IE54814B1 - Use of antibiotic ll-c23024 alpha in controlling nematodes - Google Patents

Description

USE OF ANTIBIOTIC LL-C23024 n IN CONTROLLING NEMATODES This invention relates to the use oi the antibiotic LL-C23G24 in controlling nematodes. The present application is divided out of our co-pending Application No. 8225339 (Publication No. 2108113A).

The antibiotic LL-C23024 a has the following structure: DMe w.

The antibiotic LL-C23024 a may be obtained by 20 aerobically fermenting Ac-tinomadura yumaeose sp. nov. or a mutant thereof in a liquid medium containing assimilable sources of carbon, nitrogen and inorganic salts, until substantial antibiotic activity is imparted to the fermentation broth, as described in greater detail in the 25 parent application, supra.

The antibiotic LL-C23024 a identical to the antibiotic designated X-14868A in U.S.-A-427S663 assigned to Hoffman-La Roche Inc which issued on 14-07-81. This prior U.S. patent discloses 30 that antibiotic X-14868A is active against the growth of certain gram-positive bacteria, possesses anticoccic'iiostatic activity eg when administered to fowl such as turkeys and chickens, can be used as a growth -promotant in ruminant animals such as cattle, thereby -2- also preventing and treating ketosis in such animals, and, finally, has demonstrated activity in the treatment or prevention of swine dysentery.

The present invention is based on our discovery 5 that antibiotic LL-C23024 a exhibits activity as a nematocidal agent. As is well known (see eg Webster's Third New International Dictionary, 1971), nematodes may be parasites of warm-blooded animals or free-living dwellers in soil.

·· The following tests demonstrate the nematocidal activity of antibiotic LL-C23024 a.

EXAMPLE A Evaluation of test compositions for nematocidal activity using the free-living Hermaphroditic tnicrobivorous 15 nematode Caenorhabditis elegans II In the following tests, C. elegans is used to determine the nematocidal activity of the fermentation broth and the harvest mash containing antibiotic LL-C23024 a and prepared by the procedures taught in the 20 parent application. This organism is'also used to evaluate LL-C23024 a to determine the nematocidal activity thereof. -3- Η ' In these tests, fermentation broth is the fermentation liquid end solids mixture taker, before the solids, the harvest nash, is separated i:-„n the liquid. Harvest cash is the solids remaining after separation.

For evaluation of harvest mash, the harvest cash solids are dispersed 1:1 in distilled water. The fermentation broth is used as is and contains both liquid and solids.

In the present evaluations, C. eleaans is maintained in a C. briggsae Maintenance Medium. The Maintenance 10 Medium is commercially available from Grant Island Biological Company, Grant Island, Mew 'fork, and has the following compositions: C. briggsae MAINTENANCE MEDIUM^1) Component rag/L INORGANIC SALTS CaCl2 * 2H20 220.50 CuCl2 · 2H20 b.50 Fe(MHi>)2 (S0i})2 - 6h20 58.80 KH2P0« 1225.50 KOH (a) MnCl2 - 4h20 22.20 ZnCi2 1C.20 -4- '* w i. -i OTHER COMPONENTS N-Acecy lslucosamins 1 5.00 Acer.osine-3 ' -(2 ') -chosohoric ac id · Κ·?0 355.00 C/c id ins-J'- (2 ') - ohosshor ic ac id 32 3.0·:· D-C-lucose 1315.00 Giucachions. reduced 2QA.OO Guanos ins-3'-(2') -PO^Na·?-H?0 363.00 Masnesiun citrate-5HtQ (Dibasic) 915.00 Pocassiua cicrate-K-jO £56.00 DL-Tbioctic ac.d 3.75 Thyaine 126.00 Urid ins-3 * - (2') -ohosoohoric acid 32Δ.ΟΟ AMINO ACIDS L-Alanine 1395.0!) C-Arginine 975.00 L-Aspartic acid 1620.00 L-Cysteine HCI-HyO 78.00 L-Glutanats (NaJ-HyO 550.00 L-Glutanine 1963.00 Glycine 722.00 L-Histidine 285.00 U-Isoleuc i πε 861.00 L-L^ucint* is3·- f)-0 L-Lysine HC1 1283.00 L-Methionine 389.00 L-Phenylalanine 803.00 L-Proline 653.00 L-Serine 788.00 L-Threonine 717.00 L-Tryptophan 184.00 L-Tyrosins 272.00 L-V'al ine 1020.00 VITAMINS 1 p-aainobsn-oic acid 7.50 Eiocin 3.75 Choline dihydrcsea citrate o8.50 -5- VI-AMIMS (Continued! Cyanocobalanir.e (E*2) 3.75 Folinace (Ca) 3.75 Hyo-inosicol 6^.50 Niacin: 7.50 Niacinamide 7.50 Pantechine 3.75 Pantochenace (Ca) 7.50 Pterolyelucamic acid 7.50 Pyridoxal phosohace 3.75 Pyridoxamine 2HC1 3.75 Pycidoxine HC1 7.50 Riborlavin-5‘-F0i(Na)-2HoO 7.50 Thiamine KC1 7.50 15 References: Cl) Hansen, E. L., Proc. Soc. Exp. Bio. & Med. 121: 30-393 (1966).

