JP2801178B2 - Animal feed composition - Google Patents

Description

Description: BACKGROUND OF THE INVENTION Tetracycline (including tetracycline derivatives; hereinafter the same) is a well-known broad-spectrum antibiotic, and is used to promote animal feed and weight gain in animals. Used as an additive to feed. However, the current use of antibiotic tetracyclines as an additive to animal feeds to promote improved feed conversion and weight gain, as described above, is currently under way in animals. Has been found to have the detrimental effect of causing overgrowth of resistant organisms that are considered dangerous to the health of humans. That is, an object of the present invention is to provide an animal feed composition using a tetracycline derivative that does not cause overgrowth of resistant organisms that threaten animal health, even when used to promote feed conversion and weight gain. is there. [0003] Certain tetracycline derivatives of the present invention, when used as feed conversion and weight gain promoters, do not result in the overgrowth of resistant organisms that threaten animal health. Can promote weight gain. Therefore, the animal feed composition of the present invention can be used as an improved animal nutritional supplement. In the present invention, a special non-antimicrobial or antibiotic tetracycline such as dedimethylaminotetracycline is used as an additive to animal feed.
Animal feed for ruminants, non-ruminants, and homologs of ruminants is usually obtained from cereals, proteins, amino acids, minerals, vitamins and, for example, preservatives (antioxidants, bacteriostats, fungicides). It consists of additives and supplements that help protect animals against parasites and diseases. The advantage of the use of the special tetracycline according to the invention as an ingredient or additive in animal feed is that such a use is unlikely to cause an antibiotic resistant tribe of bacteria. The primary purpose of employing the particular type of tetracycline of the invention in animal feed is as an anti-collagen dissolving agent for improving nutritional intake and body weight by enhancing animal feed conversion or growth promotion. A particularly useful tetracycline in the practice of the present invention is, but is not limited to, dedimethylaminotetracycline. For examples of the specific tetracycline concentration or amount of the invention employed in animal feed, for poultry feed for chickens, turkeys, and broilers, the amount of tetracycline incorporated in the feed is per ton of feed. It may range from about 10 grams to about 200 grams. For feed to young beef calves up to about 12 weeks of age, the amount of tetracycline in the feed may range from about 0.1 mg to about 1.0 mg per pound of calf body weight per day depending on the calf at the time of feed consumption. Should be the amount taken. For beef cattle, the amount of tetracycline present in the feed is from about 75 mg to about 300 m / day from the tetracycline-containing feed.
g should be the amount consumed or consumed. When the special tetracyclines according to the invention are used as additives in animal feed, they do not cause a bacterial antibiotic-resistant race. Thus, no overgrowth of resistant organisms is provided. EXAMPLES Preparation of Stock Solutions of Tetracycline, Minocycline, and Dedimethylaminotetracycline (CMT) Stock solutions of minocycline, dedimethylaminotetracycline (CMT), and tetracycline were prepared as aqueous solutions. These solutions are used to measure the antibacterial activity of each of these tetracyclines. Minocycline is available from Sigma Chemical Company (St. Louis, Missouri, Lot $ 60F-0)
048). Tetracycline is also a Sigma Chemical Company (St. Louis, Missouri, Lot $ 103F)
-03350) and dedimethylaminotetracycline is obtained from J. H. Booth et al. [Journal of the American Chemical Society, Vol. 80, p. 1654 (1958)] and J. R. Day. It was prepared synthetically by the method of McCormick et al., Journal of the American Chemical Society, Vol. 79, page 2849 (1957). The stock solutions were prepared as aqueous solutions containing 100 micrograms per milliliter of each of the compounds. For 10.00 mg of each of minocycline, tetracycline, or dedimethylaminotetracycline, 10 ml of distilled water was added and the suspension was swirled in a 100.0 ml volumetric flask. 10 ml of each suspension of the compound
1.0N sodium hydroxide was added to obtain a clear solution of each compound. 70 ml of distilled water was added to each solution and the pH was adjusted to pH 8.0-8.3 by adding an appropriate amount of 1.0N hydrochloric acid. Each solution was 100.000 in a volumetric flask by addition of distilled water.
