Use of natural cyclodextrins and their derivatives for the solubilization of platelet anti-aggregating agents from the family of ginkgolides

The invention relates to the use of natural cyclodextrins and their derivatives for the solubilization of platelet anti-aggregating agents from the family of Ginkgolides. The cyclodextrins used comply with the formula: ##STR1## in which n is equal to 6, 7 or 8 and the R.sup.1, which can be the same or different, represent OH or SR.sup.2 with R.sup.2 representing a group derived from a monosaccharide or an oligosaccharide. The Ginkgolide is preferably Ginkgolide B.

The present invention relates to a solubilization process in an aqueous 
medium for an antagonizing agent of the aggregation factor of platelets 
belonging to the family of Ginkgolides. Ginkgolides are chemical compounds 
having 6 cycles with 5 joined members or groups. 
FIG. 1 shows the developed formula of said Ginkgolides. As can be seen the 
Ginkgolides have a tetrahydrofuran cycle (D), 2 cyclopentane cycles with a 
common carbon (A and B) forming a spiro-(4,4)-nonane and 3 
.gamma.-lactones (C, E and F). Ginkgolides are extracted from leaves of 
Ginkgo bilobe, have a unique cage-shaped structure and a strong potential 
of biological activities. The great diversity of these activities in the 
series of Ginkgolides would appear to be solely due to substitution 
differences and conformational changes. 
In these Ginkgolides, whose formula is given in FIG. 1, R.sup.3, 
R.sup.4,R.sup.5 and R.sup.6 can represent H or OH and R.sup.4 can also 
represent a lower alkoxy group such as methoxy (OMe) or ethoxy (OEt). 
Table 1 groups certain known Ginkgolides complying with this formula with 
the nature of the substituents R.sup.3, R.sup.4, R.sup.5 and R.sup.6. 
TABLE 1 
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Compound Nomenclature 
No. Ginkgolide 
1 HB R.sup.3 
R.sup.4 
R.sup.5 
R.sup.6 
______________________________________ 
1 A BN 52020 OH H H OH 
2 B BN 52021 OH OH H OH 
3 C BN 52022 OH OH OH OH 
4 J BN 52024 OH H OH OH 
5 M BN 52023 H OH OH OH 
6 synthetic BN 50580 OH OMe H OH 
7 synthetic BN 50585 OH OEt H OH 
______________________________________ 
The majority Ginkgolides are compounds 1, 2 and 3, forms A, B and C, the 
other forms (J, M), compounds 4 and 5, being rare. The compound having the 
most interesting pharmacological properties is Ginkgolide B (compound 2) 
with 3 closed lactone cycles. 
Ginkgolide B has formed the object of a considerable amount of research in 
various fields (Bracquet, P., "Ginkgolides : Chemistry, Biology, 
Pharmacology and Clinical Perspectives", 1 and 2; J. R. Prous Science 
Publishers Barcelona 1988). Moreover, complete retrosynthesis was carried 
out by Corey (Corey, E. J., Kang M. C., Ghosh A. K., Houpis I. N.; J. Am. 
Chem. Soc., 1988, 110 649-651). Ginkgolide B has interesting 
pharmacological properties and would appear to demonstrate an antagonizing 
activity against the aggregation factor of platelets (anticoagulant), 
which plays a key part in the inflammatory process. It therefore has an 
anticoagulating activity. In vitro, pharmacological research has revealed 
that the effectiveness of Ginkgolide B was closely linked with the pH and 
that the anti-platelet activating factor or PAF activity was increased in 
an acid medium (Braquet, P, Op. Cit. 1, 11-23). This result is clearly 
related with the existence of three lactone groups which can be 
hydrolyzed, which leads to the opening of the cycles. These processes are 
reversible because, as has been demonstrated by Braquet, the acidification 
of basic solutions of Ginkgolide B always regenerates the starting 
Ginkgolide B without any deterioration in the molecular structure. This 
important point has been confirmed by NMR (cf. the thesis of Oussama 
ZEKRI, Rennes University, 1994). The authors demonstrated the presence of 
8 possible forms, whose existence is closely dependent on the pH value. 
