Amino glycoside antibiotics having antiprotozoal activity

In the fermentation production of gentamicin C a plurality of additional antibiotic compounds are obtained. Four of these compounds, gentamicin A, gentamicin B, gentamicin B.sub.1 and gentamicin X exhibit significant anti-protozoal and anthelmintic activity while gentamicin B and B.sub.1 have significant antibacterial properties with markedly reduced side effects.

FIELD OF INVENTION 
This invention relates to antibiotics which possess antiprotozoal and 
anthelmintic activity and to methods for their production and use. More 
particularly, this invention relates to antibiotics co-produced with 
gentamycin, said antibiotics either in admixture with each other or 
separated one from the other possessing antiprotozoal and/or anthelmintic 
properties. 
BACKGROUND OF INVENTION 
The preparation, isolation and purification of gentamycin is described in 
U.S. Pat. No. 3,091,572. Gentamycin, now known as gentamicin, was 
described in the patent as the principal antibiotic component and is 
referred to herein as the C-component or gentamicin C. In the patent, 
reference is made to co-produced antibiotics BA-3 (Fraction A) and BA-3 
(Fraction B) which in admixture with each other exhibited a low order of 
antibacterial activity. These were separated from gentamicin C. 
By the utilization of a particular chromatographic system described later 
herein, we have found that the substances described in U.S. Pat. No. 
3,091,572 as co-produced antibiotics BA-3 (Fraction A) and BA-3 (Fraction 
B) are not single entities unto themselves but together they represent a 
mixture of at least four components. Resolution of this mixture of the 
co-produced antibiotics into its components (hereinafter called gentamicin 
A, gentamicin B, gentamicin B.sub.1 and gentamicin X), has led to the 
discovery that each member of this group possesses surprising and 
unexpected applied-use characteristics. 
Gentamicin A has been previously isolated in pure form, investigated 
chemically and biologically and found to have rather weak antibiotic 
activity. We have discovered the rather surprising applied-use 
characteristic of gentamicin A, namely, its utility as an antiprotozoal 
agent. Gentamicin B, gentamicin B.sub.1 and gentamicin X heretofore have 
never been isolated one from the other. We have discovered that these 
substances possess certain valuable properties as antibiotics, 
anthelmintic and antiprotozoal agents. 
BRIEF DESCRIPTION OF THE INVENTION 
We have found that the entire mixture of antibiotics produced by the 
fermentaion of Micromonospora purpurea and Micromonospora echinospora as 
described in U.S. Pat. No. 3,091,572 may be separated by means of a column 
chromatographic separation process which utilizes the lower layer of a 
solvent system consisting of methanol, chloroform, and ammonium hydroxide 
as the eluting solvent. Prior to chromatography, equal volumes of the 
solvents are mixed, allowed to settle and the layers separated. Gentamicin 
C, also known as Garamycin.RTM. (Trademark Schering Corporation, 
Bloomfield, New Jersey) (known to be composed of three components, 
C.sub.1, C.sub.1a and C.sub.2) is first eluted and is followed in turn by 
gentamicin B.sub.1, gentamicin X, gentamicin B and lastly, gentamicin A. 
These antibiotics co-produced with gentamicin C and eluted after 
gentamicin C are structurally similar to each other as shown in formula I 
which depicts gross molecular arrangement without regard to spatial 
considerations. 
##STR1## 
wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are as defined above. 
Gentamicin A and gentamicin X, and to a lesser extent, gentamicin B and 
gentamicin B.sub.1, are effective against such protozoa as Entamoeba 
histolytica, Histomonas meleagridis, Trichomonas foetus and Trichomonas 
vaginalis. Histomonas meleagridis is the protozoal parasite which causes 
enterohepatitis, the so-called "blackhead disease" in turkeys. This 
disease causes serious economic problems in the turkey raising industry. 
Trichomonas vaginalis is the pathogen most frequently associated with 
cases of human vaginal infections and is usually a most difficult pathogen 
to eradicate. 
Gentamicin A, gentamicin B, gentamicin B.sub.1 and gentamicin X are also 
effective as anthelmintic agents against pinworms such as Syphacia 
obvelata. 
That these substances, gentamicin A, gentamicin B, gentamicin B.sub.1 and 
gentamicin X, possess valuable antiprotozoal properties is surprising. It 
has been reported that gentamicin C, which is structurally related to the 
aforementioned antibiotics, exhibits only slight activity against T. 
vaginalis at somewhat high dose ranges. It has been further reported by 
Albach et al. in the American Journal of Tropical Medicine and Hygiene, 
15, 885 (1965) that gentamicin C is relatively ineffective against E. 
histolytica. All of these gentamicin antibiotics are members of a chemical 
class of compounds sometimes referred to as aminoglycosides. This class 
includes such known antibiotics as neomycin, kanamycin, streptomycin and 
paromomycin. Only paromomycin is known to exhibit antiprotozoal activity 
to any substantial degree. Furthermore, with the exception of paromomycin, 
gentamicin A, gentamicin B, gentamicin B.sub.1 and gentamicin X are the 
only aminoglycosides now known to exhibit anthelmintic activity in 
reasonably useful dosage ranges. 
In view of these properties gentamicin A, gentamicin B, gentamicin B.sub.1 
and gentamicin X may be used in vitro in wash solution for sanitary 
purposes, such as in the cleaning of laboratory equipment contaminated 
with protozoa of the type named above. This is especially true of 
laboratory table tops and animal cages and pens from which it is desired 
to eradicate susceptible parasitic protozoal and bacterial species. By 
virtue of their in vivo activity, the compounds of this invention may be 
used in admixture with drinking water or animal feeds to treat laboratory 
or domestic animals having intestinal bacterial infections, intestinal 
protozoal infections, and helmintic infestations, or combinations of the 
foregoing wherein the infecting organisms are susceptible to the compounds 
of this invention. The compounds are especially useful for intestinal 
disorders in view of the fact that they are not appreciably absorbed from 
the gastro-intestinal tract. Thus, substantial concentrations of 
therapeutic agent may be ingested without causing toxic manifestations to 
the host, and at the same time be present in quantities sufficient to 
destroy, or at least to substantially inhibit, the infecting organism. For 
combatting generalized (systemic) bacterial infections administration of 
antibiotic is efffected by other than oral means, preferably by injection. 
SUMMARY OF THE INVENTION 
The invention sought to be patented in one of its process aspects resides 
in the concept of resolving a mixture consisting of gentamicin A, 
gentamicin B, gentamicin B.sub.1 and gentamicin X into the individual 
antibiotic entities of which the mixture is composed. 
