs during their training. For example, it has been found that a small molar percentage, i.e., about 5 mol% or more, of phosphatidic acid (charged lipid), added to the phospholipid component of microreservoirs can increase the affinity of the substance xenobiotic for microreservoirs. It is therefore possible, in accordance with the invention, to use one or more different phospholipids to form the phospholipid constituent of the composition of the microreservoirs. It is also conceivable to choose a single phospholipid or an optimal mixture of phospholipids as the phospholipid constituent of the microreservoirs in order to obtain a predetermined fixation of the xenobiotic substance.

It is also possible to modify the binding of a xenobiotic substance to the reservoir by the inclusion of other lipids which are soluble in the phospholipid component or slightly soluble in the cholesterol ester or the triglyceride. The modifications obtained in a system formed by a microreservoir and daunomycin are illustrated in Example 7 and in Table 1; the effect exerted by these modifications on the emission or release rates of the xenobiotic substance from the resulting microreservoirs is illustrated in Example 8 and in FIGS. 13 and 14.

Example 7:

A series of microreservoir formulations containing 105 jamol of 14C carbon-labeled egg yolk phosphatidylcholine (specific activity of 4160 dpm / | a.mol of phosphatidylcholine), 11.6 | cholesteryl oleate imol and 11, are prepared. 6 (xmol of triolein (glyceryl trioleate), containing or not a second phospholipid represented by an amount of 11.6 (imol of phosphatidic acid, and containing or not containing cholesterol as modifying agent in quantity from 0 to 75 | imol. In each formulation, 1.72 mg of daunomycin is used as the xenobiotic substance to be transported and released.

The formulations are prepared in the form of chloroform solutions which are evaporated to dryness in 10 ml flasks with a screw cap and kept under vacuum for approximately 18 h. Each formulation sample is then hydrated with 3 ml of 0.154 M NaCl and 10 mmol of trihydroxymethylamine (pH 7.2), then stirred for several minutes at room temperature. Each sample is then treated by ultrasound at the desired temperature for 20 min in a stream of nitrogen. Ultrasonic treatment of samples containing cholesteryl oleate is carried out at 51 ° C; that of samples containing triolein is carried out at 3 ° C. After the ultrasonic treatment, each sample is centrifuged for 10 min in a laboratory centrifuge, to remove any fragments of titanium.

Each sample is passed through a column of Sephadex G-50 of 2.5 x 15 cm using 0.154 M sodium chloride and 10 mmol of trihydroxymethylamine as elution buffer. The interstitial volume of each of the columns of G-50, containing the microreservoirs, is collected and analyzed in order to determine the radioactivity of the egg yolk phosphatidylcholine labeled with carbon 14C and the fluorescence coming from daunomycin. The results of these measurements, expressed in micrograms of daunomycin per micromole of phosphatidylcholine and in micrograms of daunomycin per micromole of total phospholipid and by the percentage of fixation of daunomycin by the microreservoirs, are reproduced in table 1.

In reproducing these results, the percentage of encapsulated daunomycin was reported to the sample with the highest degree of encapsulation of daunomycin per micromole of total phospholipid.

(Table at the top of the next page)

It appears from the results reproduced in Table 1 that the inclusion of a small amount of phosphatidic acid in the phospholipid component (samples 1 and 7) increases the absorption of daunomycin by the microreservoirs. Unlike imidocarb (examples 13 to 15), which requires the inclusion of phosphatidic acid, daunomycin, which has an ionizable amino functional group, benefits from the use of phosphatidic acid as a portion of the phospholipid component. It can therefore be seen that phosphatidic acid is applicable to xenobiotic substances of various natures.

