1-Cyclopropylquinolone-3-carboxylic acids of the formula ##STR1## in which R.sup.1 is hydrogen, methyl, ethyl or .beta.-hydroxyethyl, and PA1 R.sup.2 is hydrogen, chlorine or fluorine or salts thereof stimulate the immune system, especially in conjunction with an antigen.

The present invention relates to immunostimulating agents which contain 
known 1-cyclopropylquinolone-3-carboxylic acids of the general formula I 
##STR2## 
in which R.sup.1 represents hydrogen, methyl, ethyl or 
.beta.-hydroxyethyl, and 
R.sup.2 represents hydrogen, chlorine or fluorine, and/or their 
physiologically tolerated acid addition salts and alkali metal salts. 
It has already been disclosed that certain 
1-cyclopropylquinolone-3-carboxylic acids have good antibacterial effects 
(DE-OS [German Published Specification] No. 3,142,854). 
It has now been found that the known 1-cyclopropylquinolone-3-carboxylic 
acids of the general formula I and their physiologically tolerated acid 
addition salts and alkali metal salts have immunostimulating effects. 
Surprisingly, in addition to the very good antibacterial effects, they 
have the abovementioned immunostimulating effects to a greater extent than 
do known immunostimulating substances. Thus, the agents according to the 
invention represent an enrichment of pharmacy. 
The active compounds of the general formula I, according to the invention, 
are known (DE-OS [German Published Specification] No. 3,142,854, DE-OS 
[German Published Specification] No. 3,033,157) and DE-OS (German 
Published Specification No. 33 18 145). 
The following may be particularly mentioned as active compounds: 
1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarbo 
xylic acid (ciprofloxacin); 
1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(4-ethyl-1-piperazinyl)-3-quinol 
inecarboxylic acid; 
1-cyclopropyl-6,8-difluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-4-quinolinec 
arboxylic acid; 
1-cyclopropyl-6,8-difluoro-1,4-dihydro-4-oxo-7-(4-methyl-1-piperazinyl)-3-q 
uinolinecarboxylic acid. 
The following groups of patients, for example, can be treated successfully 
with the agents according to the invention: 
Patients who suffer from immune deficiencies related to infection or age, 
induced by tumors or caused by cytostatic agents. 
In addition, postoperative and post-traumatic deficiences of the 
immunological defences occur. The treatment of patients with acquired 
immune deficiencies of this type is as yet unsatisfactory. 
All these patients require medications which support or normalize the 
body's own defences. 
The present invention includes pharmaceutical formulations which, in 
addition to non-toxic, inert pharmaceutically suitable vehicles, contain 
one or more active compounds according to the invention or which consist 
of one or more active compounds according to the invention, and processes 
for the preparation of these formulations. 
The present invention also includes pharmaceutical formulations in dosage 
units. This means that the formulations are in the form of individual 
parts, for example tablets, coated tablets, capsules, pills, suppositories 
and ampules, of which the content of active compound corresponds to a 
fraction or a multiple of an individual dose. The dosage units can 
contain, for example, 1, 2, 3 or 4 individual doses or 1/2, 1/3 or 1/4 of 
an individual dose. An individual dose preferably contains the amount of 
active compound which is given in one administration and which usually 
corresponds to a whole, a half or a third or a quarter of a daily dose. 
By non-toxic, inert pharmaceutically suitable vehicles there are to be 
understood solid, semi-solid or liquid diluents, fillers and formulation 
auxiliaries of all kinds. 
Tablets, coated tablets, capsules, pills, granules, suppositories, 
solutions, suspensions and emulsions, pastes, ointments, gels, creams, 
lotions, powders and sprays may be mentioned as preferred pharmaceutical 
formulations. 
