CA1305923C - Methods and compositions for controlling protozoal infections in warm-blooded animals - Google Patents

Abstract

METHODS AND COMPOSITIONS FOR CONTROLLING
PROTOZOAL INFECTIONS IN WARM-BLOODED ANIMALS

ABSTRACT OF THE INVENTION
This invention relates to methods and compo-sitions for the control of protozoal infections in warm-blooded animals by administering to said animals a protozoacidally-effective amount of a new antibiotic designated LL-El9020.alpha., LL-E19020.beta. or a pharmaceutically and prophylactically acceptable salt thereof. The new antibiotics are produced by microbiological fermentation under controlled conditions using a new strain of Streptomyces lydicus subspecies tanzanius or mutants thereof.

Description

~305923 METHODS AND COMPOSITIONS FOR CONTROLLING
PROTOZOAL INFECTIONS IN WARM-BLOODED ANIMALS
BACKGROUND OF THE INVENTION
Protozoan parasites are known to infect both man and animals and to have exceedingly debilitating effects there upon.
Although the art is replete with reports of diseases stemming from protozoal infections, coccidiosis, malaria and babesia, are, perhaps, the most commonly encountered diseases resulting from protozoan infections in animals and/or man. Coccidiosis is an extremely severe disease that frequently afflicts swine, poultry, such as chickens, turkeys and geese, and ruminants such as cattle and sheep. This disease can strike with dev-astating suddenness and destroy an entire flock of birds or decimate a herd of cattle or flock of sheep or it may manifest itself as a mild outbreak tbat simply causes weight loss of the infected animals and reduces effi-ciency of feed utilization thereby. However, regardless of the manner in which the disease afflicts the host animals, significant monetary losses generally accompany such disease outbreaks when they occur in flocks or herds of meat producing or companion animals.
The protozoan parasites usually responsible for coccidiosic in the above-mentioned animals are generally of the genus Eimeria. Six species which have been shown to be primary causive agents for the disease are:

. ..

.' 13059~3 61109-7545 Eimeria tenella, Eimeria nec~trlx, Elmerla mivati, ~imeria maxima, Eimeria brunetti and Eimeria acervulina. In swine it has been shown that the protozoan Isospora suis is also responsible for coccidiosis.
Babesia infections, like Eimeria infections, have been a major concern to the livestock industry for many years. These infections frequently produce anemia and death in the infected animals and are responsible for significant economic lo~ses for the livestock producers.
While Babesia infections have generally been found to occur in ruminants, in recent years it has been dis-covered that the disease is also transmitted to domesticated pets such as dogs and to humans.
Importantly, it has been recognized by the livestock industry that there i8 no entirely satisfactory method for preventing, treating or controlling, Babesia infections in livestock, domestic animals or humans, presently available.
The present invention seeks to provide a method for preventing, treating or controlling Babesia infections in warm-blooded animals.
This invention also seeks to provide a method for preventing, treating or controlling coccid-iosis in wàrm-blooded animals.
Anticoccidial treatments that have met with some success and acceptance by the poultry industry in-clude the anticoccidial treatments of E. Waletzky et al.
described in Reissue U.S. patent number RE 26,833 reissued March 24, 1970; the A.S. Tomcufcik, U.S. paeent 3,769,432 issued October 30, 1973 and the W.D. Celmer et al. U.S.
patent number 4,148,882, issued April 10, 1979. The drugs described in these patents are useful for treating coccidial infections in poultry; but, new more effective treatments are needed if the industry is to successfully control the disease that plagues meat production through-out the world.
C

13~5~Z3 It is, therefore, another obiect of tha present invention to provide a novel method for preventing, treating or controlling, protozoan infections in warm-blooded anlmals.

SUMMARY OF THE INVENTION
~ses The present invention relates to ~M~*r~h~ and compositions effective for preventing, treatlng, controlling or ameliorating, protozoal infections in warm-blooded animals, particularly in poultry, cattle, sheep, swine and goats and in companion animals such as dogs and rabbits.
The antlbiotics whlch are effective when used in the composltions and methods of the present invention are antibiotics LL-E19020~, LL-E19020~ and the pharmaceutically and pharmacologically acceptable salts of said antibiotlcs. These antibiotlcs LL-E19020a and LL-E19020~ and a method for the preparatlon thereof are described in U.S. Patent 4,705,688 of G.T.
Carter M. Greensteln; J.J. Goodman; D.B. Borders; W.M. Maiese and R.T. Testa.
The abovè-said antibiotlcs and the pharmaceutlcally and prophylactically acceptable salts thereof are effective for preventlng or controlling proto70al infections in said warm blooded anlmals, when administered to said animals in protozoacidally effective amounts. More particularly, said antibiotlcs and antlbiotic salts are especially ef~ective for treatlng, preventlng, controlllng or amelloratlng protozoal lnfectlons in swlne, poultry, rumlnants and companlon animals.
The above antlblotics are also effective for controlllng protozoan infectlons caused by Eimeria and Babesia species in cattle, sheep, swine, chlckens, turkeys, ducks, geese and dogs.