The incorporation of cholesterol, a lipid miscible with phospholipids, into the composition of microreservoirs inhibits the binding of daunomycin to microreservoirs. The addition of cholesterol to the microreservoir compositions containing glycerol trioleate (samples 3 to 6) exerts a much less effect on the binding of the active substance than when the composition of the microreservoirs

8

5

10

1S

20

25

30

35

40

45

50

55

60

65

9

637,825

Table l

Effect of the composition of the phospholipid component and of the addition of lipids miscible with the phospholipid on the fixation of daunomycin by microreservoirs

Sample number

Composition of the microreservoir (mol%)

Micrograms of daunomycin by

Encapsulated proportion (%)

PC

GTO

CO

PA

Chol micromole of PC

micromole of PL

1

82

9

9

6.67

6.03

100

2

90

10

-

-

-

4.90

4.90

81

3

82

9

-

-

9

4.26

4.26

71

4

72

8

-

-

20

4.14

4.14

69

5

65

7

-

-

28

4.03

4.03

67

6

54

6

-

-

40

3.91

3.91

65

7

82

-

-

9

-

5.83

5.27

87

8

90

-

10

-

-

4.41

4.41

73

9

82

-

9

-

9

4.21

4.21

70

10

72

-

8

-

20

3.43

3.43

57

11

65

-

7

-

28

3.14

3.14

52

12

54

-

6

-

40

2.36

2.36

40

PC = phosphatidylcholine

GTO = glycerol trioleate

CO = cholesteryl oleate

PA = phosphatidic acid

Chol = cholesterol

PL = total phospholipids (PC and PA)

contained cholesteryl oleate (samples 9 to 12). Finally, the results of Table 1 show that, in the case of daunomycin, the use of glycerol trioleate (samples 1 to 6) in place of cholesteryl oleate (samples 7 to 12), facilitates the binding of this xenobiotic substance in microreservoirs. It is therefore obvious that, by the choice of the phospholipid constituent and of the immiscible constituent of the phospholipid (with or without addition of agent modifying the binding of the xenobiotic substance), it is possible to influence and predetermine the degree of absorption. of the xenobiotic substance in the micro-tanks or its degree of binding to the micro-tanks. This influence gives flexibility to the composition of the invention intended to deliver a xenobiotic substance, as regards the dose rates, the rate of release of the xenobiotic substance, etc. 45

The equilibrium binding of a xenobiotic substance can not only be affected by the composition of the microreservoirs, but the rate of emission or release of this substance can also be influenced and predetermined by the composition. This is illustrated by the use of a model which makes it possible to determine the emission rates of a xenobiotic substance from the microreservoirs in reaction to unbalanced conditions, as indicated in detail in Example 8 and as shown in fig. 13 and 14.

Example 8: 55

Microreservoirs are formulated as described in Example 7. To establish the desired non-equilibrium conditions, microreservoirs containing daunomycin as the xenobiotic substance are incubated with a large excess of dispersions of phosphatidylcholine from egg yolk not treated by ultrasound. <> 0 These dispersions are obtained by adding aliquots of microreservoirs of the various compositions containing approximately 1 (imol of phosphatidylcholine from egg yolk to 9 | imol of phosphatidylcholine from egg yolk not treated with ultrasound in 1.0 ml of 0.154M NaCl and 10 mmol of trihydroxymethylamine (pH 7.2) Comparable control dispersions are prepared which contain an equivalent amount of free daunomycin in place of the daunomycin carried by the microreservoirs.

In determining the release rate of daunomycin, one of the mixtures thus formed is used for each time chosen. At the times indicated, the sample is centrifuged at 15,000 x g for 2 min and, under these conditions, the dispersion not treated by ultrasound is easily separated from the microreservoirs remaining in the resulting supernatant liquor. Since daunomycin tends to rebalance between microreservoirs and phospholipid dispersions not treated by ultrasound, the equilibrium speed can be used as a kinetic parameter to evaluate the role played by the various constituents forming the composition of microreservoirs in determining the release speed of daunomycin contained in the microreservoirs.

An aliquot of the supernatant liquor formed is analyzed to determine the fluorescence, and the amount of fluorescence that remains is compared with that which is initially present in the mixture. This quantity of daunomycin remaining in the supernatant liquor therefore represents the quantity of active substance still contained in the microreservoirs.

The results obtained in this series of measurements are reproduced graphically in FIGS. 13 and 14 which show the decrease in fluorescence as a function of time for the microreservoirs containing glycerol trioleate (fig. 13) and for those which contain cholesteryl oleate (fig. 14). From the results graphically reproduced in FIGS. 13 and 14, it can be seen that the free daunomycin is very quickly removed from the supernatant liquor.

while that which is fixed to the microreservoirs is retained to a much greater degree. The inclusion of 9 mol% phosphatidic acid in the phospholipid component of the microreservoirs markedly reduces the rate of release, while the addition of cholesterol increases this rate. Finally, the use of a triglyceride in place of the cholesterol ester slightly reduces the rate of release.