Tablets, coated tablets, capsules, pills and granules can contain the 
active compound or compounds alongside the customary vehicles such as (a) 
fillers and extenders, for example starches, lactose, sucrose, glucose, 
mannitol and silica, (b) binders, for example carboxymethylcellulose, 
alginates, gelatin and polyvinylpyrrolidone, (c) humectants, for example 
glycerol, (d) disintegrating agents, for example agar-agar, calcium 
carbonate and sodium bicarbonate, (e) solution retarders, for example 
paraffin, and (f) absorption accelerators, for example quaternary ammonium 
compounds, (g) wetting agents, for example cetyl alcohol or glycerol 
monostearate, (h) adsorbents, for example kaolin and bentonite, and (i) 
lubricants, for example talc, calcium stearate and magnesium stearate and 
solid polyethylene glycols, or mixtures of these substances listed under 
(a) to (i). 
The tablets, coated tablets, capsules, pills and granules can be provided 
with the customary coatings and shells, optionally containing opacifying 
agents, and can also be of such composition that they release the active 
compound or compounds only, or preferentially, in a certain part of the 
intestinal tract, optionally in a delayed manner, examples of embedding 
compositions which can be used being polymeric substances and waxes. 
The active compound or compounds, optionally together with one or more of 
the abovementioned vehicles, can also be in a microencapsulated form. 
Suppositories can contain, in addition to the active compound or compounds, 
the customary water-soluble or water-insoluble vehicles, for example 
polyethylene glycols, fats, for example cacao fat, and higher esters (for 
example C.sub.14 -alcohol with C.sub.16 -fatty acid) or mixtures of these 
substances. 
Ointments, pastes, creams and gels can contain the customary vehicles in 
addition to the active compound or compounds, for example animal and 
vegetable fats, waxes, paraffins, starch, tragacanth, cellulose 
derivatives, polyethylene glycols, silicones, bentonites, silica, talc and 
zinc oxide or mixtures of these substances. 
Powders and sprays can contain the customary vehicles in addition to the 
active compound or compounds, for example lactose, talc, silica, aluminum 
hydroxide, calcium silicate and polyamide powders or mixtures of these 
substances. Sprays can additionally contain the customary propellants, for 
example chlorofluorohydrocarbons. 
Solutions and emulsions can contain the customary vehicles in addition to 
the active compound or compounds, such as solvents, solubilizing agents 
and emulsifiers, for example water, ethyl alcohol, isopropyl alcohol, 
ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene 
glycol, 1,3-butylene glycol, dimethylformamide, oils, especially 
cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and 
sesame oil, glycerol, glycerol-formal, tetrahydrofurfuryl alcohol, 
polyethylene glycols and fatty acid esters of sorbitan, or mixtures of 
these substances. 
For parenteral administration, the solutions and emulsions can also be in a 
sterile form which is isotonic with blood. 
Suspensions can contain the customary vehicles in addition to the active 
compound or compounds, such as liquid diluents, for example water, ethyl 
alcohol or propylene glycol, suspending agents, for example ethoxylated 
isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, 
microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar 
and tragacanth, or mixtures of these substances. 
The formulation forms mentioned can also contain colorants, preservatives 
and additives which improve the odor and flavor, for example peppermint 
oil and eucalyptus oil, and sweeteners, for example saccharin. 
The therapeutically active compounds should preferably be present in the 
abovementioned pharmaceutical formulations in a concentration of about 0.1 
to 99.5, preferably of about 0.5 to 95, % by weight of the total mixture. 
The abovementioned pharmaceutical formulations can also contain other 
pharmaceutical active compounds in addition to the active compounds 
according to the invention. 
The abovementioned pharmaceutical formulations are prepared in the usual 
manner according to known methods, for example by mixing the active 
compound or compounds with the vehicle or vehicles. 
The present invention also includes the use of the active compounds 
according to the invention and of pharmaceutical formulations which 
contain one or more active compounds according to the invention, in human 
and veterinary medicine for the prevention, amelioration and/or cure of 
the abovementioned illnesses. 
The active compounds or the pharmaceutical formulations can be administered 
locally, orally, parenterally, intraperitoneally and/or rectally, 
preferably parenterally, especially intravenously. 