B

:

.-1~0592;3 It is also anticipated that the antibioticcompositions of this invention will prove to be effective for controlling malaria, sarcosporidiosis and toxoplas-mosis in warm-blooded animals since the causative agents for these diseases are protozoan infections biologically related to Eimeria and Babesia.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. I shows an ultraviolet absorption spectra of LL-E19020~.
Fig. II shows an infrared absorption spectrum of LL-E19020~.
Fig. III shows a proton n~clear magnetic reso-nance spectrum of LL-E19020.
Fig. IV shows a carbon-13 nuclear magnetic resonance spectrum of LL-E19020~.
Fig. V shows an ultraviolet absorption spectra of LL-E19020~.
Fig. VI shows an infrared absorption spectrum of LL-E19020~.
Fig. VII shows a proton nuclear magnetic reso-nance spectrum of LL-E19020~.
Fig. VIII shows a carbon-13 nuclear magnetic resonance spectrum of LL-E19020~.
Fig. IX shows the effect of LL-E19020~ on the growth of B. bigemina.

130~;9;~3 DESCRIPTION OF THE PREFERRED EMBODIMENTS
Administration of the above-identified anti-biotics for control, treatment or prevention of proto-zoan infections in meat-producing and companion animals, will generally be most practical in or with the feed or drinking water of the animals. However, said antibiotics can be given to the animals on an individual basis in the form of capsules, tablets, oral gels, or the like. They may also be administered parenterally, generally by sub-cutaneous injection, as a gel, paste, pellet, solution orthe like, under the skin of the host animal.
In the practice of the present invention the antibiotics LL-E19020a, LL-F19020~ and the pharmaceuti-cally and pharmacologically acceptable salts thereof, may be employed prophylactically, pharmaceutically or thera-peutically for the control, prevention or inhibition of protozoal infections in poultry and ruminants. Generally about 0.1 ppm to 300 ppm, and preferably about 1 to 100 ppm of the antibiotic or antibiotic salt, in feed or drinking water, is effective for controlling protozoal infections, such as coccidiosis and Babesia in poultry, ruminants and companion animals.
Medicated animal feeds useful in the method of the present invention are usually prepared by thoroughly admixing about 0.00001% by weight to 0.03% by weight of the antibiotic LL-E19020a or ~ or salt thereof with a nutritionally balanced daily ration.
When using the compound of the invention for the prevention or control of protozoal infections, the active antiprotozoal agent is generally first prepared as an animal feed premix. The premix usually contains a relatively high percentage of the antiprotozoal agent and is generally blended with the animal's feed just prior to administration. If desired, the feed premix may also be applied as a top dressing for the animal's daily ration.
. .

, .. . .

Feed premixes or concentrates, useful in the practice of the present invention may be prepared by admixing about 1.0 to 15.0% by weight of the above-iden-tified antibiotic, or a pharmaceutically and pharma-cologically acceptable salt thereof, with about 99.0% to85% by weight of a suitable carrier or diluent. Carriers suitable for use to make up the feed supplement composi-tions include the following: alfalfa meal, soybean meal, cottonseed oil meal, linseed oil meal, sodium chloride, calcium carbonate, corn meal, cane molasses, urea, bone meal, corncob meal, rice hull meal, and the like. The carrier promotes an essentially uniform distribution of the active ingredient in the finished feed into which the supplement is blended. It thus performs an important function by ensuring proper distribution of the active ingredient throughout the feed.
In practice, usually one or more pounds of premix is added per ton of feed to obtain the desired level of antibiotic in the finisbed feed.
If the supplement or premix is used as a top dressing for feed, it likewise helps to ensure uniformity of distribution of the active material across the top of the dressed feed.
Since the compound of this invention and its pharmaceutically and pharmacologically acceptable salts are relatively insoluble in water, it is generally de-sirable, when administering the compound in the animal's drinking water, to dissolve the active compound in an organic solvent such as methanol, ethanol, acetone, DMS0, oleic acid, linoleic acid, propylene glycol, or the like, and admix with the solution a small amount of surfactant and/or dispersing agent to assure solution and/or disper-sion of the active ingredient in the animal's drinking water.

