US3574830A - Process for inhibiting the transfer of antibiotic resistance determinants in bacteria - Google Patents

Abstract

PROCESS FOR INHIBITING THE TRANSFER OF ANTIBIOTIC RESISTANCE DETERMINANTS FROM DONOR BACTERIA TO RECIPIENT BACTERIA BY THE USE OF AN EFFETIVE AMOUNT OF A LINCOMYCIN ANTIBIOTIC.

Description

United States Patent PROCESS FOR INHIBITING THE TRANSFER OF ANTIBIOTIC RESISTANCE DETERMINANTS IN BACTERIA I Joanne Roeser, Kalamazoo, Mich., assignor to The Upjohn Company, Kalamazoo, Mich.

No Drawing. Continuation-impart of application Ser. No. 749,584, Aug. 2, 1968. This application Oct. 17, 1968, Ser. No. 768,502

Int. Cl. A61k 21/00 U.S. Cl. 424-481 7 Claims ABSTRACT OF THE DISCLOSURE Process for inhibiting the transfer of antibiotic resistance determinants from donor bacteria to recipient bacteria by the use of an effective amount of a lincomycin antibiotic.

CROSS REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of copending application Ser. No. 749,584, filed on Aug. 2, 1968, and now abandoned.

BRIEF SUMMARY OF THE INVENTION The subject process concerns the use of a lincomycin antibiotic, as herein defined, to prevent or inhibit the transfer by conjugation of antibiotic resistance determinants from donor bacteria to recipient bacteria. Antibiotic resistance determinants are genetic factors possessed by some bacteria which make them resistant to certain antibiotics and chemical antibacterial agents, such as sulfa drugs. It is well known in the microbiological art that antibiotic resistance determinants can be transferred from donor bacteria to recipient bacteria by conjugation. This transfer of resistance determinants is believed to be mediated through the agency of a resistance-transfer factor, abbreviated RTF. For example, if bacterium A possesses a resistance determinant for tetracycline, and RTF is present, then by conjugation with bacterium B which does not possess such a resistance determinant, the latter bacterium may also become resistant to tetracycline. A recent comprehensive review of transferable drug resistance is published in Science Journal 4, pp. 71-76 (1968). The combination of resistance determinants and RTF is generally referred to as R-factor.

The process of the present invention effectively blocks the transfer of resistance determinants from donor bacteria to recipient bacteria. Thus, for example, if an effective amount of a lincomycin antibiotic, defined, is used to treat the donor and recipient bacteria, described above, then the ability of the resistance determinant, possessed by bacterium A, to be transferred to bacterium B is inhibited. The term donor bacteria or donor means a bacterium which possesses a resistance determinant and 'RTF; whereas the term recipient bacteria or recipient means a bacterium which is not a donor but is capable of receiving a resistance determinant from a donor. The transfer of resistance determinants by conjugation has been shown to occur only between Gram-negative bacteria.

It should be noted that resistance determinants possessed by a bacterium are not necessarily directed to a single antibiotic but often times a single resistance determinant will enable the bacterium to be resistant to more than one antibiotic. Also, it is known that more than one resistance determinant can be transferred during a single conjugation by the presence of agency of a single RTF. Though the mechanism of the subject process to inhibit the transfer of resistance determinants from donor to recipient bacterium is not completely understood, it is felt that the 3,574,830 Patented Apr. 13, 1971 trated by the following structural formula:

CH3 R2 OAC1 wherein R is methyl, or ethyl; R is cis or trans alkyl of 2 to 8 carbon atoms, inclusive; R is hydrogen or alkyl to 8 carbon atoms, inclusive; X is hydroxy, chlorine, bromine, or iodine, each in the (R) or (S) configuration; A0 and AC2 are hydrogen or acyl, selected independently, wherein acyl is the acyl of a hydrocarbon carboxylic acid of not more than 18 carbon atoms, or a halo-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, or loweralkoxy hydrocarbon carboxylic acid of a total of up to 18 carbon atoms; and, Ac is of the same group as Ac and Ac or phosphate, and acid addition salts thereof.