The invention sought to be patented in another of its process aspects 
resides in the concept of combatting bacterial infections in animals which 
comprises administering to an animal having such an infection a 
therapeutically effective quantity of a member selected from the group 
consisting of gentamicin B, gentamicin B.sub.1, gentamicin X or their 
non-toxic pharmaceutically acceptable acid addition salts. 
The invention sought to be patented in a further process aspect resides in 
the concept of treating bacterial infections in animals which comprises 
administering to an animal having such an infection a therapeutically 
effective quantity of a mixture containing as the principal active 
ingredients one or more of the substances of gentamicin B, gentamicin 
B.sub.1, gentamicin X or their non-toxic pharmaceutically acceptable acid 
addition salts. 
The invention sought to be patented in yet another of its process aspects 
resides in the concept of combatting protozoal infections and/or helmintic 
infestations which comprises administering to an animal having such an 
infection or infestation, a therapeutically effective quantity of a member 
selected from the group consisting of gentamicin A, gentamicin B, 
gentamicin B.sub.1, gentamicin X or their non-toxic pharmaceutically 
acceptable acid addition salts. 
The invention sought to be patented in still another of its process aspects 
resides in the concept of treating protozoal infections and/or helmintic 
infestations which comprises administering to an animal, having such an 
infection or infestation, a therapeutically effective quantity of a 
mixture containing as the principal active ingredient one or more of the 
substances gentamicin A, gentamicin B, gentamicin B.sub.1 and gentamicin X 
or the non-toxic pharmaceutically acceptable acid addition salts thereof. 
The invention sought to be patented in one of its composition aspects 
resides in the concept of the novel chemical compounds gentamicin B, 
gentamicin B.sub.1, gentamicin X having the molecular structure as 
hereinabove described and the non-toxic pharmaceutically acceptable salts 
of said compounds. 
The invention sought to be patented in another of its composition aspects 
resides in the concept of pharmaceutical compositions for eliciting an 
antibacterial, an anti-protozoal and/or anthelmintic response which 
comprises a therapeutically effective quantity a member selected from the 
group consisting of gentamicin A, gentamicin B, gentamicin B.sub.1, 
gentamicin X a non-toxic pharmaceutically acceptable acid addition salt 
thereof, or mixtures thereof, and an inert pharmaceutical carrier. 
DETAILED DESCRIPTION OF THE INVENTION 
Gentamicin A, gentamicin B, gentamicin B.sub.1 and gentamicin X have amine 
or substituted amine groups which are capable of forming non-toxic acid 
addition salts with organic and inorganic acids. Non-toxic salts are 
defined herein as those that do not give rise to detrimental side effects 
when administered at the usual dosage level. Generally, the inorganic acid 
salts may be formed by adding an amount of dilute (1.0N) acid to an 
aqueous solution of antibiotic base to adjust the pH of the solution to 
about 4.5 and lyophilizing the resulting solution. Thus, by the foregoing 
procedure acid addition salts are formed with hydrochloric acid, sulfuric 
acid, phosphoric acid and the like including the alkali metal salts of the 
dibasic and tribasic acids. In general, the salts of inorganic acids are 
water soluble but are substantially insoluble in organic solvents. 
However, some such salts (e.g. hydrochlorides) may have very limited 
solubility in polar organic solvents such as lower alcohols. Salts of 
organic acids, such as, hydrocarbon carboxylic acids including dibasic 
acids, such as succinic, tartaric, maleic, fumaric, glutaric and the like 
are usually prepared by reacting an aqueous solution of the antibiotic 
with a stoichiometric quantity of acid and either lyophilizing the 
resulting solution or precipitating the product with a miscible organic 
solvent such as acetone, dioxane, tetrahydrofuran, methanol or the like. 
Gentamicin A, gentamicin B, gentamicin B.sub.1 and gentamicin X are 
obtained from the fermentation as a complex mixture which also includes 
the major fermentation product, gentamicin C. The gentamicin "C" 
components constitute from about 60% to about 80% of the mixture and the 
co-produced antibiotics constitute the remainder. Thus, in order to obtain 
the co-produced antibiotics it is advantageous to at least partially 
remove the gentamicin C. There are a substantial number of resins that are 
suitable for this purpose. Exemplary of such resins are Amberlite IRA-400, 
Amberlite IRA-401, Dowex 1-X2, and Dowex 2-X4 (Amberlite being a trademark 
of Rohm and Haas Company, Philadelphia, Pennsylvania) and Dowex being a 
trademark of Dow Chemical Company, Midland, Michigan). In general, porous, 
strongly basic quaternary amine type resins are of utility. Applicants 
prefer to utilize Dowex 1-X2 in the hydroxyl cycle. 
At the end of the fermentation, the antibiotics produced therein are 
extracted from the broth which is accomplished by adsorbing the total 
antibiotic complex from the fermentation broth onto a cation exchange 
resin, preferably of the Amberlite type such as IRC-50. The resin is 
advantageously utilized in the ammonium cycle and the crude antibiotic 
mixture eluted with dilute ammonium hydroxide. The eluate is decolorized 
by passage through a column containing a decolorizing carbon, or, 
preferably, by passage through an anion exchange resin column, especially, 
a resin of the Amberlite IRA type mentioned above. For decolorizing the 
IRC-50 resin eluate IRA-401S resin in the hydroxyl cycle is preferred. 
After decolorization, the eluate is concentrated in vacuo to a solution 
containing from about 30% to about 60% solids and held for further 
processing. 
At this juncture, the solution contains all of the antibiotics produced in 
the fermentation and in substantially the ratio produced therein. Removal 
of the gentamicin C components is effected by adsorbing the concentrate 
prepared above on a column of a suitable anion exchange resin, such as 
Dowex 1-X2 and selectively desorbing the gentamicin C components 
therefrom. 
Elution of the gentamicin C components is effected by the use of deionized 
water having from about 50,000 to about 150,000 ohms resistance. The 
column may advantageously be connected in series and through a 
conductivity bridge to an automatic fraction collector so that a sharp 
rise in conductance of the effluent will actuate the fraction collector. 
Similarly, a drop in conductance (rise in resistance) followed by a sharp 
rise in conductance will start the collection of a new fraction. The care 
with which this column is run, to a substantial degree, controls the 
quantity of gentamicin "C", which remains in the mixture of co-produced 
antibiotics and to some degree affects the efficiency of the subsequent 
(silica gel) column which is used to separate the co-produced antibiotics 
one from the other. 
