Complexes and pharmaceutical preparations containing complexes of the pleuromutilin derivative of formula I ##STR1## in free base or in acid addition or quaternary salt form and cyclodextrin are described. They can be prepared by complexing the above pleuromutilin compound with an appropriate cyclodextrin. They are indicated for use as pharmaceuticals, e.g. as antibiotics, especially in veterinary medicine, e.g. as feed additives.

The present invention relates to pleuromutilin derivatives. In particular 
it provides complexes of a pleuromutilin derivative and cyclodextrin and 
pharmaceutical preparations containing theses complexes. 
Pleuromutilin derivatives are antibiotics with excellent micro-biological 
activity against a series of pathogenic microorganisms. In particular, the 
pleuromutilin derivative of formula I 
##STR2## 
namely 
14-O-[1-((D)-2-amino-3-methylbutyrylamino)-2-methylpropan-2-ylthioacetyl]m 
utilin, in free base or in acid addition or quaternary salt form, is highly 
effective against many gram-positive and gram-negative bacteria. Due to 
these properties the above compound may be employed as an antibiotic for 
the treatment of conditions induced by these pathogens, predominantly in 
veterinary medicine, for example on poultry, pigs, cattle, sheep and 
goats, especially in the treatment of respiratory tract disorders and 
dysentery. 
Cyclodextrins are cyclic oligosaccharides made up of 
.alpha.-(1.fwdarw.4)-linked D-glucopyranose units, e.g., for .alpha.-, 
.beta. and .gamma.-cyclodextrin, respectively of 6, 7 or 8 units, which 
are described in detail in the literature, e.g. in J. Szejtli, 
"Cyclodextrin Technology", Kluwer Academic Publishers (1988) and in D. 
Duchene "Cyclodextrins and their Industrial Uses", Editions de Sante 
(1987). In the preparations according to the invention any cyclodextrin, 
e.g. .alpha.-, .beta.- or .gamma.-cyclodextrin or a derivative thereof may 
be used. It is preferable to use .beta.- or .gamma.-cyclodextrin or a 
derivative thereof, especially .beta.- or .gamma.-cyclodextrin. .alpha.-, 
.beta.- and .gamma.-cyclodextrins are non-toxic compounds which may be 
used without risk as pharmaceutical excipients for oral preparations. 
Moreover, in many countries .beta.-cyclodextrin is also permissible as a 
food additive for human use. 
Derivatives of cyclodextrin are for example ethers with lower alcohols, 
such as methylcyclodextrin or hydroxypropylcyclodextrin. 
It is known from the literature that cyclodextrins can form inclusion 
compounds (inclusion complexes) with appropriate guest molecules, while 
the consequences on the effects obtained with the resultant complexes are 
difficult to predict. Based on the properties of the preparations 
according to the invention, it can be concluded that inclusion complexes 
similarly exist here. The observed desirable properties cannot be obtained 
solely from mere physical mixture. 
It is observed that, surprisingly, with the compound of formula I in free 
base or in acid addition or quaternary salt form the formation of 
complexes with cyclodextrins results in a marked improvement of the 
properties of the resultant pharmaceutical preparations, particularly as 
regards stability in feed mixtures upon storage. 
The compound of formula I is known from e.g. Sandoz U.S. Pat. No. 4,675,330 
as Example 12 therein. 
The preferred form of application of antibiotics to animals for therapeutic 
and prophylactic use is administration in the drinking water or feed. 
Whilst the compound of formula I in the form of its water-soluble 
hydrochloride may be administered easily and efficiently via the drinking 
water, application via the feed is difficult, because the substance is 
broken down very rapidly by constituents contained in conventional 
complete feed mixtures. However, the stability of ready feed/medicament 
mixtures is an essential requirement of practical application, since after 
stocks of such mixtures are produced it must be possible to stockpile them 
for at least several months. 