The lincomycin compounds, herein defined, can be prepared by procedures disclosed in various patents, publications and patent applications. These are as follows:

LincomycinU.S. Pat. 3,086,912 with reference to Formula I, wherein R=methyl or ethyl-U.S. Pat. 3,380,992, (specification and Example 12) |R =cis or trans alkyl to 8 carbon atoms-US. Pat.

3,380,992, (specification and Example 1) R =Hydrogen or alkyl to 8 carbon atomsU.S. Pat. 3,380,992, (specification and Examples 1E and 1G and H) X: (S)OH-U.S. Pat. 3,380,992, (specification and Example 11 D) X=(R) or (S) C1 or BrBe1gium Pat. 676,202, US. Application Ser. No. 498,989, filed Oct. 20, 1965, now abandoned X=(R) or (S) I-Application Ser. No. 696,518, filed Jan. 9, 1968, now US. Pat. 3,496,163 Lincomycin acylates-U.S. Pat. 3,326,981 Lincomycin 2-acylatesBelgium Pat. 696,412, US. Pat. application Ser. No. 568,102, filed July 27, 1966, now US. Pat. 3,426,012 7-chlorolincomycin 2-acylates-U.S. pat. application Ser.

No. 637,358, filed May 10, 1967, now abandoned 7-chlorolincomycin 2-phosphates and lincomycin 2-phosphates-US. Pat. application Ser. No. 602,116, filed Dec. 16, .1966, now U.S. 'Pat. 3,487,068

Of the above compounds, the compound 7(S)-chloro- 7-deoxy-lincomycin is also known by the generic name climmycin.

DETAILS OF THE INVENTION ple, upon contacting mating strains of Escherichia coli, one strain of which harbors a resistance determinant for tetracycline, with 7(S)-chloro-7-deoxy-4depropyl-4'-pently-l'-demethyllincomycin in varying concentrations ranging from 0.5 ,ug./ml. to ,ug./ml., there is obtained an Escherichia coli culture exhibiting a decrease in the frequency of transfer of the tetracycline resistance determinant. Control runs, i.e., wherein no lincomycin antibiotic is used, result in a high percentage of conversion of the recipient bacteria from tetracycline sensitive to tetracycline resistant due to R-factor transfer from the donor Escherichia coli bacteria.

A bacterial culture harboring Gram-negative bacteria, among which are donors and recipients, can be contacted with a lincomycin antibiotic present in various vehicles depending on the site of the bacterial culture. For example, for inhibiting the transfer of antibiotic resistance determinants in bacteria resident in an animals gut, a lincomycin antibiotic can be administered as an additive to a feed. Advantageously, the lincomycin antibiotic can be added to the feed as a liquid or solid premix composition in which the concentration of the lincomycin antibiotic is from about 100 to about 2000 times greater than the concentration of antibiotic desired in the final feed ration. The lincomycin antibiotic can be dissolved or suspended in a fluid vehicle such as corn oil, cottonseed oil, water-based vehicles, molasses, distillers solubles, and the like, or in volatile solvents such as ethanol, acetone, dimethylsulfoxide, dimethylformamide and the like, to prepare a liquid premix. Alternatively, a solid premix can be prepared by mixing the lincomycin antibiotic with an edible solid diluent such as sucrose, lactose, starch, corn meal, flour, calcium carbonate, oyster shell flour, soybean meal and the like.