The co-produced antibiotics are eluted from the Dowex 1-X2 column 
subsequent to the gentamicin C components and the aqueous eluate 
concentrated in vacuo to a suitable volume and lyophilized. At this stage, 
it is advantageous to dissolve the antibiotic mixture in hot methanol and 
cool while exposing the solution to air. Gentamicin A adsorbs carbon 
dioxide from the air forming a sparingly soluble carbonate salt which 
crystallizes from the methanol solution and is removed by filtration and 
dried in vacuo. The filtrate is concentrated to a residue and dissolved in 
about 3 volumes of the lower phase of a solvent mixture consisting of 
methanol:chloroform:ammonium hydroxide in the volume ratio of 1:1:1. 
The foregoing solution is placed atop a suitably sized column of silica gel 
and the column eluted using the abovedescribed solvent mixture and a 
controlled flow rate. Gentamicin B and gentamicin B.sub.1 may be obtained 
in crystalline form by dissolving the residues from the appropriate column 
fractions in ethanol and concentrating in vacuo to a residue, redissolving 
the residue in ethanol and re-evaporating the solution until a crystalline 
suspension is obtained. This procedure yields substantially pure 
gentamicin B and gentamicin B.sub.1, as ethanol solvates having about 1 
mole of ethanol per mole of antibiotic. Gentamicin X is usually obtained 
as an amorphous solid and, in this connection, behaves like the gentamicin 
"C" components. 
Alternatively, the total antibiotic mixture from the IRA-401S column which 
includes the gentamicin C components may be subjected to silica gel 
chromatography. However, the separation effected by this technique is not 
as sharp as that obtained when most of the gentamicin "C" and the 
gentamicin A are removed prior to silica gel chromatography. 
Gentamicin A, gentamicin B, gentamicin B.sub.1, and gentamicin X, after the 
removal of the gentamicin C components, may be lyophilized and used as a 
mixture to elicit an antibacterial, antiprotozoal and/or an anthelmintic 
effect. 
The compounds of this invention readily form hydrates and solvates which 
are extremely difficult to break. Consequently, the physical constants for 
gentamicin A, gentamicin B and gentamicin B.sub.1 are based upon 
relatively stable hydrates or solvates. Further, although gentamicin B and 
gentamicin B.sub.1 form crystalline ethanol solvates they do not give 
reproducible melting points but rather tend to soften and eventually 
become viscous liquids over quite a wide temperature range. Gentamicin X 
has not yet obtained in crystalline form; it is isolated as a white 
amorphous solid which likewise has no discrete melting point. 
The antibiotics described herein have physical properties substantially as 
set forth in Table I below: 
TABLE I 
__________________________________________________________________________ 
Physical Constants 
Elemental 
Analyses Sulfate Salt 
Compound Calculated 
Found 
##STR2## 
Analyses 
##STR3## 
Gentamicin A 
C = 43.63 
C = 43.60 
+136.degree. +96.degree. 
.sup.1 Calculated for 
H = 7.93 
H = 7.86 
(C = 1.0,H.sub.2 O) 
(C = 0.3,H.sub.2 O) 
C.sub.18 H.sub.36 N.sub.4 O.sub.10 . 1.5H.sub.2 O 
N = 11.31 
N = 11.56 
O.sup.1 = 37.13 
O.sup.1 = 37.16 
Gentamicin B 
C = 47.70 
C = 47.78 
+155.degree. 
C = 30.35 
+116.degree. 
.sup.1 Calculated for 
H = 8.41 
H = 8.38 
(C = 0.3,H.sub.2 O) 
H = 6.59 
(C = 0.3,H.sub.2 O) 
C.sub.19 H.sub.38 N.sub.4 O.sub.10 . O.sub.2 H.sub.5 OH 
N = 10.60 
N = 10.59 N = 7.49 
O.sup.1 = 33.29 
O.sup.1 = 33.25 
S = 8.48 
Gentamicin B.sub.1 
C = 47.22 
C = 48.68 
+161.degree. 
C = 31.26 
+117.degree. 
.sup.1 Calculated for 
H = 8.34 
H = 8.56 
(C = 0.3,H.sub.2 O) 
H = 6.83 
(C = 0.3,H.sub.2 O) 
C.sub.20 H.sub.40 N.sub.4 O.sub.10 . C.sub.2 H.sub.5 OH 
N = 10.41 
N = 10.32 N = 7.34 
O.sup.1 = 34.03 
O.sup.1 = 32.44 
S = 8.72 
Gentamicin X C = 43.54 
+154.degree. 
C = 31.96 
+121.degree. 
.sup.1 C.sub.19 H.sub.38 N.sub.4 O.sub.10 
H = 7.22 
(C = 0.3,H.sub.2 O) 
H = 6.57 
(C = 0.3,H.sub.2 O) 
N = 9.90 N = 8.64 
O.sup.1 = 39.34 
S = 8.70 
__________________________________________________________________________ 
.sup.1 All oxygen values are by difference 
Gentamicin A, gentamicin B, gentamicin B.sub.1 and gentamicin X have 
characteristic infrared absorption spectra in mineral oil (Nujol) as shown 
by FIG. 1 (gentamicin A), FIG. 3 (gentamicin B), FIG. 5 (gentamicin 
B.sub.1) and FIG. 7 (gentamicin X). These spectra are of the respective 
antibiotics as sulfate salts. 
These antibiotics also have characteristic nuclear magnetic resonance 
spectra (NMR) as shown by FIG. 2 (gentamicin A), FIG. 4 (gentamicin B), 
FIG. 6 (gentamicin B.sub.1), and FIG. 8 (gentamicin X). All of the NMR 
spectra are of the antibiotic free bases; however, the samples of 
gentamicin A and gentamicin X were hydrated and those of gentamicin B and 
gentamicin B.sub.1 are of the ethanol solvate. These spectra were obtained 
by the use of a Varian A-60-A spectrometer (Varian Associates, Palo Alto, 
California) on about 0.4 ml. of a solution (conc. about 20 mg/ml) of the 
antibiotic in deuterium oxide (D.sub.2 O). The spectra are recorded in 
parts per million (PPM) from 3-(trimethylsilil)-propanesulfonic acid 
sodium salt, the internal standard. 
In Table II below are set forth the more characteristic peaks from FIGS. 1, 
3, 5 and 7, respectively. 
TABLE II 
______________________________________ 
Significant Infrared Absorption Bands 
Gentamicin A Sulfate 
2.9 3.8 .mu. (S, V brd) 
6.85 .mu. (Nujol) 
3.35 .mu. (Nujol) 7.25 .mu. (Nujol) 
3.55 .mu. (Nujol) 8.50 - 10.75 .mu. 
(V, S, V brd) 
4.83 .mu. (W, brd) 12.95 .mu. 
(V, W, brd) 
6.17 .mu. (M) 13.88 .mu. 