Remarks: (a) As needed for'adjuscmenc Co pH 5.9 + 0.1. -6- 4 a j- * A well plate having a series of small veils is used for the evaluations. Twenty-five si of fermentation broth, 1:1 wacer/fcarvest cash suspension, or an aqueous/ soetor.s solution of LL-C2302-C. containing from 5 31.25 to 500 ppm of te_t compound, is asesptieaily deposited in separate veils. To each veil· is then aided £5 r.i of the C. elegans maintenance medium containing 10-20 acuit worms plus larvae of various ages plus eggs. The plates are then placed in a hooc and examined at US hours post-10 -inoculation to assess the speed and degree of nematocidal activity of the test compositions. Data obtained are reported below. Where activity is observed for the fermentation broth, said broth is further diluted to provide a 1:4 broth to C. elensns ratio.

TABLE VII Concentration ppm or Nematocicial Activity Composition 20 Fermentation Broth • Dilution at 48 hours D=l:l 3 (no motility) 0=1:4 0 0 in ppni 7 250 ppm 7 125 ppm 7 62.5 p pm 6 31-25 ppm 0 Harvest Mash LL-23024 e 25 The activity legend employed in these evalua , 30 t i 0 is as follows: Activity Legend 3 = No Motility (Apparent Death) 7 = Markedly Reduced Motility 6 =vSeduced Motility 0 = Normal Motility for Species -7- 35 EXAMPLE B Evaluation of LL-C2302-gCas a r.e.T.a tool dal arent arairst infective larvae"5* of ruminant rer.atocss Evaluation of LL-C2302- as a ner.atGcieai assn», against the infective larvae of ruminant nematodes: Haeacr.hr.u5 contortus, Ostertaaia circur.cincaa, Trichoserc-r.gy'.us axei. T. Colubriforais; and against the vinegar eel worm. Turbatrix aceti was determined using the above-mentioned nematode species were substituted for £. elgzar.s.

Data obtained are reported in the Table below. s* Third Stage Sheathed Larvae -8- l. Λ νο νο M3 νο Ο uα Η TADLH VIII η η «4 r^’ X Ο 1* 2 ο γΗ W _3 Ζ) <**. Λ Ό Ό C C 'Ό <3 *3 Ο Ο U U tz α Cu 3 ο U α CU U 3 jj r—i 3 ϋ 0 ό 0 JJ (3 c » α C c α jj tu <0 •Η α. r-1 "7 ο U L-) 3 (N JJ •S Cl Η 'O jj a 3 a >« —H 0 f3 c Jj u c —♦ G α C i—i 04 3 0 jj —4 CO Μ —4 c Jj SJ JJ G o u u X 0 JJ <3 V^J o a *v >. «-J LI a a JJ > #-! < u •J JJ C* . «*—« 3 f—1 « Ό —< H •ar >1 £ JJ —4 Cl V3 «. JJ o JJ kJ ffv O t: —· o 3 rn e 3 o C ro c? •o O d JJ 'a o #H 1 3 o o o (3 •*r •3 _j X 3 s CM Ο ““ u 3 u jj «j 0 n G 0 3 ο 0 Ul 2 X ε 3 ·“· «3 pH > !1 11 u 11 ο — JJ £3 o o s jj L*1 υ — iN < ·9· EXAMPLE C Evaluation of r.e.-natccidal activity of antibiotic LL-C2;02Ac. against her.acosoiroidss cubius ana Asoicularis ‘•-etriiters in nice = r~·. the following tests, Swiss-Webs ter female whits o.ice ere infected with iiamatosoiroides duoius end Asoicularis cetraofcera and held for three weeks to permit the infections to nature.

