JP2676093B2 - Animal feed composition - Google Patents

Description

Description: BACKGROUND OF THE INVENTION Tetracycline (including tetracycline derivatives, and so on) is a well-known broad-spectrum antibiotic and animal feed and animal for promoting weight gain. It is used as an additive to feed.
Recently, the antibiotic tetracycline has also been found to inhibit the activity of collagen-destructing enzymes such as, for example, mammalian collagenase, macrophage elastase, and bacterial collagenase. For example, tetracycline has recently been found to be useful in treating periodontal ligament disease, rapidly developing adult periodontitis, and localized child periodontitis. The antibiotic tetracycline also treats non-infectious corneal ulcers, treats pathologically excessive bone resorption,
It is useful for the treatment of rheumatoid arthritis, and for those diseases that are generally characterized by excessive collagen breakdown. In vitro studies antibiotic tetracycline (1) inhibits the collagenase activity of leukocytes and chondrocytes. (2) Reduced bone resorption in tissue culture induced by either parathyroid hormone, endotoxin, or prostaglandin E ₂ , and (3) reduced macrophage collagenase and elastase activity in cell culture. showed that. In addition, in an in vivo study, antibiotics tetracycline and minocycline in the treatment of diabetic rats (a) reduced pathologically excessive collagenase activity in gingiva or skin. (b) Reduced pathological cutaneous collagen reabsorption. And (c) Reduction of pathologically excessive hydatidiform bone loss. Was found. In addition, clinically, regular and low dose levels of antibiotic tetracyclines (minocin, acromycin, and vibramycin) have been found to reduce collagenase activity in human periodontal sac, and regular dose levels of tetracycline A refractory, non-infectious corneal ulcer, resulting in the healing of a disorder believed to be mediated by mammalian collagenase. However, although the commercially available antibiotic tetracycline is effective as an anti-collagen dissolving agent, it is continuously or, for example, 2 weeks of administration-3 months of discontinuation-2 weeks of administration-3 months. Long-term use, either in the intrusive way such as cessation, is subject to the usual complications of long-term antibiotic use, such as intestinal upset, overgrowth of yeast and fungi, and growth of antibiotic resistant bacteria. It will be easier. Also, the current use of antibiotic tetracycline as an additive to animal feeds to promote improved feed conversion and weight gain, as described above, is tolerated by the resistances currently considered to be dangerous to animal health. It has been found to have the deleterious effect of causing overgrowth of organisms having Thus, it is an object of the present invention to have anti-collagenolytic activity, especially in the animal treatment of diseases or conditions characterized by excessive collagen destruction, without simultaneously causing the usual complications associated with long-term antibiotic use. Another object of the present invention is to provide an animal feed composition using a tetracycline derivative that does not cause overgrowth of a resistant organism that threatens the health of animals. [0003] Tetracycline, which is substantially free of effective antibiotic or antibacterial activity, has been found to have anti-collagenase activity (anti-collagen degrading enzyme activity). More specifically, it has been found that tetracycline, which has substantially no effective antibiotic or antibacterial activity, has the ability to inhibit collagenolytic enzyme activity and collagen reabsorption. Tetracycline has been characterized as a compound consisting of four carbon rings and is known as an antimicrobial or antibiotic. However, not all tetracyclines have antimicrobial or antibiotic properties. Some of the tetracyclines do not substantially exhibit antimicrobial or antibacterial activity, while other tetracyclines exhibit some antimicrobial or antibiotic activity, but as chemotherapeutic or antibiotics in the treatment of disease. It has no antimicrobial or antibacterial activity to the extent that it is useful. Those tetracyclines that show no, substantially no, or poor antimicrobial or antibiotic activity are usefully employed in the practice of the present invention. A particularly useful tetracycline in the practice of the present invention is dedimethylaminotetracycline. However, as pointed out, other tetracyclines that do not have sufficient antimicrobial activity, if any, are also useful in the practice of the invention. In general, tetracycline is what is currently found, but all have anti-collagenase activity (anti-collagen destructive enzyme activity) regardless