(W, brd) 
6.53 .mu. (M) 
______________________________________ 
Gentamicin B Sulfate 
2.95 - 3.25 .mu. 
(M-S, brd) 
7.26 .mu. (Nujol) 
3.40 .mu. (Nujol) 7.75 .mu. (W) 
3.51 .mu. (Nujol) 8.75 - 9.75 .mu. 
(S, V brd) 
4.85 .mu. (W, brd) 10.28 .mu. 
(W-M) 
6.16 .mu. (M) 12.95 .mu. 
(VW, brd) 
6.55 .mu. (M) 13.90 .mu. 
(W, brd) 
6.83 .mu. (Nujol) 
______________________________________ 
Gentamicin B.sub.1 Sulfate 
2.95 -3.25 .mu. 
(M-S, brd) 
7.25 .mu. (Nujol) 
3.38 .mu. (Nujol) 7.75 .mu. (W) 
3.50 .mu. (Nujol) 8.75 - 9.75 .mu. 
(S, V brd) 
4.85 .mu. (W, brd) 10.27 .mu. 
(W-M) 
6.16 .mu. (M) 10.75 .mu. 
(W) 
6.55 .mu. (M) 13.05 .mu. 
(V-W, brd) 
6.83 .mu. (Nujol) 13.90 .mu. 
(W, brd) 
______________________________________ 
Gentamicin X Sulfate 
3.0 - 3.3 
.mu. (S, brd) 7.25 .mu. (Nujol) 
3.37 - 3.51 
.mu. (Nujol) 7.75 .mu. (W-M) 
3.65 - 4.33 
.mu. (N, brd) 8.7 - 10.0 .mu. 
(V-S, brd) 
4.82 .mu. (W, brd) 10.30 .mu. 
(Brd, shd) 
5.94 .mu. (W, shd) 11.50 .mu. 
(W, brd) 
6.17 .mu. (M) 13.03 .mu. 
(V-W, brd) 
6.54 .mu. (N) 13.90 .mu. 
(Nujol) 
6.83 .mu. (Nujol) 15.08 .mu. 
(V-W) 
______________________________________ 
Notations: M = medium, S = strong, shd. = shoulder, V = very, W = weak, 
brd. = broad. 
ANTIBIOTIC ACTIVITY - IN VITRO 
In Table III below is set forth the in vitro minimal inhibitory 
concentration of gentamicin B, gentamicin B.sub.1 and gentamicin X all in 
the free base form against a variety of gram positive and gram negative 
microorganisms. The experiments were carried out in a yeast beef broth 
medium at pH 7.4. 
Table III 
______________________________________ 
MIC (mcg/ml).sup.1 
Gentamicin Gentamicin Gentamicin 
Organism B B.sub.1 X 
______________________________________ 
Aerobacter aero- 
genes 0.08 0.08 -- 
Bacillus subtilis 
0.8 3.0 0.03 
Escherichia coli 
0.03 - 0.3 0.08 - 0.3 0.3 
Klebsiella pneumo- 
niae 3.0 3.0 0.75 
Pseudomonas aeru- 
ginosa 0.03 - 0.08 
0.08 0.3 - 0.75 
Sarcina lutea 
0.3 0.3 -- 
Staphylococcus 
aureus 0.3 - 3.0 0.3 - 3.0 0.08 - 0.75 
Streptococcus 
pyrogenes 3.0 3.0 0.3 - 0.75 
Shigella sp. M324 
5.0 5.0 -- 
Salmonella sp. 1 
0.8 0.3 0.75 
Salmonella sp. 2 
0.8 0.3 -- 
Salmonella sp. B 
0.8 0.3 -- 
______________________________________ 
ANTIBIOTIC ACTIVITY - IN VIVO 
The protective activity of gentamicin B, gentamicin B.sub.1 and gentamicin 
X is determined by administering the respective compound subcutaneously 
one-hour after administration of an infecting dose of pathogen and by 
counting the survivors 48 hours after infection. Infected non-treated 
controls die in about 18 to 24 hours. Generally 5-7 dose levels are 
administered to mice weighing about 20 grams in groups of 7-10. The 
PD.sub.50 values are set forth in Table IV below: 
Table IV 
______________________________________ 
Protective Activity of Gentamicin B, B.sub.1 and X in Mice 
Injecting PD.sub.50 (mg/kg) 
Microorganism 
Gentamicin B 
Gentamicin B.sub.1 
Gentamicin X 
______________________________________ 
Staphylococcus 
aureus Gray 
20.0 30.0 4.0 
Staphylococcus 
aureus W 50.0 50.0 -- 
Staphylococcus 
aureus Smith 
5.0 2.5 -- 
Streptococcus 
pyogenes C 
15.0 18.0 -- 
Salmonella 
paratyphi B 
5.0 4.0 -- 
Pseudomonas 
aeruginosa SC 
2.0 2.0 7.0 
Escherichia 
coli SC -- -- 1.5 
______________________________________ 
ACUTE TOXICITY 
The acute toxicity of gentamicin B, gentamicin B.sub.1 and gentamicin X is 
determined in male CF-1 (Carworth Farms) mice weighing about 20 grams 
each. The results of this test are set forth in Table V below: 
Table V 
______________________________________ 
Acute 
Toxicity of Gentamicin B, Gentamicin B.sub.1 and Gentamicin X 
LD.sub.50 (mg/kg) 
Route Gentamicin B 
Gentamicin B.sub.1 
Gentamicin X 
______________________________________ 
I.V. 228 230 225 
I.P. 1750 1600 &gt;1000 
S.C. &gt;1500 &gt;1500 
Oral &gt;250 &gt;250 
______________________________________ 
IN VITRO ACTIVITY AGAINST TRICHOMONAS VAGINALIS 
In Table IV below is set forth the in vitro activity of the compounds of 
this invention against T. vaginalis. The test procedure used was 
substantially as described by R. J. Schnitzer on pages 289-331 in 
Experimental Chemotherapy, Volume 1, Academic Press, New York (1963). 
Table VI 
__________________________________________________________________________ 
Minimal Minimal 
99% Suppression Level (mcg/ml) 
Cidal level (mcg/ml) 
Compound 24 hours 48 hours 
24 hours 
48 hours 
__________________________________________________________________________ 
Gentamicin A 
10 5-10 25 10 
Gentamicin B 
25 25 &gt;250 25 
Gentamicin B.sub.1 
25 10-25 
&gt;250 25 
Gentamicin X 
-- 2.5 -- 10 
Garamycin.sup..RTM. 
250 100 &gt;250 100 
STS - Simplified Tripticase Serum Medium, Baltimore Biological 
Laboratories, Baltimore, Maryland. 