These kinetic evaluations based on the release rates contribute to the importance of the results presented in Table 1 with regard to the equilibrium binding of daunomycin to microreservoirs; these results also confirm the fact that the composition of the microreservoirs can be chosen to predetermine and influence the pharmacodynamics of the xenobiotic substance contained in the microreservoirs used as a vehicle for distributing active substance according to the present invention.

The substance AD 32 is a very hydrophobic analogue of adria-mycin, used in chemotherapy. Since this active substance is completely insoluble in an aqueous buffer, it is necessary

637,825

<4

Please use a detergent and an organic liquid together to form an AD 32 solution suitable for clinical applications. An example of a solvent currently in use is a mixture of equal volumes of ethanol and a detergent consisting of a sulfuric ester of ethylene glycol (emulphol).

Despite the hydrophobic nature of the substance AD 32, it is possible to incorporate this substance into the microreservoirs in accordance with the present invention and, consequently, to modify in a beneficial sense the pharmacodynamics of this substance with regard to both the kinetics in the plasma that therapeutic efficacy,

as illustrated in examples 9 to 12 and in figs. 15 to 18.

Example 9:

1090 | imol of phosphatidylcholine from egg yolk, 121 μmol of 3H hydrogen-labeled cholesteryl oleate (specific activity 190,000 dpm / nmol) and 17.5 mg of AD 32 are dissolved in chloroform 1090 | imol solution dry and kept under vacuum for about 18 h. 5.5 ml of 0.154 M NaCl and 5 mmol of trihydroxymethylamine (pH 7.41) are added to the dry mixture. The liquid is treated by ultrasound at 48 ° C. under a nitrogen atmosphere and, after this operation, it is chromatographed on a Sepharose 4B column of 2.5 × 40 cm. Individual fractions thus obtained are analyzed to determine the phosphatidylcholine content, the cholesteryl oleate content and the AD 32 content, as indicated above. The elution profile, which represents a set of analyzes, is reproduced graphically in FIG. 15. From this graph, it can be seen that substance AD 32 is associated with non-vesicular microreservoirs (fractions 2-5) as well as with vesicular microreservoirs (fractions 6 to 18).

The inclusion of a small amount of phosphatidic acid in the phospholipid component of the composition of the microreservoirs has little or no effect on the rate of release of the substance AD 32 when a triglyceride is used as a component immiscible with the phospholipid . This is apparent from Example 10 and FIG. 16.

Example 10:

A basic formulation for microreservoirs is used containing 104.6 jimol of egg yolk phosphatidylcholine and 11.6 nmol of glycerol trioleate labeled with hydrogen 3 H (specific activity 1.5 x 105 dpm / | imol). 1.73 mg of AD 32 is added and 11.62 µmol of phosphatidic acid derived from egg yolk is also added to one of the formulations. These formulations are evaporated to dryness from a chloroform solution and the residues are kept under vacuum for approximately 18 h. 3 ml of 0.154M NaCl and 10 mmol of trihydroxymethylamine are added to each sample and the hydrated liquids are treated with ultrasound for 20 min at 3 ° C under a nitrogen atmosphere. Each sample is passed through a 2.5 x 20 cm Sephadex G-50 column and an amount of each sample obtained from vesicular microreservoirs equivalent to 1 jamol of phospholipid is incubated with 9 nmol of phosphatidylcholine liposomes in 0.46 ml of buffered saline to form a dispersion.

At various times, the resulting dispersions are centrifuged at 15,000 x g for 2 min and the amount of fluorescence remaining in the supernatant is determined. The results of these release rate determinations are shown graphically in FIG. 16. It can be seen that the rate of release of the substance AD 32 is relatively high and remains essentially unaffected by the inclusion of phosphatidic acid in the composition of the microreservoirs.

Although the rate of release of substance AD 32 from microreservoirs is relatively high, the use of microreservoirs produces a pronounced beneficial change in the kinematics of this active substance in plasma, compared to the dosage forms currently in use. This is illustrated in Example 11 and in FIG. 17.

