DE2101187C3 - Means for administration to poultry and pigs to protect them from Pasteurella species - Google Patents

Description

N-N

wherein R is a hydrogen atom or a tetrahydrofurylacetylamino group means contains.

The invention relates to an agent for administration to poultry and pigs in order to keep them from Pasteurella to protect species. The agent according to the invention is characterized in that it is a compound the formula

O ₁ N

CH =

N-N

wherein R is a hydrogen atom or a tetrahydrofurylacetylamino group, contains.

Domestic animals and poultry are susceptible to infection with a variety of pathogens as they grow and often get sick. The poultry or chickens are made with Pasteurella multocida infected and can be killed as a result. Pigs can also specie ^ with Pasteurelle, for example Pasteurella multocida. become infected and become subacutely or chronically ill. It was now found that the above compounds are excellent preventive and therapeutic Effect on poultry and pigs infected with Pasteurella species.

The specified compounds show excellent antibacterial activity against Pasteurella species, for example past, haemolytica, past, multocida, Past, pestis and Past, tularensis, inter alia Past, multocida.

The preparation of the connection according to the invention with R = H is from Chemical Abstracts, Vol. 72, No. 25, S. 341 (1971), known that the compound according to the invention with R = tetrahydrofurylacetylamino group is from Chemical Abstracts, Vol. 73,87,87,929.

The effective doses of the compounds can be used in conjunction with known carriers or adjuvants be applied in accordance with normal procedures. In a particularly preferred embodiment, is powdered medicine containing 2 to 10 percent by weight of any of the compounds listed as effective

Contains ingredient dispersed in fillers such as corn flour, lactose, starch, talc, etc. to the Feed of poultry and pigs added. The compounds can also be in the form of a liquid Medicine along with a humectant.

a solubility-imparting agent, a surfactant such as polyoxyethylene glycol. CMC, glycerin, etc. or in the form of a suspension such as gum arabic are useful administered.

The effective dose of the compound will generally be in the range of 10 to 200 mg, preferably 50 to 180 mg. When the compound is added to feed for poultry or pigs, one uses 0.005 to 0.02 percent by weight or 0.005 to 0.01 (weight percent or 0.01 to 0.02 percent by weight of the compound, based on the feed.

The duration of the compound administration time will depend on the severity of the symptoms. In general is a continuous administration of about 7 to 30 days is sufficient. The effect of the compound is from the 4th to the 5th day of administration on visible. The compounds thus protect poultry and pigs or sheep very effectively Diseases caused by Pasteurella species.

The in vitro melting points and minimum inhibition concentrations (IMC) against Pasteurella multocid of the compounds are given in Table I below. From the given results it can be seen that the compounds have very strong antibacterial activity.

Table 1
Minimum inhibition concentration (in vitro)

I.
O ₂ NO \
CI ο O R R) NH-COCH ₂ O '■' Τ. - '^'"'""""κ ; ^; Melting point: 164.5 to 165.5 ° C
Look: '
yellow, head-like crystals . ': ·''U ^ vSyii - "-"' ■ < ^: ' ^: - ·' ■ ·
Microorganism * ...;. rvri .... ».. ^ : Schmfelzpunkg ^; 33 ;; to: 134-5
i appearance;: ig (; ί- | j ^; j ^ -. ^ ;;> ^ν
: igeiBfsäulenärtiplKfistaiie I ^; ■: l; 0T / ml b. «, -» ^ RSfiifei-iiK ^: - *; ^: i ^: - ■. . ¹ I ¹ :; .
; »·! Ϊ́" ■ «οΛίΑΛ <■■ '- ■ λ. ■■■■ -"
.% «- ■ -ij. / Ff-ii :. '. , - ■:. . ■:.: · ^ MmM * ¹ - ■: - i§k _L. Pästeufe1iff ^ ito | i | [a ...... ^ .. > ..

: z4m

2 1Oi 187

The minimum inhibition concentration was determined in the following way: To a triptoso bean broth culture medium, obtained by adding 17 g Tripton, 3 g soybean peptone, 2.5 g glucose, Dissolve 2.5 g of potassium hydrogen phosphate and 5 g of sodium chloride in 1 1 of water which has a pH of 7.3 10 percent by volume of cow serum was given. The compound of the invention was separated in Dimethj'lsuloxyd dissolved, and then this solution was to the above medium in a ratio of 2 percent by volume, based on the medium, i.e. H. 25 μg / ml of the medium was added. The medium became diluted to various concentrations by known methods. 2 ml of the medicine of different Concentrations were measured using a syringe with a drop of the liquid pathogen inoculated, whereby the pathogens are in the medium produced first (which has no connection contained) had grown for 20 hours. It was then cultured in an incubator for 48 hours 37 ° C. Thereafter, the turbidity of each medium; examined to determine if the pathogen or germs had multiplied.