It was made up to 00 ml. The final concentration of each of these compounds was 100 micrograms per ml. The stock solutions were stored in the refrigerator until they were used. Stock solutions were made fresh daily. Evidence of loss of antimicrobial activity In vitro evidence: To demonstrate that dedimethylaminotetracycline (CMT) reduced antimicrobial activity, comparative samples were tested with a minimum inhibitory concentration (MI
C). MIC for minocycline, tetracycline, and dedimethylaminotetracycline
Was performed in the following manner. 3.0 ml of sterile brain
To a series of tubes containing cardiac immersion (BHI) broth (DIFCO), appropriate amounts of stock solutions of minocycline, dedimethylaminotetracycline, and tetracycline were added to the concentrations shown in Table 1. Each tube was then adjusted to a final volume of 4.0 ml with sterile BHI broth. Each of the tubes in the series was then inoculated with 0.1 ml of a 24-hour broth culture of Bacillus cereus that had been hatched at 37 ° C. The test tube was then swirled and incubated at 37 ° C. for 24 hours. The tubes were then judged for the presence or absence of growth as measured by turbidity. The results of these comparative evaluations for antimicrobial activity are recorded in Table 1. Bacillus dedimethylaminotetracycline
It was clear that it had no detectable antibacterial activity against B. cereus. It was 100 times less active than tetracycline, and 1,000 times less active than minocycline. Bacillus cereus was used as the test microorganism because it is so sensitive to these antibiotics that it is routinely used for tetracycline assays. The microorganism is an American Type Culture Collection (ATCC # 117)
78). [Table 1] In vivo evidence: Normal animals and animals suffering from diabetes have been tested in comparative studies to determine the in vivo activity of dedimethylaminotetracycline.
They were either untreated or treated with metronidazole or dedimethylaminotetracycline. In this study, four different groups of rats were used. Group I is an untreated control group of normal rats potentially having a normal oral flora, Group II is an untreated control group of diabetic rats suffering from diabetes by administration of streptozocin, and Group III Was the diabetic group treated orally with metronidazole and Group IV was the diabetic group of animals treated orally with dedimethylaminotetracycline. At the end of the experiment, the rats were sacrificed and samples were taken from each of these groups of animals by sterile cullet from the gingival margin of rat molars and transferred to broth and incubated for 72 hours at 37 ° C. Either the isolated microorganisms metronidazole or dedimethylaminotetracycline were administered and then compared to determine if there was a major change in the composition of the flora. The results of the isolation and identification of the microorganisms obtained from the gingival samples are as follows. (1) Untreated normal control animals showed slightly more microbial presence than animals in any other group and were easily identified by the amount of growth and bacterial composition.
(2) Diabetic animals receiving metronidazole had less growth from gingival samples than any other group and had primarily Gram-positive cocci. (3) Untreated diabetic control animals and diabetic animals receiving dedimethylaminotetracycline were essentially indistinguishable in the amount of growth obtained from gingival samples and in the bacterial composition of each sample. These results are summarized in Table 2. Although these in vivo studies are clearly expected, metronidazole preferentially inhibits Gram-negative aerobic microorganisms in crevasse-like microflora, while dedimethylaminotetracycline or CMT contrasts with the effects of antibiotic tetracycline. Showed no detectable effect on the crevasse-like microflora. Golb et al. (Periodontal Research Journal, Vol. 18, pp. 516, 1983) have shown that the antibiotic tetracycline suppresses Gram-negative anaerobic microorganisms in the crevasse flora of diabetic rats, similar to the effects described above for metronidazole. [Table 2] Evidence for anti-collagenase activity In vitro evidence: Bacterial collagenase (from C. histolithium), which is a calcium-dependent metalloprotease such as mammalian collagenase, has a CMT of 0.20.
Alternatively, the cells were incubated with [ ³ H-methyl] collagen for 18 hours at 27 ° C. in the presence of 60 μg / ml, and the results are shown in FIG. In the absence of CMT, the bacterial collagenase level in the hatch mixture was reduced to 1
Although expected to increase from 0-100 ng, the radiolabeled collagen substrate (40-9
0%). A similar pattern is 20 μg /
Seen when ml of CMT was added, but at all levels of bacterial collagenase (10-100 ng), the degradation of collagen was lower than seen in the absence of CMT. CMT concentration of 60 μg / ml
Increased inhibition of collagenase activity at essentially all of the enzyme levels tested. Other experiments were performed. In this experiment, a single level of bacterial collagenase (a) has a broad range of CMT concentrations (0.
2, 10, 20, and 60 μg / ml), and (b)
Incubated with known inhibitors of metalloproteases, EDTA, and phenanthroline. As in the first experiment, 10 ng of collagenase degraded about half of the available radiolabeled collagen substrate after 18 hours of incubation at 27 ° C in the absence of CMT (Figure 2). A very low concentration of 2 μg / ml CMT is about 20%
Inhibited collagenase activity until
The concentration of g / ml CMT reduced the activity by about 90%. These higher concentrations of CMT are consistent with the hypothesis that the metal-binding properties of tetracyclines (including CMT) are at least partially responsible for their anti-collagenolytic enzyme properties, chelating agents, EDTA, and It inhibited collagenase activity to the same extent as phenanthroline. A third experiment was performed and the results are provided in FIG. In this experiment, radiolabeled collagen molecules were incubated with an extract of rat leukocytes known to have collagenolytic enzyme activity (18).