of whether they have antimicrobial or antibiotic activity. This anti-collagenase activity is due to the unique structure of tetracycline, a property of tetracycline, and is likely due to the unique four-carbon ring that tetracycline possesses. As observed, it is believed that the carbonyl moiety in the nucleus, which is the carbon ring of tetracycline, is important for the anti-collagenolytic activity of these compounds because they form chelates with metal ions such as calcium and zinc. This is an important property because the collagen-forming enzymes mentioned are metal-dependent. The specific tetracycline derivative in the present invention is one found based on the property of the above tetracycline, and an animal feed composition containing this specific tetracycline derivative has the usual complications associated with the use of antibiotics for a long period of time. At the same time, it is possible to promote the weight gain of animals without causing overgrowth of resistant organisms that threaten the health of the animals when they are not caused simultaneously and when used as a feed conversion and weight gain promoter. Therefore, the animal feed composition of the present invention can be used not only as an anti-collagen agent for animals but also as an improved animal nutritional agent. The action of tetracycline is
When used in the practice of the present invention as an anti-collagen solubilizer, when used in the treatment of an animal suffering from a condition or illness characterized by excessive collagen breakdown, tetracycline may be applied directly to, for example, gingival tissue, such as having excess collagenase activity. In some cases, it is used locally, such as in the treatment of periodontitis, and in the treatment of ulcerative disorders such as acne ulcers, diabetic ulcers, epidermolysis bullosa, but systemically. Seems to work. In the practice of the present invention, tetracycline will be used in any of the known modes of use suitable for treating animals of associated conditions. Dosages of the non-antimicrobial or non-antibiotic tetracyclines of the invention having an anti-collagenase effective amount for reducing collagenase activity or when used as an anti-collagen lytic agent are common when used as anti-microbial agents or antibiotics. The dose of tetracycline may be similar or slightly higher. In addition, the dosage may, however, be that of a commonly used antimicrobial tetracycline such as a standard dosage of 5 to 50% with respect to the use of the antimicrobial or antibiotic tetracycline when used in the treatment of a disease. It can be substantially below, or only a fraction of, or insignificant than the standard dosage or level. The present invention uses a special non-antimicrobial or antibiotic tetracycline such as dedimethylaminotetracycline as an additive to animal feed. Animal feed for ruminants, non-ruminants, and animals that are homologous to ruminants typically includes grains, proteins, amino acids,
Minerals, vitamins, and preservatives (antioxidants,
It consists of additives such as bacteriostats, fungistats) and reinforcing agents 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. First adoption of the special type of tetracycline of the present invention in animal feed
Its purpose is as an anti-collagen dissolving agent to improve nutrition and weight by enhancing feed conversion or growth promotion in animals. For examples regarding the concentration or amount of the particular tetracycline 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 will 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 should be in the amount of about 75 mg to about 300 mg ingested or consumed per day from the tetracycline-containing feed. The specific tetracyclines according to the invention are resistant to bacteria when used in the treatment of animal diseases or conditions characterized by excessive collagen destruction or when used as additives to animal feed. It does not cause an antibiotic 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 a Sigma chemical company (St. Louis, Missouri, Lot # 60F-004).
8). Tetracycline is also used by Sigma Chemical Company (St. Louis, Missouri, Lot # 103F-0.
350), and dedimethylaminotetracycline is obtained from J. et al. 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. 1.0 ml of 1.0N sodium hydroxide was added to each 10 ml of the compound suspension 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 brought to 100.0 in a volumetric flask by addition of distilled water.
It was made up to 0 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) diminished antimicrobial activity, a comparative sample was used to compare the minimum inhibitory concentration (MIC) of each of the compounds. ) Was created from.