It is common in the art of raising domestic animals to add growth stimulating agents, usually other antibiotic substances, for example penicillins, oxytetracycline, chlortetracycline, spectinomycin, bacitracin (various forms), erythromycin, tylosin, oleandomycin, and the like, or non-antibiotic growth stimulating agents such as arsanilic acid (sodium salt), 3-nitro 4 hydroxyphenylarsonic acid, nitrofurazone, nitrofurazolidone, sulfamethazine and other sulfa drugs, trimethylalkyl ammonium stearate and the like to the feed or drinking water of the animals. Such growth stimulating agents also increase the efficiency of the conversion of feed into animal weight. The lincomycin antibiotics themselves possess growthpromoting qualities and can be added to the feeds as the only antibiotic when growth stimulation is desired. Alternatively, a second growth-promoting agent can be fed concomitantly with the lincomycin antibiotic. The second growth-promoting agent can be added to the feed as a separate premix, for example as a commercial growthpromoting premix composition such as those readily available in the marketplace. Advantageously, however, the lincomycin antibiotic and a second growth-promoting agent can be supplied in the same premix composition. Such premix compositions are then added to the animal feed to supply the lincomycin antibiotics at a rate of 10 to 1000 grams per ton of feed. In the cases where the lincomycin antibiotics are to be fed for their growthstimulating qualities in addition to their RTF-suppressive qualities, levels at the higher end of the range are desirable.

The lincornycin antibiotics, as herein defined, also can be administered to the animals in their drinking water. When administered by this route, the lincomycin antibiotics in water-dispersible form can be added to the drinking water as powders, crystals, premix solutions, premix suspensions, tablets, effervescent tablets, syrups, or other convenient forms, at a rate to administer the lincomycin antibiotic in the range of about 10 to about 1000 mg. per gal. of drinking water.

For control of resistance determinant transfer, the subject lincomycin antibiotics can be administered systematically by injection, for example by injection into the blood or musculature of the animal, or orally. In the case of injection, the antibiotics can be administered in a rapidly dispersible form, for example, the lincomycin antibiotic can be administered as readily soluble acidaddition salts, for example the hydrochloride or sulfate, or as readily soluble derivatives, for example as the ammonium 2-phosphate ester. In such administration, the lincomycin antibiotic is given in the range of about 1 to about mg. per kg. of body weight per day. Advantageously, in the case of intramuscular injection, the lincomycin antibiotic can be given in a more slowly dispersible, long-acting depot form, for example as the pamoate (2,2-dihydroxy 1,1 dinaphthylmethane-3,3- dicarboxylic acid) acid-addition salt or as the Z-O-higher acylate ester, for example the Z-O-palmitate ester. For such depot injection a level of l to 100 mg. per kg. body weight is administered at infrequent intervals, for example once or twice a week, to maintain a suitable blood level of the lincomycin antibiotic. For oral administration, the antibiotics may be given in various oral dosage forms such as powders, pills, tablets, capsules, solutions, suspensions, oil-water emulsions and the like.

Lincomycin antibiotics, as herein defined, also can be administered concurrently to animals being given a second therapeutic agent intended to combat an existing bacterial or viral infection. Such administration of lincomycin antibiotics is advantageous in forestalling the transfer of resistance determinants among surviving Gramnegative organisms. For example, the lincornycin antibiotic advantageously can be administered concurrently with agents such as penicillins, ampicillin, methicillin, nafcillin, oxacillin, phenecillin, tetracycline, chlortetracycline, oxytetracycline, demethylchlortetracycline, methacycline, rolitetracycline, doxycycline, erythromycin, oleandomycin, cephalothin, novobiocin, streptomycin, neomycin, kanamycin, cephaloglycin, cephaloridine, bacitra cin, polymyxin, spectinomycin, colistin, paromomycin, dihydrostreptomycin, dihydronovobiocin, sulfonamides, nitrofuran compounds, tylosin and moenomycin. The lincomycin antibiotic and the second therapeutic agent can be administered concurrently as. separate medications, for example by separate injections or as separate oral medicaments, or they can be administered in suitable admixture with each other, for example as a common single injection or in a single oral medicament.

The word animals as used herein with reference to feeds includes such animals as cattle, swine, sheep, chickens, turkeys, other domestic fowl, dogs, cats, horses, and the like.