Time, 27 ° C). This mammalian collagenase is prepared from 0-6
Incubated with 0 μg / ml CMT or with collagenase-inhibitor, EDTA or phenanthroline. Collagenase destroyed about 55% of the collagen substrate available by itself. In this experiment, 2-10 μg / ml CMT reduced collagenase activity to about 20%, while 20 and 60 μg / ml
Reduced the activity by about 85%. Two metal chelators, E
DTA and phenanthroline completely inhibited the activity of this mammalian collagenase, as expected, since leukocyte collagenase is known to depend on the presence of metal ions, calcium and zinc for normal activity. It is believed that the reason why this mammalian collagenase preparation required a higher CMT concentration than the bacterial collagenase preparation required twice the amount to achieve the same degree of inhibition. First, mammalian collagenase was a relatively impure enzyme compared to highly purified bacterial enzymes. Thus, CMT may have reacted with other metallo-proteases (eg, gelatinases) in leukocyte preparation in addition to the reaction with collagenase, thus effectively reducing the inhibitor CMT / collagenase ratio. Second, leukocytes contain high levels of calcium ions and earlier experiments have shown that the addition of calcium to a collagenase / tetracycline mixture can overcome the inhibition of enzyme activity. In vivo evidence: A group of four divided rats was provided. One group (control) was left untreated throughout the entire control study, and the second (diabetes or D group) was treated with streptozotocin I.D. V. The third group was made diabetic by injection, then made diabetic and then given the antibiotic metronidazole orally at 75 mg per day (D + metronide group), and the fourth group was made diabetic and the “C
"MT" was administered orally at a daily rate of 20 mg per day (D + "CMT" group). These dosages are the daily dosage of each of these drugs when administered therapeutically to humans. The value of 1/10 was identical to the dosage for diabetic rats in previous studies with minocycline and it was found to produce beneficial changes in rat connective tissue. Rats were weighed several times during the experimental control study.On the last day of the experiment (day 37), a sample of blood was taken from each rat for glucose analysis, the animals were weighed and Complete skin (except for head, paws, genital area) and oral gingiva from each rat was dissected, weighed, minced and extracted (all procedures were performed at 4 ° C). Done ) And the extract is purified by partially ammonium sulfate precipitation. Collagenase activity in the skin and gingival extracts was measured after Kae置with radiolabeled collagen as the substrate (skin extract ¹⁴ C Gingine-labeled collagen microfibers were incubated for 48 hours at 35 ° C., while the gingival extract was incubated with [ ³ H-methyl] collagen molecules at 27 ° C. for 18 hours. A gingival plaque sample was taken of each animal from the chin, and the samples were immediately placed in a preconditioned broth (to protect anaerobic microorganisms) and incubated under anaerobic conditions. , And Gram positive, Gram negative,
And a relatively large number of motile microorganisms were measured. As shown in FIG. 4, control rats gained weight in the control study over 37 days, while untreated diabetes (Group D) gradually lost weight. Daily metronidazole treatment had no effect in delaying weight loss in D rats, while daily treatment of D rats with CMT completely prevented weight loss. In addition, at the end of the experiment (day 30), the "D" rats treated with CMT began to gain weight in a situation comparable to non-diabetic controls. On the other hand, untreated D rats continued to lose weight for the same period. FIG.
Data show a similar pattern of change. D and D +
The metronide group lost more than 50% of the main skin by the end of the 37-day control study, while the C
Treating D rats with MT prevented loss of skin mass (skin loss is a secondary disease of diabetes). The data in Table 3 are for all of the diabetic groups (D group; D +
Metronide group; D + CMT group had severely hyperglycemic (6 mg) compared to normoglycemic (104 mg%) control rats.