Thus, three pKa values were determined at 7.14, 8.60 and 11.90. Under 
physiological conditions: physiological phosphate buffer solution (PBS) 
and pH 7.39, the solubility of Ginkgolide B is 0.2 mM and the NMR study 
revealed the presence of 4 different forms, the closed form (active form) 
representing 34% thereof at 25.degree. C. This percentage decreases when 
the temperature increases. 
Ginkgolide B can be parenterally administered. Due to its low solubility at 
acid or neutral pH, it is solubilized at pH=8.75 to a concentration of 20 
mg/mL (C=47 mM) in the presence of 10 mg/mL of mannitol. The NMR study of 
the proton at 500 MHz of this solution revealed the presence of 5 open 
forms having more than 99% Ginkgolide B. The totally closed form, i.e. the 
only active form, is in a negligible proportion, i.e. a concentration 
below 0.1 mM. Moreover, it was shown by conductometry, that although the 
opening of the lactones is relatively fast (.apprxeq.10 min.), their 
closing is slow (.gtoreq.3 h-cf. the thesis of Oussama ZEKRI, Rennes 
University, 1994). It can therefore be said that in the present injection 
form, the anit-PAF active form is extremely low, whereas the injected 
Ginkgolide quantity is high. To solve this problem one approach, which 
forms the object of the present invention, was to find appropriate 
molecules having a high solubility in an aqueous medium at physiological 
pH, in order to solubilize Ginkgolides such as Ginkgolide B in the aqueous 
medium, whilst retaining the maximum of the active form, i.e. the closed 
form of Ginkgolide. 
The present invention specifically relates to the use of natural 
cyclodextrins and certain of their derivatives, in order to solubilize and 
protect in an aqueous medium anti-PAF agents of the Ginkgolide type by the 
inclusion thereof in said cyclodextrins. 
Cyclodextrins or cyclomaltooligosaccharides are compounds having a natural 
origin formed by the connection of 6, 7 or 8 glucose units bonded in 
.alpha.-(1.fwdarw.4). Numerous works have revealed that these 
cyclodextrins could form inclusion complexes with hydrophobic molecules 
and thus permit the solubilization of these molecules in aqueous media. 
Numerous applications have been proposed for taking advantage of this 
phenomenon, particularly in the pharmaceutical sector, as is described by 
D. Duchene in the work entitled "Cyclodextrins and their industrial uses", 
chapter 6, pp 213 to 257, Editions de Sante, 1987. Pharmaceutical 
compositions using cyclodextrins have also been marketed in Japan, Italy 
and more recently in France, e.g. by Pierre Fabre Medicament for Brexin, 
which is an inclusion complex of Piroxicam in .beta.-cyclodextrin. 
According to the invention, the solubilization process in an aqueous medium 
of an antagonizing agent of the platelet aggregation factor belonging to 
the family of Cinkgolides consists of combining said agent with a 
cyclodextrin of formula: 
##STR2## 
in which n is equal to 6, 7 or 8 and the R.sup.1 which can be the same or 
different, represent OH or SR.sup.2, with R.sup.2 representing a group 
derived from a monosaccharide or an oligosaccharide, in order to form 
therewith an inclusion complex soluble in water. 
In the aforementioned formula (I), R.sup.2 can comply with the formula: 
##STR3## 
in which p is equal to 0 or an integer from 1 to 5. 
According to the invention, the antagonizing agents of the platelet 
aggregation factor, hereinafter called anti-PAF agents, are compounds 
belonging to the family of Ginkgolides. They can comply with the formula 
given in FIG. 1, in which R.sup.3, R.sup.5 and R.sup.6, which can be the 
same or different, represent H or OH, and R.sup.4 represents H, OH or an 
alkoxy group with 1 to 5 carbon atoms. 