Example II:

Fractions 6 to 18 of Example 9 are concentrated by ultrafiltration at a concentration of substance AD 32 of 3.2 mg / ml. Sufficient quantities of the substance AD 32 are injected intravenously into the microreservoirs in rats weighing 150 g so that the dose rate is 15 mg / kg; and a clinical formulation of substance AD 32 dissolved in a 1: 1 mixture by volume of ethanol and emulphol is injected in the same manner at the same dose rate to three control rats. Plasma samples are taken at different times and the AD 32 concentrations in these plasma samples are determined by fluorescence. The results obtained (average of three rats for each point) are reproduced graphically in FIG. 17 and express the number of equivalents of AD 32 as a function of time from the initial injection.

Fig. 17 demonstrates the clear modification of the kinetics of the substance AD 32 in the plasma, obtained by the use of the microreservoirs of the invention, because the concentrations of AD 32 in the plasma in which the medicament has been incorporated in the enclosed form in microreservoirs are significantly higher (by a factor of between 2 and 3 in all respects) than the concentrations obtained for the free AD 32 substance introduced in the form of a clinical solution. By allowing the active substance more time to find tumor cells, this change in its kinetics in plasma improves its effectiveness as a chemotherapeutic agent. This is illustrated in Example 12 and in FIG. 18.

Example 12:

Vesicular microreservoirs containing the substance AD 32 are formulated as in Example 9 and concentrated to 3.2 mg of AD 32 per milliliter. Several mice are injected intraperitoneally with 1 × 10 6 P388 tumor cells (leukemia). 24 h later, single daily intraperitoneal doses of the vesicular microreservoirs containing substance AD 32 and control saline are injected for a period of 5 d after the implantation of the tumor cells. Five mice are used for each experimental protocol and four different doses, namely 7.3 mg / kg, 4 mg / kg, 2 mg / kg and 1 mg / kg. The results obtained in these tests are reproduced graphically in FIG. 18 by the number of survivors as a function of time. In all cases, the mice to which the microreservoirs containing the substance AD 32 are injected have a higher survival rate than that of the control mice. In addition, even at very low dose rates of 1 mg / kg, the substance AD 32 delivered by the circulating microreservoirs exerts a chemotherapeutic effect and produces a marked improvement in the survival rate of the mice having received P388 leukemic cells.

As in the case of daunomycin, the same concentrations of AD 32 in the plasma can be obtained with lower initial levels of AD 32 conveyed by the microreservoirs, compared to clinical formulations. In addition, the composition of the microreservoirs has better biocompatibility with blood than the mixed solvent formed from ethanol and a detergent which must currently be used in clinical formulations of AD 32.

Adriamycin, daunomycin and AD 32, as well as carminomycin, are cancer chemotherapeutic agents that fall into the general class of anthracycline drugs. The preceding examples illustrate the effectiveness of the vehicle of the invention in the beneficial modification of the pharmacodynamics of this class of chemotherapeutic agents. The microreservoirs of the invention can also be used to deliver other classes of chemotherapeutic agents including, for example, nitrosoureas and metabolites.

Imidocarb is a drug that has been shown to be very effective as a parasiticide in the treatment of anaplasmosis in animals, by destroying parasites in the bloodstream. However, when this substance is administered at its most effective dose rates, traces of imidocarb tend to remain in animal tissue, which is undesirable if the animal is intended for consumption10

5

10

15

20

25

30

35

40

45

50

55

60

65

11

637,825

human tion. Consequently, it would be advantageous to find a dispensing vehicle capable of maintaining imidocarb in the circulating blood stream, which would allow it to assume its therapeutic function without accumulating in animal tissue.

Imidocarb and its hydrochloride are hydrophilic compounds, a characteristic by which they are distinguished from hydrophobic or both hydrophobic and hydrophilic drugs. However, as demonstrated by the following examples 13 and 14 and FIGS. 19 to 21, microreservoirs are just as effective in the beneficial modification of the pharmacodynamics of a hydrophilic xenobiotic substance such as imidocarb.