A comparison of MICs of minocycline, tetracycline, and dedimethylaminotetracycline was performed by the following method. Appropriate amounts of stock solutions of minocycline, dedimethylaminotetracycline, and tetracycline were added to a series of test tubes containing 3.0 ml of sterile Brain-Heart Immersion (BHI) Broth (DIFCO) as shown in Table 1. The concentration was adjusted. 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. It was clear that dedimethylaminotetracycline had no detectable antibacterial activity against Bacillus cereus. It is over 100 times less active than tetracycline and 1,000 times more active than minocycline
The activity was more than twice as small. 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 was obtained from the American Type Culture Collection (ATCC # 11778). [Table 1] In Vivo Evidence: Whether Normal and Diabetic Animals Are Untreated or Treated with Metronidazole or Dedimethylaminotetracycline in a Comparative Study to Determine the In Vivo Activity of Dedimethylaminotetracycline It was either. 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 of anti-collagen lytic enzyme activity] In vitro evidence: Bacterial collagenase (from C. histolithium), which is a calcium-dependent metalloprotease like mammalian collagenase, has a CMT of 0.20 or 60 μg /
Incubated with [ ³ H-methyl] collagen for 18 hours at 27 ° C. in the presence of ml and the results are shown in FIG. Increasing bacterial collagenase levels in incubation mixtures from 10-100 ng in the absence of CMT was expected, but increased decay of radiolabeled collagen matrix (40-90%) . A similar pattern was seen when 20 μg / ml CMT was added, but at all levels of bacterial collagenase (10-100 ng), collagen decline was lower than that seen in the absence of CMT. Increasing the concentration of CMT to 60 μg / ml essentially completely inhibited collagenase activity at all enzyme levels tested. Other experiments were performed. In this experiment, a single level of bacterial collagenase
(a) a wide range of concentrations of CMT (0.2, 10, 20 and 60 μg / ml), and (b) a metalloprotease,
Incubated with EDTA and known inhibitors of phenanthroline. 10n as in the first experiment
g collagenase disrupted approximately half of the radiolabeled collagen matrix available after 18 hours of incubation at 27 ° C. in the absence of CMT (FIG. 2). 2 μg / ml C
Very low concentrations of MT inhibited collagenase activity by about 20%, while concentrations of 10-60 μg / ml CMT reduced 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 Figure 3. In this experiment, radiolabeled collagen molecules were incubated with an extract of rat leukocytes known to have collagenolytic enzyme activity (18 hours, 27 ° C). The mammalian collagenase was incubated with conditioned 0-60 μg / ml CMT or with the collagenase-inhibitor, EDTA or phenanthroline. Collagenase is about 55 of the collagen matrix available by itself.
Destroyed. In this experiment, 2-10 μg / ml
CMT reduced collagenase activity to about 20%,
On the other hand, 20 and 60 μg / ml reduced the activity by about 85%. Two metal chelating agents, EDTA and phenanthroline, are known to depend on the presence of metal ions, calcium and zinc for the normal activity of leukocyte collagenase, and, as expected, complete the activity of this mammalian collagenase. Inhibited. Why this mammalian collagenase preparation has a higher CM than the bacterial collagenase preparation
It is believed that the reason why a T concentration was needed was that twice as much was needed to achieve the same degree of inhibition. First, mammalian collagenase was a relatively impure enzyme compared to highly purified bacterial enzymes. Thus CM
T may have reacted with other metallo-proteases (eg gelatinases) in leukocyte preparation in addition to its 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. Diabetes was made by injection, the third group was made diabetic and then the antibiotic metronidazole was orally administered at 75 mg per day (D + metronide group), and the fourth group was diabetic and "CMT" at 20 mg per day daily pace. Orally (D + "CMT" group). These dosages are 1 / of the daily dosage of each of these agents when administered therapeutically to humans.