__________________________________________________________________________ 
Against Entamoeba hystolytica, gentamicin X has a minimal inhibitory 
concentration of 4 mcg/ml (48 hours) and a minimal cidal concentration of 
7 mcq/ml (48 hours). 
ANTHELMINTIC ACTIVITY IN VIVO 
Gentamicin B and gentamicin X exhibit anthelmintic activity against 
Syphacia obvelata when tested in groups of seven male Millerton mice by 
substantially the procedures described by O. D. Standen on pages 701-892 
in Experimental Chemotherapy, Volume 1 
Table VII 
______________________________________ 
Mean 
%in % Worm 
Compound mg/kg/day Diet Infected 
Burden 
______________________________________ 
Gentamicin B 
259 0.25 0 0 
Gentamicin B 
100 0.0625 0 0 
Gantamicin B 
39 0.02 0 0 
Gentamicin B 
8 0.008 67 29.5 
Gentamicin X 
310 0.25 0 0 
Gentamicin X 
62 0.05 83 27 
Controls 0 0 100 53 
Controls 0 0 100 22 
Controls 0 0 75 6.8 
______________________________________ 
The data set forth in Table VII shows that gentamicin B has anthelmintic 
activity against Syphacia obvelata at a minimum dosage of 39 mg/kg/day 
when administered to male Millerton mice. Gentamicin X is totally 
effective at 310 mg/kg/day and partially effective at 62 mg/kg/day under 
substantially the same test conditions. 
DIMINISHED INCIDENCE OF ATAXIA 
Gentamicin B, and to a lesser extent, gentamicin B.sub.1 exhibit a property 
that is quite unusual in amino-glycoside antibiotics. When subjected to 
the test procedures described by Weinstein, Wagman and Taber in 
Antimicrobial Agents and Chemotherapy, 1965, pages 227-231, each of these 
compounds required substantially longer periods of administration at high 
doses to cause impairment of the "righting reflex" and the appearance of 
ataxia. There appears to be a causal relationship between high doses of 
amino-glycoside type antibiotics given over an extended period of time and 
vestibular damage wherein one of the early manifestations is ataxia. The 
test results, in Table VIII, show that gentamicin B and gentamicin B.sub.1 
as compared with gentamicin C (Garamycin) may be used at dosages that are 
substantially above the normal therapeutic dose. Moreover, they may be 
used at such dosages for a time interval substantially beyond that 
normally required to eliminate infection without causing ataxia; and with 
minimal, if any, toxic manifestations. Thus, gentamicin B and gentamicin 
B.sub.1 represent a substantial improvement over antibiotics of this class 
(amino-glycoside) that are currently in use. 
Table VIII 
______________________________________ 
Chronic Toxicity in Cats After Daily Subcutaneous Dosing 
Dose No. of Average No. Days 
Antibiotic 
(mg/kg/day) Cats to become ataxic 
______________________________________ 
Gentamicin B.sub.1 
240 1 21.0 
60 4 69.0 
40 4 108.0 
Garamycin.sup..RTM. 
60 8 13.5 
40 11 17.5 
20 5 31.4 
Gentamicin B 
240 1 21.0 
60 4 81.0 
40 4 &gt;312* 
______________________________________ 
*Study terminated 2 of the 4 cats had not become ataxic at the end of 385 
days; number given is the trucated median. 
PROPHYLACTIC ACTIVITY OF GENTAMICIN A IN TURKEYS AGAINST HISTOMONAS 
MELEAGRIDIS 
Gentamicin A was administered to groups of turkeys at dosage levels of 5, 
10, and 15 mg. per turkey per day in aqueous solution. Two days after the 
initiation of the drug, the turkeys were inoculated orally with 500 
Heterakis gallinae ova (the intermediate host carrier of Histomonas). A 
single group of turkeys received 0.035% of gentamicin A in their feed for 
six days. Untreated infected controls were also carried in each 
experiment. The test is continued for twelve days and the birds are 
autopsied on the thirteenth day. Infection is usually manifested 
principally in two areas, the cecum and the liver which are the areas 
examined during the autopsy. 
According to the results obtained in the foregoing test, gentamicin A 
exhibits prophylactic activity in turkeys against Histomonas meleagridis 
at a minimum dosage level of 10 mg. per bird, whereas the untreated 
controls became infected. 
Isolation of Gentamicin C and the Co-produced Antibiotics from the 
Fermentation 
Adjust the pH of the fermentation broth obtained as described in U.S. Pat. 
No. 3,091,572 to pH 2.0 with 6N sulfuric acid. Stir the mixture for about 
30 minutes and filter through a suitably sized filter which has been 
pre-coated with about a one (1) inch layer of filter aid. Re-adjust the pH 
of the filtrate to about 7 with 6N ammonium hydroxide and add oxalic acid 
to lower the pH to about 4. This quantity of oxalic acid is usually 
sufficient to precipitate the calcium ions in the fermentation broth as 
the insoluble calcium oxalate. Adjust the pH to about 7 with 6N ammonium 
hydroxide and stir the mixture for from about 1 to about 4 hours at about 
20.degree.-35.degree. C. Refilter the mixture using a suitably sized 
pre-coated filter and save the filtrate. Adsorb the antibiotic mixture on 
IRC-50 (ammonium cycle) using about 10 grams of resin per liter of the 
original broth volume. Wash the resin color free with deionized water and 
elute the antibiotics with 2N ammonium hydroxide. Concentrate the eluate 
in vacuo to a solution containing about 50% solids.

EXAMPLE 2 
Separation of Gentamicin C Components from Gentamicin A, Gentamicin B, 
Gentamicin B.sub.1 and Gentamicin X 
Charge 45 cu. ft. of Dowex 1-X2 resin (hydroxyl cycle) to a clean 
chromatographic column which is 3 ft. in diameter, at least 8 ft. high and 
is connected through a conductivity bridge to a fraction collector. Pump 
deionized water through the resin bed until the effluent has a resistance 
of at least 65,000 ohms. Charge an aqueous solution of about 120 liters 
containing about 60 kilograms of solids (from Example 1) to the column and 
adsorb the antibiotic mixture by permitting the antibiotic solution to 
pass through the column at the rate of about 2 liters per minute. 
Develop the column with deionized water at the rate of about 2 liters per 
minute and collect fractions in accordance with the conductance of the 
eluate. Typically, about seven fractions are collected which vary from 
about 14 to about 750 liters in volume. The first five fractions 
comprising about 500 liters contains the bulk of the gentamicin C 
components. The remaining two fractions comprising about 1500 to 1600 
liters contains the bulk of gentamicin A, gentamicin B, gentamicin B.sub.1 
and gentamicin X. The latter two fractions are combined, concentrated in 
vacuo to a small volume and either lyophilized or spray dried. 