When administered to cattle, sheep, swine, poultry or companion animals on a mg/kg of body weight/day basis, generally about 0.0001 to 15 mg/kg of animal body weight per day, is effective for preventing or controlling protozoan infections in the above said animals. For prolonged treatment of animals, rates of from about 0.0001 mg/kg body weight/day to 5 mg/kg of animal body weight/day are usually employed.
For parenteral administration, the antibiotic or antibiotic salt may be prepared in the form of a paste or pellet and administered as an implant, usually under the skin or the head or ear of the animal.
In practice, parenteral administration generally involves injection of a sufficient amount of the above said antibiotic or antibiotic salt to provide the animal with from about 0.0001 to 15 mg/kg of body weight of the active ingredient.
Paste formulations can be prepared by dispersing the antibiotic or antibiotic salt in a pharmaceutically acceptable oil such as peanut oil, sesame oil, corn oil or the like.
Pellets containing an effective level of the antibiotic LL-E19020~ or LL-E19020~ can be prepared by admixing the above-said antibiotic with a diluent, such as carbowax, biodegradable polymers, carnauba wax, or the like. A lubricant, such as magnesium stearate or calcium stearate may be added to improve the pelleting process if desired.
It is, of course, recognized that more than one pellet may be administered to an animal to achieve the desired dose level which will provide the increased growth rate and/or imPrOve efficiency of feed utiliza-tion by said animal. Moreover, it has been found that additional implants may also be introduced periodically during the treatment period in order to maintain the proper drug release rate in the animal's body.

,~, . .. .

The antibiotics of this invention,LL-E19020~
and LL-E19020~,are formed during the cultivation under controlled condition~ of a new strain of Streptomyces lydicus ssp. tanzanius.
This microorganism is maintained in the culture collection of the Medical Research Division, American Cyanamid Company, Pearl River, NY as culture number LL-E19020. A viable culture of this new microorganism has been deposited with the Patent Culture Collection Labor-atory, Northern Regional Research Center, U.S. Department of Agriculture, Peoria, Illinois 61604, and has been added to its permanent collection. It has been assigned the strain designation NRRL 18036 by said depository.
Access to said culture, under strain designation NRRL
18036, during pendency of the instant application shall be available to one determined by the Commissioner of Pat0nts and Trademarks to be entitled thereto under 37 C.F.R. 1.14 and 35 U.S.C. 122, and all restrictions on availability to the public of such culture will be ir-revocably removed upon grant of a patent on the instant application.
Culture LL-E19020 produces short spiral spore chains, 10-50 spores long, with occasional longer chains.
These tend to coalesce to form dry blackish masses on such ISP media a8 oatmeal and inorganic salts-starch.
Tbe spores have smooth surfaces as assessed by electron microscopy. The strain contains the L isomer of diamino-pimelic acit, and may thus be assigned to the genus Streptomyces.
In the ISP tests for utilization of carbohy-drates, LL-E19020 shows growth on arabinose, fructose, inositol, mannitol, raffinose, rhamnose, sucrose and xylose. Cellulose is not utilized.

~305923 The reactions of LL-E19020 in the Gordon physiological series are compared in Table I
with those of Streptomyces lydicus ISP 5461 which it most closely resembles morphologically and physiologically.
Because LL-E19020 differs from ISP 5461 in five characteristics (xanthine hydrolysis, decarboxyla-tion of oxalate, acid from erythritol, rhamnose and ~-methyl-D-xyloside) it is designated as a subspecies of StreptomYces lydicus.
TABLE I
Gordon Test Reactions of LL-E19020 and Streptomyces lydicus ISP 54~I

Reaction LL-E19020 ISP 5461 ; De~radation/Transformation of Casein + +
Xanthine - +
Hypoxanthine + +
Tyrosine + +
Adenine + +
Production of Amylase + +
Gelatinase + +
Phosphatase + +
: 25 Nitrate Reductase - _ Urease + +
. Esculinase + +
Growth on/in 5% Sodium chloride + +
Salicylate Ly~ozyme Broth trace trace Utilization Acetate + +
Benzoate Citrate + +
Lactate ' ~' TABLE I (Continued) Reaction LL-E19020ISP 5461 Malate + +
Mucate + +
Oxalate . +
Propionate + +
Pyruvate + +
Succinate + +
Tartrate Growth at 10C + +