The compositions of the present invention are preferably presented for administration to humans in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of a lincomycin antibiotic in the form of the free base, or pharmacologically acceptable salts and esters.

For oral administration, either solid or fluid unit dosage forms can be prepared. 'For preparing solid compositions such as tablets, the principal active ingredient is mixed with conventional ingredients such as talc, magnesium stearate, dicalcium phosphate, calcium sulfate, starch, lactose, acacia, methylcellulose, and functionally similar materials as pharmaceutical diluents or carriers.

For parenteral administration, fluid unit dosage forms are prepared utilizing a lincomycin antibiotic and a sterile vehicle, Water being preferred. The compound, depending on the form and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions, a water-soluble form of the lincomycin antibiotic can be dissolved in Water for injection and filter sterilized before filling into a suitable vial or ampul and sealing. Advantageously, adjuvants such as a local anesthetic, preservative and buffering agents can be dissolved in the vehicle.

To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. The dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection is supplied to reconstitute the solution prior to use. Parenteral suspensions are prepared in substantially the same manner except that the compound is suspended in the vehicle instead of being dissolved and sterilization cannot be accomplished by filtration. The compound can be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.

The dosage of a lincomycin antibiotic, as herein defined, depends on route and frequency of administration; and the age, weight, and condition of the patient. A dosage schedule of from about to 1000 mg, 1 to 4 times daily (every six hours), embraces the effective range for inhibition of resistance determinant transfer. For children, the dosage is calculated on the basis of 5 to 30 mg./kg./ day to be administered every six hours.

The lincomycin antibiotic, as herein defined, is compounded with a suitable pharmaceutical carrier in unit dosage form for convenient and effective administration. In the preferred embodiments of this invention, the dosage units contain the compounds in 15, 30, 50, 125, 250, 350, 500, 750 andv 1000 mg. amounts for systemic use; in 0.25, 0.5, 1.2 and 5% amounts for local use; and 5 to 65% w./v. for parenteral use. The dosage of compositions containing lincomycin antibiotic, as herein defined, and one or more other active ingredients is to be determined with reference to the usual dosage of each such ingredient.

The following examples are illustrative of the process and products of the present invention but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 An Escherichia coli donor strain harboring a resistance transfer factor with determinants for resistance to tetracycline (25 ng./ml.), chloramphenicol (25 ug./ml.), streptomycin (25 ag./ml.), and sulfathiazole (100 ,ag./ml.) is inoculated by a loop from a PenAssay Agar slant into PenAssay Broth with or without an appropriate concentration of one of the following lincomycins: 7(8)- chloro 7 deoxy 1'-demethyllincomycin as the hydrochloride salt (U26,285A); 7(S) chloro 7 deoxylincomycin as the hydrochloride hydrate salt (U-21,251F-); and 7 (S) chloro 7-deoxy-4'-depropyl-4'-pentyl-l'-demethyllincomycin as the hydrochloride salt (U-24,729A). U26,285A and U-21,251F are tested at concentrations ranging from 0.5 ag/ml. to 200 g./ml.; whereas U-24,729A is tested at concentrations ranging from 0.5 ,ug/ml. to ag/ml. The donor broth cultures are incubated at 37 C. for 24 hours on a reciprocating shaker.

An Escherichia coli recipient strain, which harbors no resistance transfer factor, and is sensitive to tetracycline (25 ag./ml.), and carries a chromosomal resistance to streptomycin (750 g./ml.), is inoculated by a loop from a PenAssay Agar slant into PenAssay Broth and is incubated at 37 C. for 21 hours on a reciprocating shaker. The recipient culture is diluted in PenAssay Broth and is incubated at 37 C. to achieve a logarithmically growing culture at a concentration of 2 to 5 10 cells per ml.