In general it has proved advantageous, both in human medicine and in 
veterinary medicine, to administer the active compound or compounds in 
amounts of about 10 to about 300, preferably 2.5 to 25, mg/kg of body 
weight every 24 hours, optionally in the form of several individual 
administrations, in order to achieve the desired results. 
However, it can be necessary to deviate from the dosages mentioned and in 
particular to do so as a function of the nature and body weight of the 
subject to be treated, the nature and severity of the illness, the nature 
of the formulation and of the administration of the medicament, and the 
time or interval over which the administration takes place. Thus it can 
suffice in some cases to manage with less than the abovementioned amount 
of active compound, while in other cases the abovementioned amount of 
active compound must be exceeded. 
The particular required optimum dosage and the type of administration of 
the active compounds can easily be decided by anyone skilled in the art, 
on the basis of his expert knowledge. 
The active compounds according to the invention have a pronounced effect 
increasing the defenses. They increase in an antigen-specific manner the 
humoral, antibody-mediated and cell-mediated defenses of the immune 
system. 
These results were obtained using the following design of experiment: 
1. Increase in the primary humoral immunity in vivo toward the soluble 
antigen ovalbumin using ciprofloxacin as an example. 
NMRI mice were immunized subcutaneously (s.c.) with a suboptimal dose of 
antigen (1 .mu.g/animal, day 0). With suboptimal antigenic stimulation, 
only a small number of lymphocytes of the animals are stimulated to 
synthesize antibodies. Additional treatment of the animals with 
ciproflocacin is able, on single administration of 10-80 mg/kg 
subcutaneously, to increase slightly, but significantly, the antibody 
titre in the serum of the animals (Table 1). The antibodies were 
determined by indirect haemagglutination on day 10. The effect of the 
treatment is expressed by the geometric mean of log.sub.2 titre. 
The immunostimulating effect of ciprofloxacin is dependent on the antigen, 
in contrast to other, for example, bacterial immunostimulants, such as LPS 
from Gram-negative bacteria, that is to say the substance induces 
synthesis of antibodies only in conjunction with an antigenic stimulus 
(ovalbumin in this case). 
2. Increase in the non-specific, mitogen-induced stimulation of mouse 
spleen lymphocytes (MSL). 
Balb/c mice were pretreated for 7 days with various s.c. doses of 
ciprofloxacin. SL of animals pretreated in this manner were stimulated on 
the 8th day ex vivo with the T-cell mitogens ConA (5 .mu.g/ml) and PHA (10 
.mu.g/ml). After 48 hours, the cultures were labelled with .sup.3 
H-thymidine for a further 16 hours, and were harvested. The incorporation 
of the radioactive thymidine in the newly synthesized DNA is taken as a 
measure of the lymphocyte proliferation which has occurred. The results 
show a marked increase in proliferation within the dose range 1-80 mg/kg 
(Table 2). 
TABLE 1 
______________________________________ 
Effect of ciprofloxacin on the synthesis of antibodies against the 
soluble antigen ovalbumin 
Dose (mg/kg) 
Active compound 
0 1 5 10 20 80 
______________________________________ 
Haemagglutination titre log.sub.2 
Ciprofloxacin 
4.6 4.8 4.8 5.4.sup.(1) 
5.4.sup.(1) 
5.4.sup.(1) 
______________________________________ 
.sup.(1) Statistically significant p &lt; 0.05 
TABLE 2 
______________________________________ 
Non-specific mitogen stimulation of mouse spleen 
lymphocytes in mice pretreated with ciprofloxacin 
Experiment 1 Experiment 2 
Dose (mg/kg) 
ConA PHA 0 ConA PHA 0 
______________________________________ 
.sup.3 H--thymidine incorporation (CPM) 
0 49,208 39,620 455 57,148 
32,372 
1,228 
1 122,421 37,093 424 84,374 
29,056 
2,534 
5 79,249 75,686 320 84,368 
45,400 
640 
20 57,009 46,289 342 68,826 
34,827 
416 
80 60,963 42,286 620 61,780 
22,818 
700 
______________________________________ 
A similar proliferation behavior was also achieved on treatment in vitro of 
the MSL mitogen cultures of non-pretreated mice. 