In those instances wherein a mixture of the co-produced antibiotics are to 
be utilized, either the lyophilized or the spray dried product will 
suffice. 
EXAMPLE 3 
Isolation of Gentamicin A 
Dissolve 19.8 kilograms of fermentation product from which most of the 
gentamicin C (Example 2) has been removed in 45 liters of hot methanol. 
Clarify the solution by filtration. Cool the solution at room temperature 
with exposure to air and seed with gentamicin A. Gentamicin A crystallizes 
as its colorless carbonate salt which is filtered, washed sparingly with 
methanol and dried at 80.degree. C in vacuo to obtain 1119 grams of the 
product of this example. The mother liquor and washes are combined, 
concentrated to a residue in vacuo and chromatographed as described in 
Example 4. 
EXAMPLE 4 
Chromatographic Separation of Gentamicin B Gentamicin B.sub.1 and 
Gentamicin X 
Fill two 5 foot .times. 4 inch chromatographic columns connected in series 
with silica gel (e.g. Baker Silica Gel for chromatography) which has 
previously been conditioned by contact with the lower phase of a 1:1:1 
mixture of chloroform, methanol and concentrated ammonium hydroxide. 
Prepare a solution of 750 grams of the residue from Example 3 in a minimum 
volume (about 2 liters of the lower layer of the above-described solvent 
mixture). Place the antibiotic solution stop the first column and elute 
the column at the rate of about 3 to about 4.5 liters per hour, collecting 
fractions of approximately two (2) liters. Monitor the effluent by thin 
layer chromatography on silica gel plates using the same solvent system 
that is being used on the column. Combine the fraction containing like 
antibiotics and evaporate to a residue. When the amount of gentamicin B 
eluted tapers off, the column is eluted with a mixture comprising equal 
volumes of methanol and concentrated ammonium hydroxide to strip the 
column. The fractions containing gentamicin B and gentamicin B.sub.1 are 
dissolved in ethanol and concentrated to a residue. Repetition of the 
foregoing step will result in a crystalline suspension of the ethanol 
solvate of gentamicin B and gentamicin B.sub.1. When the foregoing 
chromatographic separation is effected, the products are obtained in the 
amounts and order substantially as shown below: 
______________________________________ 
Gentamicin C 
198.6 gms 
Gentamicin B.sub.1 
Gentamicin B.sub.1 42.0 gms 
##STR4## 
Gentamicin B.sub.1 
67.8 gms 
Gentamicin X 
Gentamicin X 12.3 gms 
##STR5## 
Gentamicin X 
74.9 gms 
Gentamicin B 
Gentamicin B 85.8 gms 
##STR6## 
Gentamicin B 
165.0 gms 
Gentamicin A 
______________________________________ 
The fractions containing mixtures of antibiotics may be rechromatographed 
to afford more of the individual fractions. 
EXAMPLE 5 
Preparation of Gentamicin A Sulfate 
Dissolve 5.0 grams of gentamicin A in 25 ml. of water and adjust the pH of 
the solution to 4.5 with 1N sulfuric acid. Treat the solution with about 
50 milligrams of decolorizing carbon for about 15 minutes and filter. Pour 
the filtrate into about 300 milliters of methanol with vigorous agitation, 
continue the agitation for about 10-20 minutes and filter. Wash the 
precipitate with methanol and dry at about 60.degree. C in vacuo to obtain 
the product of this Example. 
In like manner, the sulfate salt of gentamicin B, gentamicin B.sub.1 and 
gentamicin X may be prepared. In order to prepare sulfate salts of a 
mixture of gentamicin A, B, B.sub.1 and X, the lyophilized or spray dried 
product of Example 2 is treated as described in this example. 
EXAMPLE 6 
Preparation of Gentamicin B.sub.1 Hydrochloride 
Dissolve 5.0 grams of gentamicin B.sub.1 in 1000 milliters of water. 
Prepare a column containing 500 grams of weakly basic ion exchange resin 
in the chloride cycle, preferably, IR-45 (Trademarked product of Rohm and 
Haas, Philadelphia, Pennsylvania). Pass the antibiotic solution through 
the column followed by an additional 500 milliters of water. Concentrate 
the effluent to about 100 milliters in vacuo and lyophilize to obtain the 
product of this example. 
In like manner, the hydrochloride salt of gentamicin A, gentamicin B, and 
gentamicin X may be also prepared. 
The tangible embodiments of this invention (i.e. gentamicin A, gentamicin 
B, gentamicin B.sub.1 and gentamicin X are suitable for oral, topical or 
parenteral administration. When administered orally they may be compounded 
in the form of tablets, capsules, elixirs and the like or may even be 
admixed with animal feed. It is in these dosage forms that the tangible 
embodiments of this invention are most effective as anthelminthic and 
antiprotozoal agents. This is especially true wherein they are being used 
to treat intestinal amoebiasis or non-specific diarrheas. The compounds of 
this invention may be incorporated into a dosage unit as the sole active 
ingredient or they may be compounded into dosage units in combination with 
other active ingredients, such as anti-anxiety agents, it being known that 
infections and infestations often cause anxiety reactions in animal 
species. When the animal species is being treated orally, the tangible 
embodiments of this invention are administered at from about 10 to about 
100 mg. per kilogram of body weight per day, preferably divided into from 
about 2 to about 4 doses. 
For topical administration, the tangible embodiments of this invention may 
be compounded into creams, ointments, suppositories and the like. Such 
dosage forms should contain from about 1 to about 10% of antibiotic and 
should be administered to the site of the infection from about 2 to about 
4 times a day. 
As shown hereinabove, (see Table VIII) gentamicin B, and gentamicin 
B.sub.1, by virtue of their low ataxic potential, may be used to treat 
deep-seated bacterial infections especially those which have proved to be 
refractory to other antibacterial agents. It is preferred that gentamicin 
B and/or gentamicin B.sub.1 be administered parenterally in dosages 
ranging between about 2 and about 10 mg. per kilogram of body weight per 
day in divided doses. 
The examples below set forth some of the various dosage forms into which 
the tangible embodiments of this invention may be compounded. 
Example 7 
______________________________________ 
ENTERAL SOLUTION 
mg/ml mg/10 ml. 
______________________________________ 
Gentamicin B (as the sulfate) 
40 to 200 mg* 
400-2000 
Methylparaben 1.8 mg. 18 mg. 
Propylparaben 0.2 mg. 2 mg. 
Water for Injection q.s. 
1.0 ml. 10.0 ml. 
______________________________________ 
*Based on theoretical 100% purity. 