Mating mixtures are prepared by adding 0.5 ml. of donor culture to 5.0 ml. of recipient culture to which an appropriate concentration of the lincomycin antibiotic, as given previously, was added in order to maintain the same concentration in the mating mixture as in each pre-treated donor culture respectively. The mating mixtures are incubated at 37 C. for 1 hour with gentle shaking.

Viable counts of the donor and recipient cultures are made at 0 time of the mating experiment by diluting samples in Spizizens minimal salts solution and by plating aliquots of each solution on PenAssay Agar plates. The numbers of converted recipients [recipients resistant to tetracycline (25 g./ml.)] are determined by making dilu tions of each mating mixture in Spizizens minimal salts solution and by plating aliquots of each dilution on Pen- Assay Agar plates containing both tetracycline (25 ,ug./ ml.) and streptomycin (750 ,ag./ml.). The plates are incubated at 37 C. for 24 hours before the number of colonies is ascertained.

Calculations are made to determine the number of converted recipients per input donor cell. A negative number is obtained even for control cultures since few cells in the total donor population are capable of transfer at any given time.

The data presented in the following table give the number of converted recipients per input donor cell at each concentration of the lincomycin compound tested. These same data when graphed show that an exponential decrease in the frequency of transfer of the tetracycline resistance deteminant occurs with a linear increase in the concentration of each of the lincomycins tested.

The PenAssay Agar disclosed above is prepared by using PenAssay Broth (Difco Antibiotic Medium No. 3, Difco, Detroit, Mich.) plus 15 grams/liter of agar (Difco).

PenAssay Broth consists of Difco antibiotic Medium No. 3.

Spizizens minimal salts solution contains the following in g./liter of deionized water:

K HPO 14.0; KH PO 6.0; sodium citrate, 5H O, 1.0;

and (NH4)2SO4, 2.0.

TABLE I.-EFFEC[ OF LINCOMYOIN ANTIBIOTICS ON RESISTANCE TRANSFER Number of converted recipients per initial donor roll U24,720A U-21,251F U26,285A

2. 1X10" 1. 3X10- 1. 2 (l.0 8. 3X10- 1. 9X10- 4. 9X10- 2. 2X10- 8. 9X10- *The degree to which low frequencies of transfer can be measured is dependent both on the initial donor viable count and the number of converted recipients which can be detected. Thus the limitation of the measurement varies from one culture to another.

EXAMPLE 2 Clinimycin, (7(S)-chloro 7 deoxylincomycin) (U- 21,251F) and 7(.S)-chloro 7 deoxy-4-depropyl-4- pentyl-1-dimethyllincomycin, (U-24,729A) have been shown to inhibit resistance transfer in vivo in the chick using the following experimental system:

Escherichia coli CSH-2 which harbors a resistance transfer factor with determinants for resistance to tetracycline 25 .ag./ml.), chloramphenicol 25 g./ml.), streptomycin (25]ag./ml.), and sulfathiazole mg./ ml.) is employed as the donor strain. An Escherichia coli strain which harbors no resistance transfer factor, is sensitive to tetracyline 25 ,ug./Inl.), and carries a chromosomal resistance to streptomycin (750 ,ag/ml.) is used as the recipient strain. The donor and recipient strains are grown overnight at 37 C. in PenAssay Broth (Difco). The recipient culture is concentrated six-fold. Both the bacterial cultures and the compounds, clinimycin and 7(S)-chloro-7- deoxy-4-depr0pyl 4 pentyl-l'-demetl1yl1incomycin dissolved in deionized water, are administered to the chicks in 0.5 ml. doses by oral intubation. Groups of chicks, each chick weighing approximately 0.035 kg., are treated with various concentrations of the compounds.

On Day 1, oneday old chicks are given the donor strain of E. coli orally. This is followed hours later by the first oral dose of clinimycin or 7(S)-chloro-7-deoxy-4'- depropyl-4'-pentyl-1-demethyllincomycin. On Day 2, two doses of either clinimycin or 7(,S)-chloro 7 deoxy-4'- depropyl-4'-pentyl 1' demethyllincomycin are administered approximately 7 hours apart. Midway between these two doses, the chicks are given the concentrated suspension of the recipient strain. The chicks are sacrificed on Day 3. Both donor and recipient E. coli are present simultaneously in the gut for approximately 20 hours.