3. Increase in the specific T-cell-mediated, allogeneic immune response 
(mixed lymphocyte culture--MLC) of MSL 
C 57 bl mice were pretreated for 7 days with various s.c. doses of 
ciprofloxacin. Spleens of animals pretreated in this manner were suspended 
on the 8th day after removal, and T-lymphocytes from each of these 
suspensions were enriched by passage through a nylon wool column (modified 
method of S. A. Eisen, H. J. Wedner and C. W. Parker, Immunological 
Communications 1, 571 [1972]). 
These T-cell fractions were used as responders in the one-way MLC. The 
stimulator cells used were MS cells of D8A mice treated with mitomycin C. 
After 72 hours, .sup.3 H-thymidine was added to the cultures which were 
harvested after a further 16 hours (modified method of D. B. Amos and F. 
K. Bach, J. Exp. Med. 128, 623 [1968]). Measurements of the incorporation 
of .sup.3 H-thymidine in this test system again showed a significant 
increase in proliferation compared with the control in the dose range 1-80 
mg/kg. 
Treatments in vitro of cultures of this type from non-pretreated animals 
likewise result in an increased proliferation of T-lymphocytes in the dose 
range 0.01-10 .mu.g of substance per ml. 
4. Mitogenic and allogeneic T-lymphocyte activation of human blood 
lymphocytes (PBL). 
Lymphocytes for this purpose were isolated by density gradient 
centrifugation from the heparinized blood of healthy donors. The cells 
thus obtained were cultured in parallel batches with the mitogens PHA (5 
.mu.g/ml) and ConA (5 .mu.g/ml).+-.the selected substance, reacted with 
.sup.3 H-thymidine after 72 hours, and harvested after a further 16 hours. 
Increases in response to the mitogen were observed in the dose range 1-30 
.mu.g of substance per ml (Table 3). 
Isolated, unfractionated PBL which were simultaneously activated in the 
one-way MCL using the substance, selected as an example, of the 
derivatives described above, at various doses, show, after culturing for 
136 hours, increases in proliferation compared with the controls in the 
dose range 0.1-30 .mu.g of substance per ml (Table 4). 
In contrast to other, conventional immunostimulants, such as, for example, 
LPS mentioned above, these compounds have no mitogenic properties, that is 
to say they act only in conjunction with an additional induced stimulus. 
TABLE 3 
__________________________________________________________________________ 
Effect of ciprofloxacin on the non-specific, mitogenic activation of 
human blood lymphocytes 
Dose Experiment 1.sup.1 
Experiment 2.sup.1 
Experiment 3.sup.2 
(.mu.g/ml) 
PHA ConA 
0 PHA ConA 
0 PHA ConA 
0 
__________________________________________________________________________ 
.sup.3 H--thymidine incorporation (CPM) 
0 102,566 
41,592 
330 
133,182 
57,604 
693 
44,028 
17,866 
1,081 
1 109,750 
46,576 
335 
125,109 
56,166 
479 
111,379 
31,276 
844 
3 105,730 
45,919 
386 
112,918 
65,598 
447 
122,275 
34,096 
884 
10 144,867 
52,455 
333 
143,780 
73,484 
460 
145,795 
36,080 
1,061 
.sup. 30.sup.3 
171,005 
36,546 
437 
176,089 
56,454 
344 
129,103 
13,790 
463 
__________________________________________________________________________ 
.sup.1 With lymphocytes from fresh blood 
.sup.2 With thawed lymphocytes which had been stored in nitrogen 
.sup.3 Cytotoxic effects already present 
TABLE 4 
__________________________________________________________________________ 
Effect of ciprofloxacin on the specific, allogeneic activation of 
human blood lymphocytes 
Dose (.mu.g/ml) 
Experiments 
0 0.1 0.3 1 3 10 30.sup.2 
__________________________________________________________________________ 
.sup.3 H--thymidine incorporation (CPM) 
No. 1 CO.sup.1 
4,890 