Example 13:

900 nmol of egg yolk phosphatidylcholine, 100 (jmol of phosphatidic acid, 100 | imol of cholesteryl oleate labeled with 3H (specific activity 1.6 × 104 dpm / nmol) and 9.16 mg are dissolved. imidocarb labeled with 14C and the solution is evaporated to dryness under vacuum, the resulting dry mixed residue is then hydrated by adding 45 ml of 0.154M NaCl and 290 mM of trihydroxyethylamine (pH 8.0) to the lipid / mixed drug residue. The liquid is then treated by ultrasound for 15 min at 51 ° C. under a nitrogen atmosphere.

The liquid treated with ultrasound is then chromatographed on a Sepharose 4B column of 2.5 x 40 cm. Individual chromatographic fractions are then analyzed to determine the amount of phosphatidylcholine and phosphatidic acid by the Gomori method, and to determine the amounts of 3 H-labeled cholesteryl oleate and 14 C-labeled imidocarb. radioactivity. The results of these analyzes are shown graphically in FIG. 19 as a function of the number of fractions, the analytical results being superimposed as in FIG. 8. The diameter of the vesicular microreservoirs is in the range of about 200 to 300 Å.

It can be seen that the elution profiles of the three constituents of the microreservoirs agree from fraction 5 to fraction 9, which indicates that imidocarb is associated with the microreservoirs.

The beneficial modification of the kinetics of imidocarb in plasma, resulting from the use of the vehicles of the present invention, is illustrated in Example 14 and in FIGS. 20 and 21.

Example 14:

100 | tmol of phosphatidylcholine and 10 | imol of phosphatidic acid derived from egg yolk, 10 nmol of cholesteryl oleate and 2 mg of 14C-labeled imidoacarb are dissolved in chloroform and the solution is evaporated to dryness under vacuum. . The resulting mixed residue is suspended in 5 ml of 0.154 M NaCl and 10 mmol of trihydroxymethylamine (pH 8.0) and the resulting suspension is treated by ultrasound for 15 min at 5 ° C. under a nitrogen atmosphere. The liquid is then chromatographed on a column of Sepharose 4B gel, 2.5 × 40 cm and the fractions having a coincidence of the 14 C-labeled imidocarb, phospholipids and cholesteryl oleate are combined and concentrated by ultrafilter. tration to a final concentration of 0.6 mg imidocarb per milliliter.

A free imidocarb solution is also prepared as a control at a concentration of 0.6 mg / ml in 0.154 M NaCl and 10 mmol of trihydroxymethylamine (pH 8.0), intended for use in the administration of free imidocarb.

Microreservoirs containing imidocarb as well as free imidocarb are injected into the tail veins of rats at two dose rates, namely 5 mg / kg and 4.4 mg / kg. Plasma samples are taken at various intervals and the amount of imidocarb in these samples is determined by measurements of their radioactivity. The results of these measurements, reproduced graphically by the variation of the imidocarb equivalents as a function of time, are given in FIGS. 20 and 21.

The beneficial modification of kinetics in plasma by the use of circulating microreservoirs to transport imidocarb in the blood stream of a living mammal is clearly shown in FIGS. 20 and 21. For example, at a dose of 5 mg / kg, the concentration of imidocarb in microreservoirs containing imidocarb in the blood stream is approximately 140 times greater than that of free imidocarb after 4 h ; and at the 4.4 mg / kg dose, it is approximately 200 times higher after the same period. In addition, as shown in fig. 21, the blood stream still contains a substantial amount of imidocarb in the microreservoir / imido-carb form after 24 h, whereas it practically no longer contains it when the imidocarb is used in the free form.

The variation in the ratio of imidocarb to microreservoirs as a function of time is also reproduced for each dose rate in FIGS. 20 and 21; these graphic representations clearly show the fact that the active substance is released at a speed which can be influenced and predetermined for any given set of conditions. It also appears from FIGS. 20 and 21 that it is possibly possible to reduce the dose rates of the active substance to a value much lower than the doses currently considered effective in the destruction of the blood parasites which cause anaplasmosis in animals.

One of the main aims of the beneficial modification of the pharmacodynamics of a xenobiotic substance is the ability to modify the distribution of this substance in the tissues. Example 15 and Table 2 show that this result can be obtained for imidocarb when using the microreservoirs of the invention.