The large intestine is removed from each chick into a sterile Petri dish. The gut is perforated with a dissecting knife and washed with 2 ml. of PenAssay Broth. The resulting suspensions are diluted and plated on PenAssay Agar with tetracycline (25 ,ug/ml.) to determine the number of donor cells; on PenAssay Agar with strep-tomycin (750 ,ug./ ml.) to determine the number of recipient cells; and on PenAssay Agar with tetracycline (25 g/ ml.) and streptomycin (750 ig/ml.) to determine the number of converted recipients (recipients resistant to tetracycline) in each gut sample. The plates are incubated at 37 C. for 24 hours before colony counts are made.

The data are given in the following Table II for clinimycin and in Table III for 7(S)-chloro-7-deoxy-4-de propyl-4'-pentyl-1-demethyllincomycin. The degree of inhibition of resistance transfer is dependent on the concentration of each compound used. A concentration of 100 mg./ kg. of clinimycin completely suppresses resistance transfer in the chicks as does a 50 mg./kg. dosage level of 7(S)-chloro-7-deoxy 4 depropyl-4'-pentyl-l'-demethyllincomycin.

*Base equivalent of clinimycin.

TABLE lII.7(S)-OHLORO-DEOXY-47-DEPROPYL-4.-PEN- TYIrY-DEMETHYLLINCOMYCIN (II-24,729A) Converted Donors Recipients recipients Mean IA 2. 9x10 7. 7x10 4. 3x10 IB 3. 5x10 3. 7X10 1. 3x10 1 Ding/kg IC 6.8)(10 3. 0X10 1.0)(10 1. 6x10 ID 2.3)(10 2.8Xl0 3.8)(10 J IE 1. 6X10 5. 6X10 2. 5X10 HA 1. 1X10 2. 8X10 1. 1X10 HR 3. 6X10 1. 1X10 2X10 3.125 mg./kg HO 4. 6X10 1. 4X10 4. 5X10 2. 4x10 IID 6. 4X10 3.1)(10 7. 0X10 HE 4. 1X10 3. 9x10 1. 2x10 IIIA 1.2)(10 5. 2x10 5 1X10 IIIB 1. 2x10 6. 2X10 1. 4X10 6.25 mg./kg H10 7. 6x10 4. 1X10 2. 6X10 9. 6X10 IIID 2. 4X10 2. 8X10 9 4x10 1 IIIE 6. 4X10 24x10 1 7X10 IVA 8. 4x10 2. 6X10 2. 5X10 IVB 7. 5x10 2. 2X10 1. 2X10 12.5 lug/kg"--. WC 9. 7X10 2. 2X10 1. 0X10 5. 5X10 IVD 5. 6x10 5. 2x10 2. 0 10 J IVE 8. 3x10 1. 9X10 8. 5X10 VA 3. 9X10 5. 4X10 1. 0X10 VB 3. 5x10 8. 1x10 1. 0X10 25 rug/kg VC 4.2)(10 1.6)(10 7. 5X10 1. 0X10 VD 2. 7X10 2. 3X10 1. 0X10 VE 1. 9x10 5. 5X10 4. 1X10 [A 2. 8X10 1. 9X10 1. OXlO VIB 4. 3X10 8. 0x10 1. 0X10 50 mg./kg VIC 4. 0X10 2. 4X10 1. 0X10 1. 0X10 VID 4.1)(10 7. 1X10 1. 0X10 VIE 1. 5X10 1. 9 l0 1.0X10

EXAMPLE 3 Sterile powder A completely soluble sterile powder of clinimycin hydrochloride which, on reconstitution, can be administered either parenterally (I.M., I.V. or subcutaneously) or topically is put up in sterile vials, each vial containing 1 gm. of antibiotic. For use, water for injection or sterile normal saline is added to give a final volume of 10 ml. This results in a 10% W./v. concentration or mg. of antibiotic per m1. of reconstituted product. The preparation of clinimycin hydrochloride is disclosed in Belgium Pat. 676,154.