Experiments on mice revealed the evidence; provided that the active ingredients, if they are administered to animals, have an invariable activity. If the compounds were administered orally to mice of the C3H type, each weighing about 18 g and which had been artificially infected with Pasteurelh multocida by inoculation into the abdominal cavity, the mean effective doses (ED ₅₀ ) can be determined as shown in Table II are given. The table clearly shows that the action of the compounds is noticeably better than that of analogous compounds, which are in the 2-position! of the oxadiazole ring - NH ₂ - or - NHCOCH ₃ 'groups.

Table II ED ₅₀ in mice infected with Pasteurella multocida

connection

Compound I

O ₁ N

CH = C

Compound II

O ₂ N-

^V O

CH - C

NHCOCH,

Comparison connection

0, N-

Ν — Ν

CH = C-

Comparison connection

O, N

CH = C

NHCOCH ;, surviving mice

Examined mice

ED ₅₀ (mg / kg)

surviving mice

Examined mice

ED ₅₀ (mg / kg)

surviving mice

Examined mice

ED ₅₀ (mg / kg)

surviving mice

Examined mice

ED ₅₀ (mg / kg)

Dose administered (mg / kg)

800 I 400 I 200 I 10ol 50

5/5

3/5

1/5

168

5.5

15.5

68

5/5

2 5

0/5

P / 5

0/5

0/5

466

0.5

> 800

The determination of the ED ₅₀ was as follows: Pasteurella multocida P-1059 was in Triptosojabohnenbouillon containing 5% rabbit blood contained, for 20 hours at 37 ° C grown, and then were each 0.2 ml thereof in a 10 ^-6 dilution in the Abdominal cavity of a mouse (4 weeks old, weight 15 to 18 g) inoculated.

Immediately after vaccination, the amount of each compound to be administered was determined triturated in an agate mortar, slurried in gum arabic, and 0.4 ml of it was applied to jec Mouse administered orally. A test group consisted of five mice.

_{The ED 50} values na (the Behrens-Kaerber method) were determined from the number of mice still alive 5 days after the vaccination.

It was further found that the compounds have various excellent properties.

In general, nitrofuran derivatives are only sparingly soluble in water and when they are administered orally their absorption through the intestines is low. Accordingly, only a small one arrives Percentage of compound administered in the animals' blood. In contrast, the active ingredients have the Agents according to the invention have a higher solubility in water than the known nitrofuran derivatives, and can therefore enter the blood of the animals that have received the compound in higher concentrations reach. In the following Table III are the solubilities of the active ingredients and their concentrations indicated in the blood of rats when administered orally. At the same time, the corresponding Values of comparative liabilities listed.

Table III Concentration in rat blood (- // ml)

connection solubility
Si
O unden na
2 : h the Ve
3 rabreichu
8th ng the verb
24 indung
4R Connection 1
^l = f ^/ NN 28.2 O 0.12 0.08 0.02 <0.016 O: Nl _o Ji-CH = CA _o J 11.0 O 0.15 5.6 2.1 <0.06 <0.06 Connection 11
_ W Ν — Ν
O ₂ N ^ _o > - CH = C 1 _o j -NHCOCH ₂ -A "J 3.2 (I. 6.3 0.027 0.04 0.024 < 0.016 Comparable
W NN
O ₂ NI ' _O JL- CH-C \ ₀ ) - NH, 7.5 O 0.03 0.02 0.016 <0.016 <0.0 lh Comparison connection
U 'Ν — Ν
υ, Ν-Ι; ₀ JU Ch - ■ = C '< _O JUNHCOCH, 0.025

45

The concentration in the blood was determined as follows:

Each prebond was suspended in 5% gum arabic at a ratio of 10 mg / ml and administered to male rats weighing approximately 150 g each at a dose of 100 mg / kg. After 2, 3, 8, At 24 and 48 hours after the administration, the rats were anesthetized with ether and their blood was removed extracted from the heart with a syringe. One test group contained three rats. The blood was after centrifuged after coagulation, and the supernatant serum was separated and stored at -20 ° C.