After the holding period, the nice are randomly 10 divided into groups of four, and· the groups placed in separate cages. Medicated feeds containing from about 31-25 ppm to 4,000 ppm of test compound are then offered ad libitum to the mice for one week. Water is also provided ad libitum throughout the test period. During the 15 treatment period, the mouse droppings are examined to determine uhether worms are being passed. Ail treatment groups were found to be passing worms, thus indicating •nematocidal activity at all compound -concentrations against both nematodes. At*the-end of the one-week medicated feed 20 treatment, the mice are necrcpsied, and the contents of the intestinal tracts thereof examined for worms. Data obtained are reported below as percent reduction of worms as compared with infec.ted, unmedicafced controls.

In these tests, crude fermentation broths obtain-25 ed before harvest of tha mash, and containing from 1,000 ppm to 4,000 ppm of the nematocidal antibiotic were evaluated. Also evaluated was mouse feed treated with from 31.25 to 500 ppm of LL-C23024d dissolved in an acetone/ •water 1:1 mixture. -10- (¾ .-.eduction). *’* Test Distsrv Composition· Cor.c, ?cm 1 *2 a * M. cubits Γ. S ·- A. terrsoti 5 *? smsnt.E" tion Broth. 4,000 73 with. LL-C23034O 2,000 44 53 1L-C23024a 1,000 65 33 500 70 S a * * 10 250 63 SA 125 61 SA 62.5 29 SA 31.25 32 SA 15 * = Amount of IiL~C23024a ' undetermined ** = Sligh.t activity: Increased number ox pinworms . recovered in fecal examination = Compared to infected unnedicated controls * * * The above examples show that antibiotic LL-C23024 a (or fermentation broth or harvest mash containing the antibiotic) is active against nematodes. Accordingly, the present invention in one aspect, provides a method of controlling nematodes in soil containing the nematodes by applying to nematode-infested soil a nemotocidally effective amount of antibiotic LL-C23024 « , or a pharmaceutically acceptable salt of the antibiotic, or a fermentation broth or harvest mash solids from which the nematocidal antibiotic is obtained.

In a further aspect, the invention directed to the use of antibiotic LL-C23Q24 a, or a pharmaceutically acceptable salt thereof, or a fermentation broth or harvest mash solid from which the antibiotic is obtained, in the preparation of a nematocidal agent for controlling ^nematodes in’warm-blooded animals.

Antibiotic LL-C23024 “is an organic carboxylic acid and this is capable of forming salts with non-toxic pharmaceutically acceptable cations. Thus, salts formed by admixture of the antibiotic free acid with stoichiometric amounts of cations, suitably in a neutral solvent, may be formed with cations such as sodium, potassium, calcium, magnesium and ammonium, as well as organic amine cations such as tri(lower alkyl)amine (eg triethylamine, triethanolamine), procaine and the like. For the purposes of this invention, the antibiotic free acid is equivalent to its pharmaceutically acceptable non-toxic salts.

Mixtures of the compound or salts may be used.

Claims (3)

1. A method of controlling nematodes in soil containing· nematodes by applying to nematode-infested soil a nematocidally effective amount of antibiotic LL-C23024 a , or a pharmaceutically acceptable salt of 5 the compound, or a fermentation broth or harvest mash solid from which the nematocidal antibiotic is obtained.

2. The use of antibiotic 11,-023024 a , or a pharmaceutically acceptable salt thereof, or a fermentation broth or harvest mash solid from which the antibiotic is 10 obtained, in the preparation of a nematocidal agent for controlling nematodes in warm-blooded animals. Dated this the 21st day of July 1989. 15 BY: TOMKINS & CO., Applicants’ Agents, (Signed)

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