Test 17,252 
21,310 
26,964 
28,982 
27,841 
32,733 
28,900 
No. 2 CO 
946 
Test 42,292 
64,461 
82,315 
87,12 
82,933 
72,662 
34,988 
No. 3 CO 
245 
Test 11,679 
21,768 
26,564 
20,314 
13,964 
15,142 
4,632 
__________________________________________________________________________ 
.sup.1 Autologous control: lymphocytes treated with mitomycin C as the 
stimulator 
.sup.2 Already cytotoxic effects present

PREATION EXAMPLES 
EXAMPLE 1 
Ciprofloxacin 
##STR3## 
A mixture of 19.7 g of 
7-chloro-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic 
acid, 30.1 g of anhydrous piperazine and 100 ml of dimethyl sulphoxide is 
heated at 135.degree.-140.degree. C. for 2 h. The solvent is removed by 
distillation under high vacuum, and the residue is suspended in H.sub.2 O, 
filtered off with suction and washed with water. For further purification, 
the moist crude product is boiled with 100 ml of water, filtered with 
suction at room temperature, washed with H.sub.2 O and dried to constant 
weight in a vacuum drying oven over CaCl.sub.2 at 100.degree. C. 19.6 g of 
1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-quinoline-3-car 
boxylic acid of decomposition point 255.degree.-257.degree. C. are 
obtained. 
The compound prepared in Example 1 is dissolved in 50 ml of hot 10% 
hydrochloric acid. The solution is filtered, 150 ml of ethanol are added, 
then it is cooled in ice, and the product is filtered off with suction, 
washed with alcohol and dried in vacuo at 100.degree. C. 
18.5 g of 
1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-quinoline-3-car 
boxylic acid hydrochloride are obtained as colorless crystals of 
decomposition point 308.degree.-310.degree. C. 
EXAMPLE 2 
##STR4## 
A mixture of 1.2 g of 
1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-quinoline 
-3-carboxylic acid, 1.13 g of ethyl iodide, 0.73 g of triethylamine and 20 
ml of N,N-dimethylformamide is heated at 70.degree.-80.degree. C. for 2.5 
h. The solvent is removed by distillation in vacuo, and the residue is 
suspended in water. The product is filtered off with suction, washed with 
H.sub.2 O and pressed on a clay plate. 1.15 g of 
1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(4-ethyl-1-piperazinyl)-quinoli 
ne-3-carboxylic acid hydroiodide of decomposition point 306.degree. C. are 
obtained. 
EXAMPLE 3 
##STR5## 
A mixture of 2.83 g (0.01 mol) of 
1-cyclopropyl-6,7,8-trifluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic 
acid, 4.4 g (0.051 mol) of anhydrous piperazine and 30 ml of dry pyridine 
is refluxed for 6 hours. The solvent is removed in vacuo, the residue is 
taken up in 25 ml of water, the pH is adjusted to 1 with concentrated 
hydrochloric acid while cooling in ice, the precipitate is filtered off 
cold with suction, and washed with cold 10% strength hydrochloric acid and 
ethanol. After drying in vacuo at 100.degree. C., 3.05 g of 
1-cyclopropyl-6,8-difluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline 
carboxylic acid hydrochloride of decomposition point 
354.degree.-355.degree. C. are obtained. 
EXAMPLE 4 
2.83 g (0.01 mol) of 
1-cyclopropyl-6,7,8-trifluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid 
are reacted with 4 g (0.04 mol) of N-methylpiperazine in analogy to 
Example 3, and 3.6 g of 
1-cyclopropyl-6,8-difluoro-1,4-dihydro-4-oxo-7-(4-methyl-1-piperazinyl)-3- 
quinolinecarboxylic acid hydrochloride of decomposition point 
300.degree.-303.degree. C. are isolated. 
It will be understood that the specification and examples are illustrative 
but not limitative of the present invention and that other embodiments 
within the spirit and scope of the invention will suggest themselves to 
those skilled in the art.