Example 15:

The animals used in Example 14 are sacrificed 4 h after the injection of free imidocarb or microreservoirs containing the imidocarb. Various tissues such as muscle, spleen, liver and kidney are removed from the animals. These tissues are burned in a Searle combustion apparatus and the resulting carbon dioxide 14CO2 is collected and subjected to radioactivity counting. We count the number of disintegrations per gram of tissue; the results are reproduced in table 2.

Table 2

Distribution in tissues of imidocarb and imidocarb transported in microreservoirs

Tissue

Imidocarb

Microreservoir / imidocarb

Change (%)

(dpm / g of tissue)

Muscular

3,985

3511

-12

Kidney

40194

59984

+49

Missed

9498

15339

+ 61

Liver

27059

29991

+ 11

The ability to maintain a xenobiotic substance in the blood stream and in certain organs is particularly important in the case of an active substance such as imidocarb. Thus, the 12% reduction in the amount of imidocarb in muscle tissue that can be obtained by using microreservoirs is important for the use of this parasiticide in the treatment of animals intended for human consumption. In addition, the higher concentration of the drug in the liver and spleen, organs that contain a higher proportion of the microorganism responsible for anaplasmosis, is another important parameter in the use of imidocarb.

Among the pharmacodynamic activities of a xenobiotic substance which can be beneficially modified by the delivery vehicle of the invention, mention is made of oral absorption, the modification being obtained by the fact that the xenobiotic substance is made capable of crossing the tract gastrointestinal and to penetrate

5

10

15

20

25

30

35

40

45

50

55

60

65

637,825

12

into the blood stream for efficient circulation. Currently, several solutions are available for administering xenobiotic substances into the blood stream which do not cross the gastrointestinal tract or which cross it only to a very limited degree. One of these solutions lies in the chemical modification of the xenobiotic substance, for example the formation of the undecanoic acid ester of estradiol which is essentially insoluble in water and its suspension in an oil, its transformation into a microcrystalline dispersion or the preparation of a solution in an organic solvent such as ethanol.

The incorporation of estradiol undecanoate (used as fertility limiting agent) in microreservoirs in accordance with the present invention eliminates the need to use liquid media such as oils and organic solvents and at the same time makes it possible to obtain a much more efficient passage of the xenobiotic substance into the bloodstream. The incorporation of estradiol undecanoate in the microreservoirs of the invention is illustrated in Example 16 and in FIG. 22.

Example 16:

300 µmol of egg yolk phosphatidylcholine, 30 µmol of cholesteryl oleate and 6 (3H hydrogen-labeled estradiol undecanoate imol (specific activity 4.6 nCi / nmol) are dissolved in benzene. and the solution is lyophilized, then 5 ml of 0.154M NaCl and 5 mmol of trihydroxymethylamine are added to the resulting anhydrous mixture, followed by ultrasound treatment of the resulting liquid for 17 min at 48 ° C. under a nitrogen atmosphere. treated with ultrasound is then fractionated by chromatography on a Sepharose 4B column to produce the elution profile of Fig. 22. Fractions 12 to 30 constitute the vesicular microreservoirs containing the xenobiotic substance used to carry out an in vivo evaluation of the vehicle dispenser of the present invention.

The ability of the microreservoirs of the invention to facilitate the oral absorption of estradiol undecanoate is further illustrated in Example 17, in FIG. 23 and in Table 3.

Example 17:

3H hydrogen-labeled estradiol undecanoate is encapsulated in microreservoirs formed from egg yolk phosphatidylcholine and 14C-labeled cholesteryl oleate. Equivalent amounts of 3H-labeled estradiol undecanoate are dissolved in ethanol. Doses of 1.63 mg / kg are administered orally to two groups of 9 rats. After 1, 4 and 24 h, three rats from each group are sacrificed and the blood is collected. The plasma is separated from the erythrocytes by centrifugation and 0.5 ml of each plasma sample is added to 9.5 ml of a 2/1 by volume mixture of chloroform and methanol containing 1.6 jimol of estradiol and oes - unmarked throne, as vehicles. The resulting liquid is filtered and a sufficient amount of saline is added to the filtrates to form a two-phase system. The lower phase is evaporated to dryness, then redissolved in 2.0 ml of methanol. 0.2 ml of this solution is subjected to a radioactivity count. The remaining volume (1.8 ml) is deposited on a thin layer chromatography plate and developed in a mixture of benzene and ethyl acetate at 3/2 by volume. The plates are briefly exposed to iodine vapor to allow visualization of the support steroids, then the iodine vapor is removed by moderate heating. The spots which correspond to estradiol and estrone are cut out and subjected to a radioactivity count. The equivalents of estradiol undecanoate labeled with 3H in the plasma are shown graphically in FIG. 23. It can be seen that, during the first hour, the vesicular microreservoirs favored the appearance of equivalents of 3 H-labeled estradiol undecanoate in the plasma compared to the ethanolic control solution. After 4 h, the amount of estradiol undecanoate equivalents decreased for both formulations.