A form of preparation is therefore desirable which has adequate stability 
in ready feed mixtures and can be administered orally to animals. Apart 
from the stability requirement of the feed, other essential requirements 
for application are good absorption, inexpensive production costs, a 
simple production process and acceptance by the treated animal. 
It has now been found that preparations containing complexes of the 
compound of formula I in free base or in acid addition or quaternary salt 
form with a cyclodextrin fulfill these requirements to a large extent. 
The invention thus concerns complexes of the pleuromutilin derivative of 
formula I as defined above in free base or in acid addition or quaternary 
salt form and a cyclodextrin, hereinafter briefly named "the complexes of 
the invention". 
The invention further concerns pharmaceutical preparations comprising a 
complex of the pleuromutilin derivative of formula I as defined above in 
free base or in pharmaceutically acceptable acid addition or quaternary 
salt form and a cyclodextrin, together with at least one pharmaceutically 
acceptable carrier or diluent, hereinafter briefly named "the preparations 
of the invention". 
The complexes of the invention can be prepared by a process which comprises 
complexing the compound of formula I in free base or in acid addition or 
quaternary salt form with an appropriate cyclodextrin. 
The process of the invention is effected in conventional manner, preferably 
by contacting with an appropriate cyclodextrin under conditions suitable 
for complex formation. Various conventional method variants may be used 
thereby. It is possible to simply bring to dryness an aqueous solution of 
the compound and a cyclodextrin. To that effect lyophilization, 
spray-drying and/or evaporation can be employed. The complexes may further 
be obtained by moistening a physical mixture with water and kneading 
intensively. The complexes may also be produced by trituration of a such 
physical mixture of the two components, e.g. in a ball mill. Furthermore, 
it is possible to obtain the complexes by crystallization from a solution 
or a solvent mixture. 
The ratio of compound of formula I to cyclodextrin may vary within a wide 
range. The molar ratio of components can e.g. be in the range of from 
about 1:0.25 to about 1:2, preferably a ratio of from about 1:0.75 to 
about 1:1.25 is employed, especially a ratio of about 1:1. 
It is demonstrated by e.g. .sup.1 H-NMR spectroscopy (see under 3. below) 
that a chemical bond comprising at least hydrogen bonding or Van der Waals 
forces is formed between the compound of formula I and the cyclodextrin. 
The preparations of the invention are manufactured in conventional manner, 
e.g. by a process which comprises mixing a complex of the pleuromutilin 
derivative of formula I as defined above in free base or in 
pharmaceutically acceptable acid addition or quaternary salt form and an 
appropriate cyclodextrin together with at least one pharmaceutically 
acceptable carrier or diluent. 
The preparations of the invention can be manufactured with the compound of 
formula I in free base form or in water-soluble or water-insoluble salt 
form. Preferably the hydrochloride is employed. 
The resultant inclusion complexes are described in more detail in the 
following experiments: 
1. Phase-solubility 
Based on the phase-solubility diagram (see "Methods of investigating and 
preparing inclusion compounds and their industrial uses" in D. Duchene, 
"Cyclodextrins and their Industrial Uses", Editions de Sante [1987]), the 
following types of inclusion compounds are obtained with the compound of 
formula I in the form of the base and 
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cyclodextrin: type A.sub.L 
.gamma.-cyclodextrin: type B.sub.S. 
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For the compound of formula I the complex binding constant is 1050M.sup.-1 
with .beta.-cyclodextrin and 1400M.sup.-1 with .gamma.-cyclodextrin. 
2. Calorimetry 
In micro-calorimetric examination of the complex of the compound of formula 
I in the form of the hydrochloride and .beta.-cyclodextrin (L. E. Briggner 
et al. Microcalorimetric titration of .beta.-cyclodextrine with 
adamantane-1-carboxylate, Thermochimica Acta 109 [1986] 139-143), a 
complex binding constant of 1004M.sup.-1 is obtained. 