50C _ _ Acid from Adonitol + +
Arabinose +
Cellobiose + +
Dextrin +
Dulcitol - _ Erythritol . +
Fructose + +
Galactose + +
Glucose + +
Glycerol + +
Inositol + +
Lactose + +
Maltose + +
Mannitol * +
Mannose -~ +
Melibiose ~ +
a-Methyl-D-Glucoside ~ +
Raffino~e + +
Rhamnose +
Salicin + +
Sorbitol + +
Sucrose + +
Trehalose + +
Xylose + +
~-Metbyl-D-Xyloside +

~. , t3059Z3 It is to be understood that for the production of these new antibacterial agents the present invention is not limited to this particùlar organism or to organ-isms fully answering the above characteristics which are given for illustrative purposes only. In fact, it is desired and intended to include the use of mutants pro-duced from this organism by various means such as ex-posure to X-radiation, ultraviolet radiation, N'-nitro-N-nitrosoguanidine, actinophages and the like.
General Fermentation Conditions .
Cul~ivation of Streptomyces lydicus ssp.
tanzanius NRRL 1803~ may be carried out in a wide variety of liquid culture media. Media which are useful for the production of LL-E19020~ and LL-E19020~ include an as-similable source of carbon, such as dextrin, sucrose,molasses, glycerol, etc.; an assimilable source of nitrogen such as protein, protein hydrolycate, polypep-tides, amino acids, corn steep liquor, etc.; and in-organic anions and cations, such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, etc. Trace elements such as boron, molyb-denum, copper, etc., are supplied as impurities of otber constituents of the media. Aeration in tanks and bottles is supplied by forcing sterile air through or onto the surface of the fermenting medium. Further agitation in tanks is provided by a mechanical impeller.
An antifoam a8ent such as silicon oil may be added as needed.
General Procedure for the Isolation of LL-ElgO20~ ant LL-E19020B
The LL-E19020~ and LL-E19020B antibiotics are recovered from the fermentation broth by pH adjustment to 4.5-5.5, filtration through diatomaceous earth, ex-traction into a solvent such as ethyl acetate, concen-tration, dissolution in a solvent such as dichloro-methane and purification by column chromatograpby on :

,..... .

silica gel using successively, dichloromethane and methanol:dichloromethane (1:4), giving a crude product.
The crude product is then separated into the a and ~ components and further purified by high pe~formance liquid chromatography on a reverse-phase column using the system acetonitrile, O.lM ammonium acetate buffer pH
4,3 (1:1).
The physico-chemical characteristics of LL-E19020~ are as follows:

1. Approximate elemental analysis: C 62.73; H 7.60;
N l.00; 0 28.67 (by difference);

2. Molecular weight: 1225 (FABMS);

3. Apparent molecular formula: C64H91N22;

4. Specific rotation: [~]D6~ O (C O.385, methanol);

5. Ultraviolet absorption spectra: as shown in Figure I
WCH~OH 3 233nm (~ 49,800) MA 290nm (~ 36,600) 6. Infrared absorption spectrum: as shown in Figure II
(K8r disc): 3420, 2970, 2925, 1717, 1695, 16 7, 1617, 1525, 1445, 1365, 1092, 1018 cm~l;

7. Proton nuclear magnetic resonance s~ectrum: as shown in Figure III (300 MHz, CDC13~;

8. Carbon-13 nuclear magnetic resonance spectrum: as shown in Figure IV (75 MHz, GDC13, ppm downfield from TMS), significant peaks as listed below:
173.3 129.0 97.3 74.2 55.4 17.2 171 4 128 6(2x) 97.0 72.0 49.8 17.0 170 1 128 43 89.2 71.9 41.8 14.8 145 7 128.38 83.3 69.1 39.8 13.5 140 3 128.1(2x) 81.6 67.5 39.1 10.8 137 0 127 5 77.6 66.4 38.8 10.0 134 4 127 1 77.0 66.1 32.9 133 9 126.3 76.4 63.5 31.0 ; 132 0 120.8 74.6 56.5 29.9 130.1 100.6 74.5 56.0 23.8 129.5(2x) g9.0 74.4 55.6 18.1 2x - two overlapping signals .....

. .