EXAMPLE 4 Intramuscular depot-form injectable for animals A sterile aqueous suspension suitable for intramuscular injection and containing in each ml. 400 mg. of clinimycm pamoate is prepared from the following ingredients:

Gm. Clinimycin pamoate, micronized 400 Polyvinylpyrrolidone 5 Sodium carboxymethylcellulose, low visc. 1 Sodium citrate, hydrous 8 Methylparaben 1.5 Propylparaben 0.1

Water for injection q.s. 1000 ml.

The preparation of clinimycin pamoate is disclosed in Belgium Pat. 676,154.

EXAMPLE 5 T ablets for large animals 10,000 tablets for oral administration to large animals such as cattle or horses and containing in each tablet 1000 mg. of clinimycin hydrochloride are prepared from the following ingredients:

The ingredients are mixed thoroughly and slugged. The ingredients are broken down by passing through a number sixteen screen. The resulting granules are then compressed into tablets each containing 1000 mg. of clinimycin hydrochloride.

EXAMPLE 6 Premix compositions PREMIX A Clinimycin hydrochloride20 gm. Lactose-qs. 1 lb.

PREMIX B 1-demethylclinimycin hydrochloride 60 gm. Corn mealq.s. 1 lb.

1 Preparation disclosed in Belgian Pat. 676,154.

PREMIX C 4'-pentyl-1'-demethyl analog of clinimycin hydrochloride gm. Wheat flourq.s. 1 lb.

PREMIX D Clinimycin base- 20 gm. Cottonseed oil-1 lb.

PREMIX E 4'-pentyl-1-demethyl analog of clinimycin hydrochloride20 gm. Waterl liter PREMIX F Lincomycin-Z-acetate --50 gm. Ethyl acetate-1 liter Disclosed in U.S. Pat. 3,326,891, Example 6.

PREMIX G Clinimycin pamoate20 gm. Bacitracin zinc-25 gm. Cornq.s. 1 lb.

PREMIX H 4'-penty1-1'-demethyl analog of clinimycin hydrochloride-10 gm. Oxytetracycline quaternary salt-50 gm. Sucrose-q.s. 1 lb.

PREMIX I l-demethylclinimycin hydrochloride50 gm. Chlortetracycline hydrochloride50 gm. Oyster shell flourq.s. 1 lb.

PREMIX J 4' pentyl 1' demethyl analog of clini'mycin, 2-

palmitate ester --10 gm.

Procaine penicillin G20 gm.

Preparation disclosed in U.S. patent application Ser. No. 637,358, filed May 10, 1967.

The foregoing premix compositions are prepared from finely ground solid materials by mixing all ingredients 10 together in an appropriate manner. In the case of liquid premix compositions the solids are dissolved or suspended in the liquids by methods known in the art.

Following the preceding formulas, premixes are simi larly prepared substituting other compounds of formula 1 for those given. In the premixes where a second antibiotic is present, as for growth promoting or therapeutic purposes, the second antibiotic may be taken from those known for growth promoting or therapeutic purposes in the art, for example those listed in the specifications above.

The above premix compositions are added to the feed of the animal, or (premix E) to the drinking water, in an amount to supply the antibiotics at the rate specified above.

EXAMPLE 7 Swine growing diet for hogs of 40 to pounds body weight 0.08; Fe, 5.0 Cu, 0.4;1, 0.24 Zn, 0.7.

2 Contains 800 USP units of Da/gm. and 1500 LU. of A/gm.

Contains per 1b.: Riboflavin, 200 mg.; calcium. pantothenate, 4000 mg.; niacin, 9000 mg.; and choline chloride, 10,000 mg.