3. .sup.1 H-NMR-spectrum: 
In the .sup.1 H-NMR-spectrum of a solution of the compound of formula I in 
the form of the hydrochloride and .beta.-cyclodextrin in D.sub.2 O, marked 
changes in the signal position appear when compared with a pure solution 
of the compound of formula I, and these are attributable to the following 
functional groups: 
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Compound of Complex of 
formula I .beta.-cyclodextrin and compound 
(hydrochloride) 
of formula I (Example 3) 
Proton no. 
(ppm) (ppm) 
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16 d 0.73 s broad 0.69 
17 d 0.97 s broad 0.96 
18 s 1.45 s 1.49 
8e d 1.99 d 1.72 
4 s 2.56 s broad 2.58 
20.20 dd 5.24 dd broad 5.35 
19 dd 6.37 s broad .about.6.5 
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4. Differential scanning calorimetry: 
A preparation of .gamma.-cyclodextrin and the compound of formula I in 
hydrochloride form corresponding to Example 1 shows in differential 
scanning calorimetry (10.degree. K./min) an endothermic signal at 
193.degree. C. 
5. Complex analysis: 
Determination of the content of a preparation corresponding to Example 1 
resulted in 
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compound of formula I (hydrochloride) 
28.6% 
H.sub.2 O 5.9% 
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This content corresponds to a molar ratio of 1:1 of compound of formula I 
(HCl) to cyclodextrin. 
In the following non-limitative Examples all temperatures are in degrees 
Centigrade:

EXAMPLE 1 
5.76 g .gamma.-cyclodextrin are dissolved in 18 ml of water under heating 
and 2.5 g compound of formula I (hydrochloride) are added. The solution is 
slowly cooled to 5.degree. whilst stirring. The resultant white 
crystalline precipitate is decanted and dried in a vacuum drying chamber. 
EXAMPLE 2 
7.8 g .beta.-cyclodextrin are dissolved in 60 ml of water under heating and 
3.87 g compound of formula I (hydrochloride) are added. The solution is 
evaporated to dryness under reduced pressure. The dry residue is crushed 
through a 1 mm sieve. 
EXAMPLE 3 
The procedure is effected in a manner analogous to Example 2, with the 
exception that the solution is spray-dried at an air temperature of 
180.degree.. 
EXAMPLE 4 
The procedure is effected in a manner analogous to Example 2, with the 
exception that the solution is frozen to -40.degree. during 3 hours and 
lyophilized. 
EXAMPLE 5 
2.9 g .gamma.-cyclodextrin are dissolved in 10 ml of water under heating 
and 2.5 g of compound of formula I (hydrochloride) are added. The solution 
is spray-dried at an air temperature of 180.degree.. 
EXAMPLE 6 
7.7 g compound of formula I (hydrochloride) are mixed with 15.5 g 
.beta.-cyclodextrin and 15 ml of water are added in a kneading machine. 
The moist mass is kneaded intensively for 3 hours. It is subsequently 
dried in a vacuum drying chamber and crushed through a sieve. 
EXAMPLE 7 
3.5 g compound of formula I (hydrochloride) are mixed with 14.1 g 
.beta.-cyclodextrin and ground in a ball mill for 6 hours. 
EXAMPLE 8 
Pre-mix 
The preparations corresponding to Examples 1 to 7 are mixed with a meal 
feed mixture to a 2% pre-mix. The pre-mix is diluted to a concentration of 
200 ppm of compound of formula I by mixing in a freefall mixer with 
further meal feed mixture. 
EXAMPLE 9 
Feed pellets 
A feed mixture according to Example 8 is pressed into feed pellets using a 
appropriate pellet press and steam. 