The concentration of each compound in the serum was determined by biological testing. The biological test medium (prepared by dissolving 0.6% peptone, 0.3% yeast extract, 0.15% meat extract, and 1% agar in distilled water and adjusting the pH of the solution to 7.0) was sterilized and kept at 50 ° C. To 100 ml of this medium were added 2 ml of 1% NaNO ₃ , 3.8 ml of O, 1% methylene blue and 0.2 ml of a liquid containing the pathogens (which was prepared by mixing Escherichia coli 8057 in normal Buillon cultivated for 20 hours at 37 ° C ¹ ).

The medium and additives were mixed, and each 2 ml of the mixture were placed in test tubes having an inner diameter \ o \\ 7 optionally mm. The culture medium was allowed to coagulate by keeping it at room temperature for about 30 minutes. The medium was added to each test tube with 0.2 ml of a standard dilution of the compound to be tested at various concentrations (16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.062, 0.031, 0.016 and 0.008 - // ml ), that is, each sample was overlaid in three test tubes. Thereafter, the compound was allowed to permeate the medium while refrigerating for 2 hours, and then the samples were kept at 37 ° C. overnight. The length of the zone of inhibition in which the methylene blue was not reduced was determined in all test tubes to obtain a standard curve, and from this the concentration of the compound in each serum was calculated.

The mean lethal dose (LD ₅₀ ) of the compounds is extremely high, which means that the high safety of the middle! is shown convincingly.

The mean lethal dose of the compounds slurried and suspended in a 5% aqueous solution

Gum arabic solution was determined by _{orally administering the compounds to C 3} H species mice each weighing about 18 g. The values obtained are given in Table IV. The low toxicity of the compounds according to the invention can be seen from the values.

Table IV
toxicity

connection Ν — Ν LD ₅₀ (mg / kg) -V Ν — Ν O ₂ N- = c— I ₀ JJ-NHCOCH ₂ -TO 1.373 O ₂ N- 1.646 -U- -CH -CH

The compounds not only show a significant antibacterial activity in vitro, but they also prevent infection with Pasteurella multocida when administered to poultry, pigs and sheep, and they are much more effective than the analogous nitrofuran derivatives. The compounds show a direct effect, they have low toxicity unc ¹ therefore have a broad view of security.

The following examples illustrate the invention without, however, restricting it.

Attempt 1

The compounds were added to the feed of 14 day old chicks. A test group consisted of 10 chicks. On the second day of administration, each chick was infected by intramuscular injection in the left leg with 0.2 ml of an ^{inoculum of Pasteurella multocida diluted to 10 4} , the inoculum in a tryptoso bean broth containing 10% rabbit blood.

to had been grown for 24 hours. The administration the compound was continued for 5 days after vaccination, and then the effect of the Linkage is determined by counting the number of chicks that survived. The results are given in Table V below. From this table it can be seen that chicks fed with feed, that contained 0.01% of the active ingredients, were protected from infection, and that 100% and 90% survived, while chicks fed feed containing 0.005% of any of the active ingredients increased 60% survived. In clear contrast to this, all animals were infected and died, the known comparison compounds had been administered.

Table V

connection Encore
to the feed
(%) number of
examined
chick Number of chicks,
the 5 days after the
administration
survived
(%) Compound I
· = - / NN
O ₂ N-gU CH = CI ₀ J 0.01 10 100 Compound II
O 0.005 10 60 ι = / Ν — Ν
O ₂ N - \ ^ - ^{CH = C} ^ O "~~ NHCOCH ₂ 4 ^ p 0.01 10. 90 0.005 10 60

409 621/366

continuation

connection

Comparison connection

NH,

Comparison connection

O ₂ N -Λ ^ JJ-CH = C

-O'

N-N

NHCOCH ₃

im (request \ ov \ Number of chicks, chick the 5 days after the administration
survived 10 (%) 10 0 10 0 10 0 0 Encore to the feed 1%) 0.01 0.005 0.1 0.005

Attempt 2

Young Berkshire Bastard Pigs being sold should, as soon as their body weight had reached 90 kg, were continuously with the invention Fed means containing 0.015% active ingredient.

Thirty pigs were selected from those that had been littered by 4 pigs on the same day and they were divided into 10 study groups, each group consisting of 3 pigs.

The experiment was started on the 73rd day after she was born. The days that took that Pigs reached a weight of 90 kg, the conversion of the feed and the lung damage were examined. The pigs that had received the agents according to the invention showed in comparison with the control group that had been given comparison compounds in the feed, excellent results, with the group fed only the forage alone fell sharply.