Place 70% of the water for injection into a suitable mixing vessel and heat 
to 70.degree. C. Add the methylparaben and propylparaben and mix until 
dissolved. Cool the above solution to 25.degree.-30.degree. C. Pass a 
stream of nitrogen gas through the solution. Add the gentamicin B (as the 
sulfate) and mix until dissolved. Bring the solution to final volume. Pass 
the solution through a suitable sterilizing filter, employing appropriate 
aseptic techniques. Fill the solution into suitable sterile containers 
employing appropriate aseptic filling techniques. 
Example 8 
______________________________________ 
ENTERAL SOLUTION 
mg/ml mg/10 ml. 
______________________________________ 
Gentamicin A (as the Sulfate 
40 to 200 mg* 
400-2000 
Methylparaben 1.80 18.00 
Propylparaben 0.20 2.00 
Sodium Bisulfite 3.20 32.0 
Disodium Edetate, Dihydrate 
0.10 1.00 
Glacial Acetic Acid 
1.25 12.50 
Sodium Acetate 2.50 25.00 
Propylene Glycol 100 to 400 1000-4000 
Water for Injection q.s. 
1.0 ml 10.00 ml 
______________________________________ 
*Based on theoretical 100% purity. 
Place 70% of the water for injection into a suitable mixing vessel and heat 
to 70.degree. C. Add the methylparaben and propylparaben and mix until 
dissolved. Cool the above solution to 25.degree.-30.degree. C. Pass a 
stream of nitrogen gas through the solution. Add the disodium edetate, 
dihydrate, sodium acetate and glacial acetic acid; mix thoroughly. Stop 
the nitrogen sparging, add the sodium bisulfate and mix until dissolved. 
Add the propylene glycol and mix thoroughly. Add the gentamicin A (as the 
sulfate) and mix until dissolved. Bring the solution to final volume. Pass 
the solution through a suitable sterilizing filter, employing appropriate 
aseptic techniques. Fill the solution into suitable sterile containers 
employing appropriate aseptic filling techniques. 
Example 9 
______________________________________ 
ENTERAL SOLUTION 
mg/ml mg/100 ml 
______________________________________ 
Gentamicin B.sub.1 (as the Sulfate) 
40 to 200 mg* 
4000-20,000 
Methylparaban 1.80 180 
Propylparaben 0.20 20 
Sodium Bisulfite 3.20 320 
Disodium Edetate, Dihydrate 
0.10 10 
Glacial Acetic Acid 
1.25 125 
Sodium Acetate 2.50 250 
Water for Injection q.s. 
1.00 ml 100 ml 
______________________________________ 
*Based on theoretical 100% purity. 
Place 70% of the water for injection into a suitable mixing vessel and heat 
to 70.degree. C. Add the methylparaben and propylparaben and mix until 
dissolved. Cool the above solution to 25.degree.-30.degree. C. Pass a 
stream of nitrogen gas through the solution. Add the disodium edetate, 
dihydrate, sodium acetate and acetic acid; mix thoroughly. Stop the 
nitrogen gas, add the sodium bisulfate and mix until dissolved. Add the 
gentamicin B.sub.1 (as the sulfate) and mix until dissolved. Bring the 
solution to final volume. Pass the solution through a suitable sterilizing 
filter, employing appropriate aseptic techniques. Fill the solution into 
suitable sterile containers employing appropriate aseptic filling 
techniques. 
Example 10 
______________________________________ 
ORAL SYRUP PER LITER 
______________________________________ 
Gentamicin A (as the sulfate) 
100 g.* 
Standard Granulated Sugar 
550 
Sorbitol Solution 200 
Water, to make 1.0 liter 
______________________________________ 
*Based on theoretical 100% purity. 
Add the gentamicin A (as the sulfate), granulated sugar and sorbitol 
solution, to approximately 350 mls of water contained in a suitable mixing 
vessel. Mix until a solution is obtained. Add sufficient water to make 1 
liter. Pass the solution through a suitable clarifying filter. 
Example 11 
______________________________________ 
ORAL SYRUP PER LITER 
______________________________________ 
Gentamicin B (as the sulfate) 
100 g.* 
Granulated sugar 100 
Glucose Syrup 44 
Sorbitol Solution 400 
Glycerin 50 
Alcohol 50 
Water to make 1.0 liter 
______________________________________ 
*Based on theoretical 100% purity. 
Add the gentamicin B (as the sulfate), granulated sugar, glucose syrup, 
sorbitol solution, glycerin, alcohol to approximately 500 ml. of water 
contained in a suitable mixing vessel. Mix until a solution is obtained. 
Add sufficient water to make 1 liter. Pass the solution through a suitable 
clarifying filter. 
Example 12 
______________________________________ 
TOPICAL CREAM PER KG. 
______________________________________ 
Gentamicin A (as the sulfate) 
10 g.-100 g. 
Stearic Acid 60 g. 
Propylene Glycol Monostearate 
100 
Isorpopyl Myristate 80 
Polyoxyethylene (20) Sorbitan 
60 
Monopalmitate 
Sorbitan Solution 20 
Water to make 1.0 kg. 
______________________________________ 
*Based on theoretical 100% purity. 
Add the stearic acid, propylene glycol monostearate, isopropyl myristate 
and polyoxyethylene (20) sorbitan monopalmitate to a suitable mixing 
vessel. Heat to 80.degree. C. to melt. Mix. 
Example 13 
______________________________________ 
TOPICAL CREAM PER KG. 
______________________________________ 
Gentamicin B (as the sulfate) 
10 g. - 100 g.* 
Ethoxylated Cetyl/Stearyl Alcohol 
20 g. 
Cetyl Alcohol 35 
Stearyl Alcohol 35 
Petrolatum 200 
Mineral Oil 50 
Water to make 1.0 kg. 
______________________________________ 
*Based on theoretical 100% purity. 
Add the cetyl alcohol, stearyl alcohol, ethoxylated cetyl/stearyl alcohol, 
petrolatum and mineral oil to a suitable mixing vessel. Heat to 80.degree. 
C. to melt. Mix. Add the gentamicin B (as the sulfate) in approximately 
95% of the water heated to 80.degree. C. in a suitable mixing vessel. Mix. 
Add the melted wax phase to the aqueous phase and mix while cooling to 
approximately 40.degree. C. Add sufficient water to make 1 kg. Mix until 
cool. 
Example 14 
______________________________________ 
TOPICAL OINTMENT PER KG. 
______________________________________ 
Gentamicin B.sub.1 (as the sulfate) 
10 g. - 100 g.* 
White Petrolatum, to make 
1.0 g. 
______________________________________ 
*Based on theoretical 100% purity. 