Some user-relevant properties of the preparations according to the 
invention are described in the following tests: 
1. Feed stability 
As model feed a ready feed for piglets of the following composition is 
used: 
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oats 10.0% 
corn 29.0% 
barley 26.7% 
wheat 8.0% 
soya scraps 23.0% 
calcium carbonate 1.05% 
dicalcium phosphate 
1.5% 
iodized cattle salt 
0.25% 
pre-mixture 0.5% 
(mineral-vitamin) 
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Various forms of the preparation are mixed with the ready feed in a 
concentration of 200 ppm of compound of formula I and stored for several 
weeks at 30.degree.. Both the starting value of the fresh mixtures and the 
content values after storage for 2, 4 and 8 weeks are analysed by high 
pressure liquid chromatography (HPLC). 
Table 1 shows the improvement in storage stability when using the complexes 
of the invention in a feed mixture, as compared with a corresponding 
preparation containing no cyclodextrin: 
TABLE 1 
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Content (in %) 
Feed mixture initial after after after 
with value 2 weeks 4 weeks 
8 weeks 
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Compound of formula I 
100 15 8 6 
(hydrochloride) alone 
Complex of Example 3 
100 89 87 90 
Complex of Example 6 
100 91 92 88 
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For determination of the optimum ratio of compound of formula I to 
cyclodextrin, investigations are carried out using various concentrations 
of cyclodextrin. Results as regards stability in feed mixtures are 
summarized in Table 2 [in the first column "ratio" means the ratio of 
compound of formula I (hydrochloride) to .beta.-cyclodextrin]: 
TABLE 2 
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Content (in %) 
Initial after after after 
Ratio value 1 month 2 months 
3 months 
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1:0 100 9 9 7 
1:2 100 86 89 91 
1:1 100 90 89 96 
1:0.75 100 49 45 38 
1:0.5 100 18 9 8 
1:0.25 100 14 8 9 
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2. Oral absorption: 
Absorption following administration to rats and pigs of 25 mg/kg body 
weight of a preparation according to Example 3 is tested and compared with 
pure compound of formula I (hydrochloride) in the drinking water. Blood 
samples are taken after a period of 24 hours and the concentration of 
active substance in plasma determined by microbiological analysis. The 
results in Table 3 show the absorption of the compound of formula I when 
administered in the form of a complex of the invention: 
TABLE 3 
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C.sub.max (.mu.g/ml) 
AUC (.mu.g/ml.h) 
rat pig rat pig 
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Compound of formula I 
8.6 1.2 139 21.4 
(hydrochloride) alone 
Complex according to 
11.7 1.6 294 24.3 
Example 3 
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C.sub.max = maximum plasma concentration 
AUC = area under curve 
The complexes of the pleuromutilin derivative of formula I as defined above 
in free base or in pharmaceutically acceptable acid addition or quaternary 
salt form are therefore indicated for use as pharmaceuticals, particularly 
as antibiotics in the treatment of conditions induced by gram-positive and 
gram-negative bacteria, such as respiratory tract disorders and dysentery. 
For this use the dosage to be employed will vary, of course, depending on 
the particular complex used, the mode of administration, the subject to be 
treated and the treatment desired. However, in general, satisfactory 
results are obtained when the complexes are administered at a daily dosage 
in e.g. pigs of from about 1 mg/kg to about 100 mg/kg body weight, 
conveniently from about 5 mg/kg to about 10 mg/kg, if desired given in 
divided dosages 2 to 4 times daily. 
The complexes of the invention may be administered in similar manner to 
known standards for use in such indications. The complexes may be admixed 
with conventional pharmaceutically acceptable carriers and diluents and, 
optionally, further excipients, and administered e.g. orally in such forms 
as feed pellets and premixes. 
The invention further includes a method of treatment of bacterial 
infections which comprises administering a therapeutically effective 
amount of a complex of the pleurcmutilin derivative of formula I as 
defined above in free base or in pharmaceutically acceptable acid addition 
or quaternary salt form and a cyclodextrin to subjects, particularly to 
animals such as poultry, pigs, cattle, sheep and goats, in need of such 
treatment.