The alkoxy groups used can in particular be methoxy and ethoxy groups. 
The invention preferably applies to the anti-PAF agent constituted by 
Ginkgolide B complying with formula (III), in which R.sup.3, R.sup.4 and 
R.sup.6 represent OH and R.sup.5 represents H. 
According to a first embodiment of the invention, the cyclodextrin is a 
natural cyclodextrin complying with the formula (I) with all the R.sup.1 
representing OH. Preferably, in this embodiment, n is equal to 7. 
According to a second embodiment of the invention, use is made of a 
branched cyclodextrin, i.e. a cyclodextrin complying with formula (I), in 
which a single R.sup.1 represents SR.sup.2, the other R.sup.1 being 
identical and representing OH. 
In this second embodiment, R.sup.2 is preferably the .alpha.-maltosyl or 
.beta.-maltosyl group, namely the group complying with formula (II) with p 
equal to 1. 
As in the first embodiment, preference is given to branched cyclodextrins 
of formula (I) in which n is equal to 7 or 8, the best results being 
obtained with n equal to 7 or 8, preferably 7. 
The cyclodextrin derivatives used in this second embodiment of the 
invention can be prepared in the manner described in EP-A-403 366 and 
FR-A-2 715 307. 
The invention also relates to the inclusion complexes of an antagonizing 
agent of the platelet aggregation factor belonging to the family of 
Ginkgolides, in a cyclodextrin complying with the formula (I). 
In these inclusion complexes, preference is given to the cyclodextrin of 
formula (I) being .beta.-cyclodextrin (n=7) or .gamma.-cyclodextrin (n=8) 
with all the R.sup.1 identical and representing OH, or a 
.beta.-cyclodextrin derivative in which a single R.sup.1 represents 
SR.sup.2, with R.sup.2 representing the .alpha.-maltosyl or 
.beta.-maltosyl group, the other R.sup.1 being identical and representing 
OH. 
The antagonizing agents included in the cyclodextrin of this complex can be 
Ginkgolides of formula (III) described in FIG. 1 and in table 1, in 
particular Ginkgolide B corresponding to formula (III with R.sup.3, 
R.sup.4 and R.sup.6 representing OH and R.sup.5 representing H. 
These inclusion complexes can be prepared in conventional manner, e.g. by 
adding to a solution of the cyclodextrin of formula (I), whose pH is below 
5, a concentrated Ginkgolide solution in an appropriate, organic solvent, 
e.g. acetone. It should be noted that the pH value of the aqueous solution 
is vital. Thus, at a pH below 5, the Ginkgolides, e.g. Ginkgolide B, are 
solely in an entirely closed form, which is the only active form in the 
case of Ginkgolide B. It is then possible to isolate the thus formed 
inclusion complex by lyophilization. 
To avoid the use of an organic solvent, during the preparation of the 
inclusion complex, it is possible to disperse the anti-PAF agent to be 
included in an aqueous solution of the cyclodextrin used, whose pH is 
below 5, for the same reasons as described hereinbefore, and stir the 
suspension obtained until a clear solution is obtained. It is then 
possible to isolate the thus formed inclusion complex by lyophilization, 
as hereinbefore. 
These inclusion complexes can in particular be used in pharmaceutical 
compositions, which also incorporate a pharmaceutically acceptable 
vehicle. 
These pharmaceutical compositions which can be administered orally or 
parenterally are e.g. solutions, powders, suspensions and in particular 
injectable solutions. The inclusion complexes formed with Ginkgolide B and 
the natural or branched cyclodextrins have a solubility at an acid pH 
value greater than that of Ginkgolide B alone. Moreover, at higher pH 
values, Ginkgolide B in the form of an inclusion complex undergoes an 
increase in the proportion of its closed form to a significant extent 
compared with Ginkgolide B alone, under the same conditions. Thus, the use 
of natural or branched cyclodextrins makes it possible to reduce the total 
quantity of Ginkgolide B injected, whilst increasing the quantity of the 
active form, i.e. the proportion of the closed form. In other words, the 
inclusion complex is more efficient with respect to the PAF, whilst 
minimizing the risks of side effects.