~3(~5923 LL-E19020~
1. Approximate elemental analysis: C 63.33; H 7.72;
N 1.16; 0 27.79 (by difference);
2. Molecular weight: 1225 (FABMS);
3. Apparent molecular formula: C64H91N22;
4. Specific rotation: [~]26 = -17~2tC 0.455, methanol);
5. Ultraviolet absorption spectra: as shown in Figure V
WCH3H , 233nm (~ 47,000~
MAX 290nm (~ 34,100) Uv0.lN HCl = 234nm (~ 46,000) MAX 301nm (~ 32,800) Uv0.1N NaOH = 217nm ( 77,800) MAX 290nm (~ 39,700) 6. Infrared absorption spectrum: as shown in Figure VI
(KBr disc): 3430, 2970, 2930, 1712, 1648, 1620, 1543, 1454, 1367, 1265, 1098, 1020, 980 c~
7. Proton nuclear magnetic resonance spectrum: as shown in Figure VII (300 MHZ, CDC13~;
8. Carbon-13 nuclear magnetic resonance spectrum, as shown in Fi~ure VIII (75 MHz, CDC13, ppm downfield TMS), 8igni~icant peak8 a~ listed below:
173.6 9g.0 55.4 170.6 98.4 ~9.6 170.0 97.2 41.6(2x) 14S.6 89.2 39.8 140 2 83.3 39.1 136 7 81.6 38.0 134 4 77.6 32.9 133 9 77.5 31.1 132 0 76.2 29.9 130 1 75.5 23.7 129 1(2x) 74.6 18.1 128 9 74.5 17.2 128.6(2x) 74.2 17.0 128.5 69.1 16.2 128.4 68.9 13.5 128 3 67.5 10.8 128 2 66.6 10.0 127 8 66.1 127 2 64.1 ~oo'69 55~5.56 2x ~ two overlapping signals .
.

1305g~3 Evaluation of test compounds as anticoccidial agents -Eimeria tenella Anticoccidial Cell Culture Screen (E. tenella) Monolayers are initiated from primary kidney cells obtained from 7 day old chickens. Generally, 50.000 cells are administered in 1 mL volumes of culture medium inro 24 well cluster plates. The medium of preference is M199 buffered with 0.125% NaHC03. 5%
fetal bovine serum is addet to complete the medium.
Monolayers are permitted to grow to 50% con-fluency prior to ino_ulation and drug medication. Thisusually requires 48 hours in a humidified incubator held at 41C in an atmosphere of 5% C02-95% air.
Inocula containing 60,000 E. tenella sporozoites per 1 mL (2 mL total volume) is utilized to infect adequately confluent monolayers. Initial medium is aspirated off prior to administering 1.9 mL of sporozoite containing maintenance medium.
Medication is administered at predetermined concentrations in 0.1 mL volumes immediately after the introduction of sporoæoites. As a rule synthetic compounds are testet at 1 ppm and crude fermentation products at a dilution of 1:200. The former are dissolved in DMS0 and the latter in phosphate buffered saline. Penicillin and streptomycin are added to all medium to control possible contamination.
Treatments are observed by inverted phase contrast microscopy at 96-120 hours post-inoculation Eor cytotoxicity and anticoccidial activity. No staining of monolayers is required. Agents which prevent or markedly reduce the development of second generation asexual stsges oE the parasite liEe cycle are considered active.

.
,.,: : , 13()592;~

Medications which prevent an anticoccidial reading due to gross cytotoxicity are diluted 2 fold until end-points are achieved. Actives are generally diluted to inactivity to determine relative potency.
Monensin and robenidine are included in all experiments as positive standards. Data obtained are reported in Table 2 below.

Table 2 Evaluation of Test Compounds Using In Vitro Anticoccidial Screen - Eimeria tenella Results in ppm of Culture Medium Compound 5 2.5 1.25 0 6 0.3 Q.15 LL-E19020~ T T A A a 0 LL-E19020~ T T T T A A