It is known that oestradiol is the active form of the drug and oestrone is the inactive form. In addition, it is assumed that estradiol esters are inactive until they have been hydrolyzed to estradiol. Therefore, the ratio of oestradiol to oestrone in plasma should be an indication of the ability of microreservoirs to promote the active form of the drug.

The results of the thin layer chromatographic analysis of the labeled derivatives of oestradiol in plasma are shown in Table 3. It can be seen from these results that the ratio of oestradiol to oestrone when using microreservoirs is 60% higher than their value when using an ethanolic solution.

Table 3

Ratio of 3H-estradiol to 3H-estrone in plasma 1 h after oral administration of estradiol undecanoate

Formulation

Estradiol / estrone *

In ethanolic solution In micro-tanks

1.14 1.84

* This report is based on the average of three rats.

The microreservoir used as a dispensing means in accordance with the invention also offers the possibility of oral administration of xenobiotic substances hitherto unsuitable for this mode of administration. Due to the passage of these drugs through the gastrointestinal tract, it is possible to deliver them indirectly to the blood stream, thereby eliminating their metabolism in the gastrointestinal tract and the need for injections, as well as discomfort and the complications that accompany this form of administration.

The microreservoirs can be used in their vesicular or non-vesicular form or in a combination of these two forms. This is illustrated by example 18 and by figs. 24 and 25.

Example 18:

It is dissolved in chloroform (imol of egg phosphatidylcholine, 75 nmol of cholesteryl oleate labeled with 14C and 2 (tmol of estradiol undecanoate labeled with 3H and the solution is evaporated to dryness. The mixture of lipids is evaporated to dryness under vacuum for approximately 18 h, then hydrated with 4.4 ml of 0.154 M NaCl and 5 mmol of trihydroxymethylamine (pH 7.2). The resulting liquid is treated by ultrasound at 40 ° C. for 20 min under a nitrogen atmosphere, then loaded onto a Sepharose 4B column The resulting elution profile is shown in Fig. 24. It can be observed that estradiol undecanoate has a greater affinity for the vesicular form of microreservoirs than for the non-vesicular form, according to the ratio of estradiol undecanoate to cholesteryl oleate in the two forms of microreservoirs. The substance which is in the interstitial volume of the column consists of the non-vesicular form , while the substance that is in the volume internal is the vesicular form.

Samples of the two forms, non-vesicular and vesicular, containing estradiol undecanoate and about 1 µmol of total lipids are treated by incubation with 9 µmol of phosphatidylcholine dispersion from egg not treated with ultrasound. At various times, the samples are centrifuged at 15,000 x g for 2 min and the supernatant liquor containing the microreservoirs is subjected to a radioactivity count. Since cholesteryl oleate is a non-exchangeable body in this system, the ratio of 3H-labeled estradiol undecanoate to 14C-labeled cholesteryl oleate is an indication of the rate of release of l estradiol undecanoate by non-vesicular or vesicular microreservoirs in the phosphatidylcho- dispersion

5

10

15

20

25

30

35

40

45

50

55

60

65

13

637,825

line. The results are shown in fig. 25. It should be noted that estradiol undecanoate remains associated with the two forms of microreservoirs, which indicates that these two forms are satisfactory for the transport and release of xenobiotic substances.

The microreservoirs of the invention containing xenobiotic substances can be presented in various dosage forms. Thus, for example, they can be dispersed in a physiologically compatible liquid, they can be used dry to form tablets or they can be enclosed in capsules of a suitable biocompatible material.