Melt and heat the petrolatum to 50.degree. C. in a suitable mixing vessel. 
Remove a portion of the melted petroleum and make a slurry of the 
gentamicin B.sub.1 (as the sulfate). Pass the slurry through a suitable 
colloid mill and mill until a uniform dispersion is obtained. Add the 
milled slurry to the remainder of the melted petrolatum and mix until 
cool. 
Example 15 
______________________________________ 
A B C 
TABLETS 10 mg Tab. 
25 mg Tab. 
100 mg Tab. 
______________________________________ 
Mixture of 
Gentamicin A, B, 
B.sub.1 and X (as the 
17.50 mg.* 
43.75 mg.* 
175.00 mg.* 
Sulfate) 
Lactose, Impalpable 
Powder 197.50 171.25 126.00 
Corn Starch 25.00 25.00 35.00 
Polyvinylpyrrolidone 
7.50 75.0 10.50 
Magnesium Stearate 
2.50 2.50 3.50 
250.00 250.00 350.00 
______________________________________ 
*Based on 60% potency, plus 5% excess. 
Prepare a slurry consisting of the mixture of gentamicin A, B, B.sub.1 and 
X (as the sulfate) from Example 2, lactose and polyvinylpyrrolidone. Spray 
dry the slurry. Add the corn starch and magnesium stearate, and mix. 
Compress into tablets using suitable tableting equipment. 
Example 16 
______________________________________ 
HARD GELATIN A B C 
CAPSULES 10 mg Cap. 
25 mg Cap. 
100 mg Cap. 
______________________________________ 
Gentamicin A, B, B.sub.1 
and X 
(as the sulfate) 
17.50 mg* 43.75 mg.* 
175.00 mg..sup.a 
Lactose, Impalpable 
Powder 231.50 205.25 124.25 
Magnesium Stearate 
1.00 1.00 0.75 
______________________________________ 
*Based on 60% potency, plus 5% excess. 
Place the mixture of gentamicin, A, B, B.sub.1 and X (as the sulfate) and 
lactose into a suitable mixing bowl and mix. Pass the mixed powders 
through a mill. Add the mixed milled powders to a suitable mixing vessel 
and mix again. Pre-mix the magnesium stearate with a portion of the batch 
from above. Pass the premixed magnesium stearate to the batch and mix. 
Fill into empty gelatin capsules using suitable encapsulating equipment. 
Example 17 
______________________________________ 
BOLUS 100 mg Bolus 
______________________________________ 
Mixture of 
Gentamicin A, B, B.sub.1 and X 
175.00 mg.* 
(as the sulfate) 
Lactose 4335.00 
Corn Starch Pregelatinised 
1080.00 
Magnesium Stearate 90.00 
Water 100.0 ml. 
10,000.00 
______________________________________ 
*Based on a 60% potency, plus 5% excess. 
Place the mixture of gentamicin A, B, B.sub.1 and X (as the sulfate), 
lactose and a portion of the corn starch into a suitable mixing bowl and 
mix. Prepare a paste of a portion of the corn starch and use this paste to 
prepare a damp mass of the above powders. Screen the mass to produce 
granules. Dry the granules. Reduce the dried granules to specific particle 
size. Add the magnesium stearate, mix and compress the granulation into 
tablets using suitable tableting equipment. 
Example 18 
______________________________________ 
BOLUS 5 g. Bolus 
______________________________________ 
Mixture of 
Gentamicin A, B, B.sub.1 and X (as the sulfate) 
8250.00 mg.* 
Lactose, Impalpable Powder 
8560.00 
Polyvinylpyrrolidone 900.00 
Magnesium Stearate 90.00 
Corn Starch 200.00 
18,000.00 mg. 
______________________________________ 
*Based on 60% potency, plus 5% excess 
Prepare a slurry consisting of the mixture of gentamicin A, B, B.sub.1 and 
X (as the sulfate), lactose and polyvinylpyrrolidone. Spray dry the 
slurry. Add the corn starch and magnesium stearate, and mix. Compress into 
tablets using suitable tableting equipment. 
Example 19 
______________________________________ 
ENTERAL SOLUTION mg/ml 
______________________________________ 
Gentamicin X (as the sulfate) 
40 to 200 mg.* 
Methylpraben 1.8 mg. 
Propylparaben 0.2 mg. 
Water for Injection q.s. 
1.0 ml. 
______________________________________ 
*Based on theoretical 100% purity. 
Place 70% of the water for injection into a suitable mixing vessel and heat 
to 70.degree. C. Add the methylparaben and propylparaben and mix until 
dissolved. Cool the above solution to 25.degree.-30.degree. C. Pass a 
stream of nitrogen gas over the solution. Add the gentamicin X (as the 
sulfate) and mix until dissolved. Bring the solution to final volume. Pass 
the solution through a suitable sterilizing filter, employing appropriate 
aseptic techniques. Fill the solution into suitable sterile containers 
employing appropriate aseptic filling techniques. 
Example 20 
______________________________________ 
ANIMAL FEED gm 
______________________________________ 
Mixture of 
Gentamicin A, B, B.sub.1 and X (as the sulfate) 
10* 
Soybean meal 400 
Fish meal 400 
Wheat Germ Oil 50 
Sorghum Molasses 140 
______________________________________ 
*Based on 100% purity 
Mix all of the above ingredients thoroughly and press into suitably sized 
pellets, the size of the pellet depending upon animal being treated. Mix 
the pellets with the animal's regular diet in such a quantity that from 
about 10 to about 100 mg. per kgs. of body weight is consumed by the 
animal each day. This composition may be fed to laboratory animals, such 
as mice, rats, rabbits, cats, dogs and the like. 
Alternatively, the antibiotic mixture or a single member thereof may be dry 
mixed with the animal's regular diet so as to provide a therapeutic dose 
in a normal day's ration. The method is particularly advantageous when the 
antibiotics are used to provide a prophylactic effect such as has been 
previously described for turkeys. 
Example 21 
______________________________________ 
5 gm- 
SUPPOSITORY Suppository 
______________________________________ 
Mixture of 
Gentamicin A, B, B.sub.1 and X (as the sulfate) 
5 gm.* 
Water 10 ml. 
Gelatin Granula 20 gm. 
Glycerin 65 gm. 
______________________________________ 
*Based on theoretical 100% purity 
Dissolve the mixture of gentamicin A, B, B.sub.1 and X (as sulfate) in the 
water and add the glycerin with stirring. Finally, add the gelatin and 
warm the mixture with stirring until homogeneous. Transfer the mixture to 
pre-chilled molds and allow to congeal. This formulation is sufficient to 
prepare twenty 5 gram suppositories.