It is 10. The value of 1/10 was identical to the dose for diabetic rats in previous studies with minocycline and it was found to produce beneficial changes in connective tissue in rats. Rats were weighed several times during the experimental control study. On the last day of the experiment (day 37), blood samples were taken from each rat for glucose analysis, the animals were weighed and killed. Complete skin from each rat (excluding head, legs and genital area)
Oral gingiva was dissected, weighed, minced, extracted (all procedures were done at 4 ° C.) and the extract was partially purified by ammonium sulfate precipitation. The collagenase activity of skin and gingival extracts was measured after incubation with radiolabeled collagen as a substrate (skin extracts were incubated with ¹⁴ C glycine labeled collagen microfibers at 35 ° C. for 48 hours. Meanwhile, the gingival extract was incubated with [ ³ H-methyl] collagen molecules for 18 hours at 27 ° C. An accessory gingival plaque plaque sample was taken from each chin for each animal prior to killing the rat. Samples were immediately placed in preconditioned broth (to protect anaerobic microorganisms) and incubated under anaerobic conditions, and relatively free of gram-positive, gram-negative, and motile microorganisms. A large number were measured: As seen in Figure 4, control rats gained weight in a 37 day control study, while untreated diabetic (D group) rats. The daily treatment with metronidazole had no effect in delaying the weight loss in D rats, whereas the daily treatment of D rats with CMT completely prevented the weight loss. 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, while untreated D rats continued to lose weight for the same period. The data in Figure 5 shows a similar pattern of changes: D and D.
+ Metronido group lost more than 50% of skin bulk by the end of the 37-day control study, while treating D rats with CMT at a daily pace prevented skin bulk loss (skin loss was Diabetes is a residual disease).
The data in Table 3 are all in the diabetes group (D group; D
+ Metronido group; D + CMT group) are severely hyperglycemic (638-850 mg% blood glucose levels) compared to normoglycemic (104 mg%) control rats. 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 this disclosure and the emphasis in the practice of the present invention are on dedimethylaminotetracycline as the non-antimicrobial or non-antibacterial tetracycline, other substantially non-antimicrobial or non-antibacterial Tetracyclines such as 6α-benzylthiomethylene tetracycline are also useful, such tetracyclines having, for example, the following structural formulas: Embedded image Also useful are nitrile-substituted tetracyclines having the following structural formula: Embedded image Further useful are the mono N-alkyl amides of tetracycline having the structural formula: Embedded image Also useful is 6-fluorodemethyldeoxytetracycline, which has the following structural formula: And 7,11α having the following structural formula
-There is dichlorotetracycline. Embedded image The particular non-antimicrobial or non-antibacterial tetracycline according to the invention is suitably used per se 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, polyurethanes, ethylene cellulose, and these, after tetracycline has been incorporated or dispersed therein, fibers, A sheet, film or particles, or a particulate substance that can be formed or shaped into a suitable shape for the treatment of periodontal disease or the like, or for direct application to an affected area showing pathological collagen destruction or resorption, or Molded. 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. [0018] Therefore, according to the present invention, an animal characterized by excessive collagen growth can be safely characterized without causing overgrowth of a tolerant organism that is considered to be dangerous to animal health. Treat illness,
It can promote the weight gain of animals.

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.

   ────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Thomas F. McNamara               United States 11777 New York City,               Port Jefferson, Fairway                 Drive 34 (72) Inventor Nungabaram S. Llama Marcy               United States 11787 New York City,               Smithtown, Lyman Court 10 (72) Inventor Lone M. Gorb               United States 11787 New York City,               Smithtown, Witney Gate               29

Claims (1)

(57) [Claims] What is claimed is: 1. A composition comprising a tetracycline derivative having substantially no antibacterial activity, but having anticollagenase activity, and an animal feed, wherein the tetracycline derivative has the following structural formula: Dedimethylaminotetracycline having the following structural formula: A substituted nitrile of tetracycline having the following structural formula: A 6α-benzylthiomethylene tetracycline derivative having, a mono-N-alkylamide of tetracycline, having the following structural formula: At least one member selected from the group consisting of 6-fluorodemethyldeoxytetracycline and 7,11α-dichlorotetracycline having the following: An animal feed composition, characterized in that it is present in an effective amount. 2. The tetracycline derivative is 10 to 1 ton of feed.
The animal feed composition of claim 1, present in the range of 200 g. 3. The animal feed is for young calves up to about 12 weeks of age, and the amount of tetracycline derivative in the feed composition is 0. The animal feed composition according to claim 1, wherein the amount is such that 1 to 1.0 mg is ingested. 4. The animal feed composition according to claim 1, wherein the animal feed is for beef cattle and the amount of the tetracycline derivative in the feed composition is such that the animal will ingest 75-300 mg per day in the feed administration.