In the test group that had been fed with agents according to the invention, the number of days was required for the pigs to weigh 90 kg is lower. A macro test the lungs for pathological changes in the pigs after slaughter confirmed the effect the agents according to the invention, since the lung damage in the treated groups was less. as in the comparison groups.

Furthermore, in the bacteriological examination of the pigs in the comparison groups, the Pigs discovered Pasteurella species, but these could be found in the lungs of the test groups that received the Funds received could not be found.

Table Vl

Added to the feed
Crowd (%)

Number of examined
Pigs

Average number
the days after 'the
birth

initially

at the end

Compound I

jo

O ₂ NO CH = C

^ NN

Il

O R

R: H R: NHCOCH, O 0.015 0.015 10
(S 6, c? 4) 10
as indicated on the left 73 73 168 ± 6.2 169 ± 7.0

Comparison connection NH ₂ Forage ^ö -1 L = / / \ Av ι η-η
O ₂ NO CH = C v ^ J)
O 0
10
as well
like left 0.015
10
as well as left 73
183 ± 7.4 73
172 ± 7.5

continuation

Number of days leading to
A pig body weight of 90 kg was required

Average body weight (kg)

initially

on the last day

Average weight gain per day
(g)

Average feed consumption per pig
(kg)

Feed conversion

Lung damage

serious

medium

Little

normal

Compound I

O ₂ NO CH = C

O R

R: H

95 ± 6.3

20.6 ± 3.5 90.2

732.63

21 !, 68 3.04

1 1

3 5

HR: NHCOCH, O.

96 ± 6.8

20.5 ± 3.8 90.2

726.04

218.60

3.14

2 1

Comparison connection

O ₂ NO CH =

O NH,

99 ± 7.5

20.5 ± 3.6
90.1

703.03

219.94
3.16

4th
2
1

Forage

110 ± 7.4

20.5 ± 4.2
90.3

634.54

242.90
3.48

0
3

Examples of possible forms of preparation

example 1

0.94 kg each of soybean oil and soybean lecithin were added to 88.120 kg of powdered grain flour, which passed through a sieve with a mesh size of 0.638 mm and mixed for 20 minutes. After that 10.00 kg of compound I, finely divided to a size of max. 20 μ, were added, and that The whole was further mixed for 30 minutes. Thereafter, the resulting mixture was passed through a sieve Mesh size 0.638 mm given, a pharmaceutical was obtained which 10% active ingredient contained.

This pharmaceutical can be mixed with feed.

Example 2

80 kg heavy calcium carbonate, which was ground to a size of max. 100 μ, and 20 kg of compound II, which were finely divided to a size of max. 20 μ, were placed in a mixer and mixed for 30 minutes, after which the mixture obtained through a sieve of mesh size 0.30 mm was given to obtain a pharmaceutical containing 20% active ingredient.

This pharmaceutical can be mixed with feed.

Example 3

The connection I in finely divided to max. IO μ The mold was added to 85.40 kg of lactose and mixed by means of a kneader for 10 minutes, after which 0.50 kg of millet pulp, dissolved in 5 kg of water, was added and kneaded for 10 minutes. Thereafter, 10.00 kg of polyoxyethylene (20) sorbitan monolaurate and 0.04 kg of silicone resin were used as antifoam agents was added, and kneading was continued for 20 minutes, followed by a granulator Granules of 0.8 mm in diameter were obtained, which were dried so as to be a pharmaceutical to obtain that contains 4% active ingredient.

This pharmaceutical will disintegrate instantly in water to form a stable suspension, and can dem Drinking water must be added.

Example 4

15 kg of compound II in finely divided to max. IO μ Form and 0.5 kg of sodium carboxymelhyl cellulose were added to 34.60 kg of glycerin and mixed with it. The obtained mixture became

dissolved, with 0.10 kg of methyl p-hydroxy-benzoate and 0.04 kg of n-propyl-p-hydroxy-benzoate as antiseptic Funds were added. In another vessel, 1 kg of bentonite was added to 40 kg of water and heated to 80 ° C, after which, after cooling once to room temperature, with the addition of 0.10 kg Saccharin sodium was dissolved. By adding

the previously obtained glycerol liquid to the one obtained Bentonite solution and mixing, after which more water was added, for a total of 100 kg To obtain a pharmaceutical suspension containing 15% active ingredient was obtained.

This pharmaceutical can be administered directly orally.

Claims (1)

Claim: Means of administration to poultry and pigs to protect them from Pasteurella species, characterized in that it is a compound of the formula O, N CH = C V ⁰ V ⁷