38-850 mg% blood glucose level). Treating diabetic rats with metronidazole is pathological in diabetic skin (Table 3), or diabetic gingiva (FIG. 6), even though the blood glucose in metronidazole (antimicrobial) -treated rats is slightly reduced (Table 3). Has no absolute effect on excess collagenase activity. In marked contrast, this modified tetracycline showed up to about 50% cutaneous and gingival collagenase as shown in FIGS. 3 and 6, even though CMT treatment appears to have a lesser effect on blood glucose levels than metronidazole. Activity was dramatically inhibited. These data indicate that CMT inhibits mammalian collagenase activity (and collagen reabsorption) in vivo, similar to the standard antibiotic tetracycline, but achieves this therapeutic effect without significantly affecting the crevasse-like microflora. It shows what you can do. [Table 3] Although the emphasis in this disclosure and in the practice of the present invention is on dedimethylaminotetracycline as a non-antimicrobial or non-antibacterial tetracycline, other substantially non-antimicrobial or non-antibacterial Tetracyclines, such as, for example, 6α-benzylthiomethylenetetracycline, are also useful, such as those having the following structural formula: Embedded image Also useful are nitrile-substituted tetracyclines having the following structural formula: Embedded image More useful are tetracycline mono-N-alkylamides having the following structural formula: Embedded image Also useful are 6-fluorodemethyldeoxytetracyclines having the following structural formula: ## STR4 ## And 7,11α- having the following structural formula:
There is dichlorotetracycline. Embedded image The particular non-antimicrobial or non-antibacterial tetracycline of the present invention may be used as such or in the form of a salt, for example, the hydrochloride salt. Other water-soluble salts, such as sodium or potassium salts, may also be used. The tetracyclines of the invention can also be encapsulated for oral administration, as described above, because the tetracyclines of the invention are believed to be totally effective as anti-collagen dissolving agents. The tetracyclines of the invention can also be formulated in tablets, capsules, panacea and the like. The tetracyclines of the invention can be formulated in solution or suspension for intramuscular or peritoneal administration. In addition, the tetracycline may also be incorporated or prepared in a polymeric carrier or delivery system for local or topical use, for example, for delivery directly into the periodontal sac, for example, in the treatment of periodontal disease. Can be done. Suitable polymeric materials used for incorporation into the tetracyclines of the present invention include ethylene vinyl acetate, polycaprolactam, polyurethane, ethylene cellulose, which are incorporated after the tetracycline is incorporated or dispersed therein.
Appropriate for fibers, sheets, films or particles, or particulate matter that can be formed or shaped into a form suitable for treatment of periodontal disease, etc., or for direct application to the affected area showing pathological collagen destruction or resorption Formed or shaped. Many adjustments, modifications and substitutions are possible in practicing the invention without departing from its spirit or scope, in light of the foregoing disclosure, as will be apparent to those skilled in the art. Thus, the present invention can promote weight gain of animals without causing overgrowth of resistant organisms considered to be dangerous to animal health.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 graphically illustrates the effect of the tetracycline of the present invention on bacterial collagenase activity in vitro, with respect to increasing levels of bacterial collagenase activity in vitro (10-100 ng). Two different concentrations of CMT (20-60 μg / m
The effect of l) is shown. FIG. 2 graphically illustrates the effect of increasing concentrations of tetracycline of the invention on in vitro bacterial collagenase activity, with the “CM” versus in vitro bacterial collagenase (10 ng) activity.
FIG. 3 shows the effect of increasing concentrations of T ″ (2-60 μg / ml). FIG. 3 graphically illustrates the effect of increasing concentrations of tetracycline of the invention on rat granulocyte collagenase activity in vitro. Figure 4 shows the effect of increasing concentrations of “CMT” (2-60 μg / ml) on rat granulocyte collagenase activity in vitro in vitro Figure 4. Weight of diabetic rats receiving oral administration of tetracycline of the present invention was compared FIG. 4 is a graphical illustration of the drug compared to other rats receiving the drug, in which diabetic rats were treated with metronidazole or “CMT” (20 mg / day).
1 shows the effect on body weight gain when orally administered. FIG. 5 graphically illustrates the effect on skin weight of diabetic rats to which the tetracycline of the present invention was administered, in which diabetic rats were treated with metronidazole or “CM”.
FIG. 6 shows the effect of oral administration of T ″ (20 mg / day) on skin mass after inducing diabetes and starting drug treatment 37 days after starting drug treatment. FIG. 6 in gingival tissue after administration of tetracycline of the invention 1 graphically illustrates the effect of collagenase activity in diabetic rats on oral administration of metronidazole or "CMT" to diabetic rats on collagenase activity in gingival tissue.

──────────────────────────────────────────────────の Continued on the front page (72) Thomas F. Inventor. McNamara United States 11777 New York, Port Jefferson, Fairway Drive 34 (72) Inventor Nungabalam S. Rama Marcy United States 11787 New York, Smithtown, Lyman Court 10 (72) Inventor Lone M. Golb United States 11787 New York, Smithtown, Whitney Gate 29 (58) Fields studied (Int. Cl. ⁶ , DB name) A23K 1/17 A23K 1/18 A61K 31/65

Claims (1)

(57) [Claims] A composition comprising a tetracycline derivative having substantially no antibacterial activity but having anticollagenase activity and an animal feed, wherein the tetracycline derivative enhances an anti-collagenase effective amount and / or feed conversion in the composition. An animal feed composition used for enhancing feed conversion and growth promotion to improve animal nutrition and increase animal weight, characterized in that it is present in an amount effective to enhance growth promotion.