EXAMPLE 1 
Preparation of an inclusion complex of Ginkgolide B with 
.beta.-cyclodextrin 
The starting product is a concentrated Ginkgolide B solution in a 
water-miscible solvent, constituted by acetone, and addition takes place 
of the concentrated solution quantity corresponding to 1 .mu.mole of 
Ginkgolide B to 1 mL of a 10 mmole/L .beta.-cyclodextrin solution in 
sterile water at pH=4.9 and at ambient temperature. The acetone is 
eliminated under nitrogen bubbling and the solution is lyophilized. The 
residual solid containing 10 .mu.mole of .beta.-cyclodextrin derivative 
and 1 .mu.mole of Ginkgolide B is redissolved in a minimum of water at 
25.degree. C. This minimum corresponds to 1 mL of water, which indicates a 
maximum solubility of 1 mmole/L of Ginkgolide B in water in the presence 
of .beta.-cyclodextrin at a concentration of 10 mmole/L. 
The partial nuclear magnetic resonance (NMR) spectrum of the proton of this 
complex at a concentration of 5 mmole/L in D.sub.2 O, at 298 K and 500 
MHz, is shown in FIG. 2 (spectrum c). FIG. 2 also shows for information 
purposes the partial spectrum of Ginkgolide B only (spectrum a). On 
comparing the two spectra, it is possible to see the modifications 
confirming the inclusion of Ginkgolide B in the cavity of the 
l3.beta.-cyclodextrin. 
It can also be seen that Ginkgolide B only exists in the inclusion complex 
in the closed form at pH=4.9. 
It should be noted that the solubility of the inclusion complex is the same 
at physiological pH, namely a maximum solubility of 1 mmole/L of 
Ginkgolide B in the presence of 10 mmole/L of .beta.-cyclodextrin. At 
physiological pH, Ginkgolide B alone exists in four different forms, 
whereof the closed form only represents 34% at 25.degree. C. With the 
inclusion complex prepared in the manner described hereinbefore, only two 
different forms are observed in identical proportions including the closed 
form. By comparison, the maximum solubility of Ginkgolide B alone, in its 
closed form, is 0.2 mmole/l at pH-4.9 and 0.68 mmole/L at pH=7.39. When 
Ginkgolide B is in the inclusion complex form at pH 4.9 with a 
concentration of 1 mmole/L in the presence of 10 mmole/L of 
.beta.-cyclodextrin, it has a closed form concentration which is 10 times 
higher than that contained in the injectable formulation (1 and 0.1 
mmole/L respectively), whereas the total Ginkgolide B concentration is 40 
times lower (1 and 47 mmole/L respectively). 
EXAMPLE 2 
Preparation of an inclusion complex of Ginkgolide B with 
.gamma.-cyclodextrin 
The starting product is a concentrated Ginkgolide B solution in a 
water-miscible solvent, constituted by acetone, and addition takes place 
of the concentrated solution quantity corresponding to 1 .mu.mole of 
Ginkgolide B to 2 mL of 5 mmole/L .gamma.-cyclodextrin solution in sterile 
water, at pH=4.9 and at ambient temperature. The acetone is eliminated 
under nitrogen bubbling and the solution is lyophilized. The residual 
solid, which contains 10 .mu.mole of .gamma.-cyclodextrin derivative and 1 
.mu.mole of Ginkgolide B, is redissolved in a minimum of water at 
25.degree. C. This minimum corresponds to 2 mL of water, which indicates a 
maximum solubility of 0.5 mmole/l of Ginkgolide B in water in the presence 
of .gamma.-cyclodextrin at a concentration of 5 mmole/L. 