Performance Ratin~
T - Toxic A - Active a - Marginal Activity 0 - Inactive ~3059~3 Evaluation of test c~po~nds as anticoccidial agents -Eimeria mitis -Anticoccitial Embryonated Egg Assay - E.mitis Chick embryros which are 10 days of age are candled for viability and placed into groups of 5 eggs each. A small hole is punched into the chorioallantoic cavity as an avenue for chemical agents. Synthetic compounds are screened at 1 mg per embryo and crude natural products at 0.2 mL per embryo. Penicillin/
streptomycin is utilized to control contamination. After administration of drugs via sterile tuberculin syringe, the pin hole is sealed with collodion. The embryos are returned to a humidified incubator set at 103F and held for 24 hours. All embryos are then recandled to remove any dead due to toxicity of drug treatment. All surviving embryos are then inoculated through the original pin hole with 80,000 E.mitis sporozoites in a volume of 0.1 mL. Embryos are resealed and returned to the incubator.
After 6 days all embryos are again candled and the dead removed and recorded. The chorioallantoic (CAM) membranes from each treatment are pooled and homogenized in 50-70 mL of tap water. Oocyst counts are then performed with a hemocytometer. A treatment is considered active if there is a 80% or greater reduction of oocysts compared to the numbers detected in the 4 replicates of nonmedicated inoculated controls. Two or more embryos must survive for a valid anticoccidial reading. In groups where one or no eggs remain after 6 days two fold dilutions are performed until an anticoccidial reading can be realized.
Robenidine is used as a positive drug at 0.1 mg per embryo.

~30S923 The E.mitis utilized has been made adaptable to this assay through repeated passages in embryos.
(P.L. Long).
Data obtained are reported in Table 3 below.

Table 3 Evaluation of Test Compounds as Anticoccidial Agents - Eimeria mitis Milligrams of Drug Per Embryo Compount 1 0.5 0.25 LL-E19020~ A A 0 LL-El9020~ 0 :~' Performance Rating i T - Toxic A ~ Active a - Marginal Activity 0 - Inactive ,:
' 30 ;
':

, i .
" , ~
~ .
..,. _"..~.,, .. .: , , , ,.

Evaluation of test compounds for controlling Babesia bovis and Babesia bigemina For these evaluations laboratory cultures of Babesia bovis and Babesia bigemina are continuously maintained. A normal donor animal is maintained as a source of normal RBC's and serum.
Babesia cultures are maintained as follows:
1. The percent of Babesia growing in each flask is determined. If the percent is under 10% the cultures are fed.
2. When the percent of Babesia in a flask is between 10-20% and the cells and Babesia look healthy the culture should be split or used for the drug screening.
A. Media for Babesia cultures 30 mL 199tEarls) Media 20 mL serum 1 mL TES
Check pH (7.00) and adjust if necessary Filter sterilize B. Feeding of Babesia cultures Always put exactly the same amount of media in a culture as you remove.
C. Splitting Babesia cultures Remove media from flask but do not remove any of the RBC. Add the exact amount removed of media + serum + tes. Mix gently.
Add 10 mL of the above gently mixed Babesia culture to 30 mL of 199 media/serum/tes and 3 mL RBC. Split Babesia culture 1:4.

130592;~

Depending on the size of the flask use the amount of split Babesia culture necessary to maintain a viable culture.
200 mL - 45 mL split Babesia culture 30 mL - 15 mL split 8abesia culture Preparation of Babesia Cultures for Drug Screening 1. Make up 50 mLs Media 199 + serum + tes (pH
and filter).
2. Make slides of infected cultures and determine the % of infected cells.
3. Make up a 5% suspension and a 6.7% suspension or normal bovine RBC's in Media 199 + serum + tes.
4. Dilute infected cells in the 6.7% suspension ' or normal RBC to obtain a final suspension containing 4%
infected cells.
Preparation of microtiter plates.
1. Label plate as follows:
a. Row A ~ 200 ~1 5% suspension normal ' RBC
,~ b. Row B - 150 ~1 4% suspension infected cells + 50 ~1 media.
;~- c. Row C - 150 ~1 4% suspension infected ` cells + 50 ~1 drug conc.
d. Row D - 150 ~1 4% suspension infected cells + 50 ~1 drug conc.
- e. Row E - 150 ~1 4% suspension infected cells + 50 ~1 drug conc.
f. Row F - 150 ~1 4% suspension infected cells + 50 ul drug conc.
g. Row G - 150 ~1 4% suspension infected 3S cells + 50 ~1 drug conc.
h. Row H - 150 ~1 4% suspension infected cells + 50 ~1 drug conc.
, I
, . .

., .