It follows from the above description and detailed examples that the dispensing vehicle of the invention is capable of modifying, in a beneficial sense, the pharmacodynamics of xenobiotic substances having a wide range of chemical and physical characteristics as well as a wide range of applications and biological properties.

10 sheets of drawings

Claims (15)

637,825 1. Distributor vehicle biologically compatible with a host mammal, for introducing and releasing into the host organism a xenobiotic substance whose pharmacodynamics is modified in a beneficial way by the use of said vehicle, characterized in that it is in the form of microreservoirs containing said xenobiotic substance, these microreservoirs being formed of a phospholipid constituent and of a lipid constituent immiscible with phospholipid, stable and essentially immiscible with a physiologically compatible liquid. 2. Dispensing vehicle according to claim 1, characterized in that the microreservoirs are in a vesicular form having diameters from 190 to 300 Å; in a non-vesicular form having diameters from 250 to 1000 Å; or both in non-vesicular and vesicular forms. 2 3. Dispensing vehicle according to claim 1, characterized in that the microreservoirs are formed in a proportion of 50 to 97 mol% of said phospholipid constituent. 4. Dispensing vehicle according to claim 1, characterized in that the microreservoirs comprise an agent modifying the binding of the xenobiotic substance or an agent limiting the rate of release of said substance. 5 in the vesicular form, having diameters from 190 to 300 Å, fractions containing the microreservoirs in the non-vesicular form, having diameters from 250 to 1000 Å. 5. Dispensing vehicle according to claim 1, characterized in that the phospholipid component is phosphatidyl-choline and can contain phosphatidic acid in an amount equivalent to at least 5 mol%. 6. Dispensing vehicle according to claim 1, characterized in that the lipid component immiscible with the phospholipid is a cholesterol ester of a fatty acid having 10 to 18 carbon atoms or a triglyceride. 7. Dispensing vehicle according to claim 1, characterized in that the xenobiotic substance is a medicament such as a chemotherapeutic medicament against cancer, a parasiticide or an agent limiting fertility. 8. Dispensing vehicle according to claim 1, characterized in that the xenobiotic substance is a medicament whose kinetics in plasma, toxicity, chemotherapeutic efficacy or oral absorption are modified in a beneficial direction. 9. A method of producing the dispensing vehicle according to claim 1, characterized in that it consists in forming microreservoirs of a composition comprising a phospholipid component and a lipid component immiscible with the phospholipid which is stable and essentially immiscible with the liquid physiologically compatible and to incorporate into said microreservoirs the xenobiotic substance to be delivered. 10. Method according to claim 9, characterized in that the step of forming the microreservoirs consists of: a) preparing with a solvent a solution of the phospholipid component and of the lipid component immiscible with the phospholipid; b) removing the solvent to produce a dry residual mixture of the phospholipid component and of the lipid component immiscible with the phospholipid; c) hydrating the residual mixture with a physiologically compatible liquid to form a suspension; d) treating said suspension by ultrasound in a non-oxidizing atmosphere at a temperature which is at least equivalent to the melting point of said lipid component immiscible with phospholipid to form microreservoirs, and e) separating the microreservoirs thus formed. 11. The method of claim 10, characterized in that the step of incorporating the xenobiotic substance in the microreservoirs consists in adding said substance to said solution in step a; or to add the xenobiotic substance to the suspension of step c before the ultrasound treatment. 12. Method according to claim 10, characterized in that the step of separating the microreservoirs thus formed consists in centrifuge the suspension treated with ultrasound and chromatograph the clear phase resulting from the centrifugation to form a series of chromatographed fractions; and, if necessary, to separate the chromatographed fractions which contain the microreservoirs 13. Method according to claim 10, characterized in that it consists in adding a substance modifying the binding of the subs- xenobiotic substance or an agent influencing the rate of release of the xenobiotic substance to said solution. 14. Method according to claim 9, characterized in that it consists in suspending the microreservoirs containing the xenobiotic substance in the physiologically compatible liquid. 15 ble so that said substance is in a liquid dosage form or to encapsulate the microreservoirs containing said xenobiotic substance in a capsule formed of a physiologically acceptable substance.