The partial nuclear magnetic resonance (NMR) spectrum of the proton of this 
complex at a concentration of 5 mmole/l in D.sub.2 O, at 298 K and at 500 
MHz is given in FIG. 2 (spectrum b). By comparing this spectrum b with 
spectrum a of Ginkgolide B alone, it can be seen that there are 
modifications to the spectrum confirming the inclusion of Ginkgolide B in 
the cavity of .gamma.-cyclodextrin. It can also be seen that Ginkgolide B 
only exists in the closed form at pH=4.9 in the inclusion complex. 
It should be noted that the solubility of the inclusion complex is the same 
at physiological pH, namely 0.5 mmole/L of Ginkgolide in the presence of 5 
mmole/L of .gamma.-cyclodextrin. With the inclusion complex prepared in 
the aforementioned manner, there are four different forms, the closed form 
representing 70%. When Ginkgolide B is in the form of an inclusion complex 
at pH 4.9 with a concentration of 0.5 mmole/L in the presence of 5 mmole/L 
of .gamma.-cyclodextrin, there is a closed form concentration 5 times 
higher than that contained in the injectable formulation (0.5 and 0.1 
mmole/L respectively), whereas the total Ginkgolide B concentration is 80 
times lower (0.5 and 40 mmole/L respectively). 
EXAMPLE 3 
Preparation of an inclusion complex of Ginkgolide B with 6-S- 
.alpha.-maltosyl-6-thiocyclomaltoheptaose. 
The starting product is a concentrated Ginkgolide B solution in a water 
miscible solvent, constituted by acetone, and addition takes place of the 
concentrated solution quantity corresponding to 1 .mu.mole of Ginkgolide 
B, to 1 mL of a 10 mmole/L solution of .beta.-cyclodextrin derivative, 
6-S- .alpha.-maltosyl-6-thiocyclomaltoheptaose, in sterile water, at 
pH=4.9 and at ambient temperature. The acetone is eliminated under 
nitrogen bubbling and the solution is lyophilized. The residual solid 
containing 10 .mu.mole of .beta.-cyclodextrin derivative and 1 .mu.mole of 
Ginkgolide B is redissolved in a minimum of water at 25.degree. C. This 
minimum corresponds to 500 ul of water, which indicates a maximum 
solubility of 2 mmole/L of Ginkgolide B in water, in the presence of said 
.beta.-cyclodextrin derivative at a concentration of 20 mmole/L. 
The partial nuclear magnetic resonance (NMR) spectrum of the proton of this 
complex at a concentration of 5 mmole/L in D.sub.2 O, at 298 K and 500 MHz 
is shown in FIG. 2 (spectrum d). On comparing spectrum d with spectrum a, 
the spectrum modifications can be seen which confirm the inclusion of the 
Ginkgolide B in the cavity of the .beta.-cyclodextrin derivative. It can 
also be observed that Ginkgolide B only exists in the inclusion complex in 
the closed form at pH=4.9. 
It should be noted that the solubility of the inclusion complex is the same 
at physiological pH, namely 2 mmole/L of Ginkgolide B in the presence of 
20 mmole/L of the .beta.-cyclodextrin derivative. With the inclusion 
complex prepared in the aforementioned manner, only two different forms 
are observed in identical proportions, including the closed form. When the 
Ginkgolide B is in the form of an inclusion complex, at pH 4.9, and with a 
concentration of 2 mmole/L in the presence of 20 mmole/L of 
6-S-.alpha.-maltosyl-6-thiocyclo-maltoheptaose, there is a closed form 
concentration 20 times higher than that contained in the injectable 
formulation (2 and 0.1 mmole/L respectively) whereas the total Ginkgolide 
B concentration is lower (2 and 40 mmole/L respectively).