-;

~30~;9;~3 2. After plate is complete place in the incu-bator over night (18 hrs) 3. Add 25 ~1 of 3H hypoxanthine to each well the next morning.
4. Place the plate in the freezer at the end of the day.
5. Harvest the plate, after incubating for 2 hrs, and prepare for counting in the scintillation counter.
Preparation of Drug 1. Weight out 10 mg of drug - place in sterile 15 mL tube. Add 10 mL of media 199 + serum + tes to make a concentration of 1 mg/mL or 1000~g/mL.
Note: if not soluble use 10 mL of 70%ETOH
+ 30% H2O
2. Filter.
End Conc.
Drug Conc. Dil factor in culture a.1000 ~g/mL none 150 ~g/mL
b.500 ~g/mL 1:2 of a 125 ~g/mL
c.200 ~g/mL 1:5 of a 50 ~g/mL
d.100 ~g/mL 1:10 of a 25 ~g/mL
e.50 ~g/mL 1:10 of b 12.5 ~g/mL
f.20 ~g/mL 1:10 of c 5 ~g/mL
then dilute the 1000 ~glmL 1:50 - 20 ~g/mL
a.20a ~g/mL none 5 ~g/mL
b.10 ~g/mL 2:2 of a 2.5 ~g/mL
c.S ~g/mL 1:2 of b 1.25 ~g/mL
d.2.5 ~g/mL 2:2 of c 0.625 ~g/mL
e.1.25 ~g/mL 2:2 of d 0.31 ~g/mL
fØ625 ~g/mL 2:2 of e 0.155 ~g/mL

.

' Anti-Babesia bovis Activity Procedure: Babesia bovis is maintained in in vitro culture. The cultures were diluted to a para-sitemia of 4% and cultured with various dilutions of the drugs. After incubation for 18 hours 3H hypoxanthine was added and the cultures incubated for an additional 18 hours. The plates were harvested and the samples counted. Growth inhibition results in a decreased in-corporation of 3H hypoxanthine. The drugs were evaluated on duplicate plates and at 2 different tim intervals.
With this procedure, the 50% inhibitory concentration of LL-E19020a was found to be 0.625 ~g/mL.
Anti-Babesia bigemina Activity Procedure: Babesia bigemina is maintained in in vitro culture. The cultures were diluted to a para-sitemia of 4% and cultured with various dilutions of the drugs. After incubation for 18 hours 3H hypoxanthine was added and the cultures incubated for an additional 18 hours. The plates were harvested and the samples counted. Growth inhibition results in a decreased in-corporation of 3H hypoxanthine can be seen in Fig.IX.
The drugs were evaluated on duplicate plates and at 2 different time intervals.

Inoculum Preparaeion A typical medium used to grow the primary inoculum was prepared according to the following formula:
Dextrose ........................ 1.0%
Dextrin ......................... 2.0%
Yeast extract ................... 0.5%
NZ Amine A~l .................... 0.5%
Calcium carbonate .... .......... 0.1%
Water qs ............. 100.0%

1 [A pancreatic digest of casein, registered trademark of Sheffield Chemical, Norwich, NY~
This medium was adjusted to pH 7.0 and then sterilized. A 100 ml portion of this sterile medium in a 500 ml flask, was inoculated with mycelial scrapings from an agar slant of Streptomyces l~dicus ssp. tanzanius NRRL 18036. The metium was then placed on a rotary shaker and incubated at 28C for 48 hours. This primary inoculum was then used to inoculate 10 liters of the same sterile medium in a bottle. This medium was grown for 24 hours providing secondary inoculum. This secondary inoculum was thén used to inoculate 250 liters of the 8ame sterile medium in a tank. This medium was grown at 28C for 48 hours with a sterile air flow of 200 liters per liter of mash per minute and agitation by an impeller driven at 220 rpm, providing tertiary inoculum.

....
- ~ , ..

EXAMPLE S
Fermentation A fermentation medium of the following formula-tion was prepared:
Dextrin ...................... ..3.0%
Molasses ..................... ..2.0%
Soy peptone .................. .Ø75%
Yeast extract ................ .Ø25%
Calcium carbonate ............ .Ø2%
Water qs ..................... 100.0~

This medium was sterilized and 2700 liters was tben inoculated with 300 liters of tertiary inoculum from Example 4. The fermentation was contucted at 28C, with a sterile air flow of 0.55 liters of air per liter of mash per minute and agitation by an impeller driven at 100 rpm for 113 hours, at which time the mash was harvested .

I~olation and Purification of LL-EI~0~ ana LL-~17V2CP
The harvest ma~h from two fermentations conducted as described in Example 5 were combined, making a total of 6000 liter~, adjust2d to pH 5 with hydrochloric acid and filteret through tiatomaceous earth. The filtrate was extracted with ethyl acetate and the extract concen-trated to a syrup.
This syrup was dissolved in dichlormethane and applied to 100 g of silica (60-200 mesh) on a sintered glass funnel. The ~ilica column was first eluted with dichloromethane, collecting four 2 liter fractions and then with methanol:dichloromethane (1:4) collecting a 4 liter fraction. This 4 liter fraction was evaporated to dryness, giving 120 g of residue. The resitue was re-dissolved in 4 liters of dichloromethane and applied to ' ''' ~305923 500 g of silica on a sintered glass funnel. The silica 5 was eluted with methanol:dichloromethane (1:4) collecting 2 liter fractions. Fractions 1 and 2 were combined and evaporated, giving 99 g of crude LL-E19020~ and 3.
This crude product was tissolved in methanol and applied to a 12 liter reverse-phase column (C18 10 bonded ph~se 40 micron). The column was eluted with acetonitrile, O.lM ammonium acetate buffer pH 4.3 (1:1) at a rate of 1.0 liter per minute. Thirteen 24 liter fractions were collected. Fraction 7 contained LL-E19020~ and fractions 11-13 contained LL-E19020~.
The antibiotics were extracted from the mobile phase using dichloromethane followed by evaporation and freeze drying from t-butanol, giving 10 g of LL-E19020 and 14 g of LL-E19020~, both as white solids.

Claims (16)

1. Use of a therapeutically effective amount of antibiotic LL-E19020.alpha., antibiotic LL-E19020.beta. or a pharmacologically acceptable salt thereof for controlling protozoan infections in infected warm-blooded animals comprising administering to said warm-blooded animals.

2. A use according to claim 1 wherein said warm-blooded animals are meat producing or companion animals-.

3. A use according to claim 1 wherein said animals are cattle, sheep, swine, rabbits, poultry or dogs.

4. A use according to claim 1 wherein the protozoan parasite causing the infection of said warm-blooded animals is of the Eimeria or Babesia species and said antibiotic or antibiotic salt is present in animal feed containing from about 0.1 to 300 ppm of said antibiotic or antibiotic salt.

5. A use according to claim 1 wherein the protozoan parasite causing the infection in said warm-blooded animals is of the species of the genus Eimeria or Babesia and said antibiotic is in doses sufficient to provide the host animal with from about 0.0001 mg/kg of animals body weight per day to about 15 mg/kg of animal body weight per day of said antibiotic or antibiotic salt.

6. Use of a coccidicidally effective amount of an antibiotic selected from LL-E19020.alpha., LL-E19020.beta. or a pharmacologically acceptable salt of said antibiotics for controlling coccidiosis in infested poultry, rabbits, cattle, swine or sheep, comprising orally or parenterally administering to said infested animals.

7. Use of a protozoacidally effective amount of the antibiotic LL-E19020.alpha., LL-E19020.beta. or a pharmacologically acceptable salt thereof for controlling protozoan infections in Babesia infected cattle, sheep, dogs, or cats comprising administering to said infected animals.

8. A composition for controlling or preventing protozoan infections comprising a therapeutically or prophylactically effective amount of the antibiotic LL-E19020.alpha., LL-E19020.beta. or a physiologically acceptable salt thereof.

9, A composition according to claim 8 for warm-blooded animals containing from about 0.1 ppm to 300 ppm of antibiotic LL-E19020.alpha., LL-E19020.beta. or a physiologically acceptable salt.

10. A composition according to claim 9 wherein the warm-blooded animals are cattle, sheep, swine, goats, horses, poultry or rabbits.

11. A composition for controlling coccidiosis in coccidiosis infected animals comprising a coccidicidally effective amount of the antibiotic LL-E19020.alpha., LL-E19020.beta. or a physiologically acceptable salt thereof.

12. A composition according to claim 11 containing from about 0.1 ppm to 300 ppm of antibiotic LL-E19020.alpha., LL-E19020.beta. or a physiologically acceptable salt.

13. A composition according to claim 12 wherein the infected animals are poultry, rabbits, cattle, swine and sheep.

14. A composition for controlling protozoan infections in Babesia infected animals comprising a protozoacidally effective amount of the antibiotic LL-E19020.alpha., LL-E19020.beta. or a physiologically acceptable salt thereof.

15. A composition according to claim 14 for warm-blooded animals containing from about 0.1 ppm to 300 ppm of antibiotic LL-E19020.alpha., LL-E19020.beta. or a physiologically acceptable salt.

16. A composition according to claim 15 wherein the warm-blooded animals are cattle,sheep, dogs or cats.