4 Contains 6 mg. B12 per lb.

To 99 parts of the preceding feed is added 1 part of Premix A to provide a feed with 200 mg. of the lincomycin antibiotic per lb. of feed.

Substituting Premixes B through I, feeds are prepared with varying amounts of lincomycin antibiotics, with or without a second growth-stimulating or therapeutic antibiotic. The foregoing composition is usefully fed to hogs for increased weight gain and improved feed conversion efficiency, and inhibition of transfer of resistance determinants.

EXAMPLE 8 A fattening feed for 800 pound yearling cattle is prepared from the following types and amounts of ingredients:

Ground ear corn 89.75 Soybean oil meal, 44% 9.0 Ground limestone 0.7 Sodium chloride 0.5 Trace mineral mixture 1 0.05

Contains the following percent of minerals: Mn, 12; Co, 0.08 Fe, 5.0; Cu, 0.4 I, 0.24 Zn, 0.7.

To 99 parts of the preceding feed is added 1 part of Premix A to provide a feed with 200 mg. per lb. of antibiotic.

Substituting Premixes B through I, inclusive, feeds are prepared with varying amounts and ratios of antibiotics.

Cattle are to receive the foregoing feed ad libitum together with 5 lb. of hay per head per day for an increased rate of weight gain, increased feed efficiency, and inhibition of transfer of resistance determinants.

1 1 EXAMPLE 9 A chicken feed for broilers is prepared from the following types and amounts of ingredients:

Percent Yellow corn meal 67.35 Soybean oil meal, 50% 24.00 Menhaden fish meal, 60% 6.00 Steamed bone meal 1.00 Ground limestone 1.00 Iodized salt .34 25% Choline chloride .13 Vitamin B supplement (6 mg./ lb.) .10 Manganese sulfate .02 Supplemental vitamin mix 1 .06

Consisting of 16.0 gm. vitamin A supplement (10,000 units/gm.); 3.6 gm. vitamin D3 supplement (15,000 units/ gm.); 7.1 gm. riboflavin supplement (1 gm. riboflavin per ounce) 500 mg. niacin.

To 99 parts of the preceding feed is added 1 part of Premix A to provide a feed with 200 mg. per lb. of antibiotic.

Substituting Premixes B through I, inclusive, feeds are prepared with varying amounts and ratios of antibiotics.

The foregoing composition is usefully fed to chickens for increased rate of weight gain, improved utilization of feed, and inhibition of transfer of resistance determinants.

EXAMPLE 10 Drinking water for chickens, turkeys, ducks, geese or other domestic fowl Premix E is added to the drinking water of the animals to provide 10 to 1000 mg. of trans-4-pentyl-l-demethyl analog of clinimycin hydrochloride antibiotic per gal.

Other lincomycin antibiotics of Formula 1 may be substituted for that given in Premix E, to provide protection against transfer of resistance determinants.

EXAMPLE 1 1 Mastitis ointment One thousand grams of an ointment for the inhibition of transfer of resistance determinants in dairy cattle being treated for mastitis is prepared from the following types and amounts of ingredients:

Gm. Clinimycin hydrochloride 25 Methylprednisolone acetate 0.5 Light liquid petrolatum 300 Chlorobutanol, anhydrous 5 Polysorbate 80 5 2% aluminum monostearate-peanut oil gel 400 White petrolatum q.s. 1000 gm.

The clinimycin hydrochloride and methylprednisolone acetate are milled with the light liquid petrolatum until finely divided and uniformly dispersed. The chlorobutanol, polysorbate 80, peanut oil gel and white petrolatum are heated to 120 F. to form a melt and the liquid petrolatum dispersion stirred in. With continued stirring, the dispersion is allowed to cool (and congeal) to room temperature and is filled into disposable mastitis syringes in gm. doses.

The above formulation can be administered to cattle exposed to cattle afiiicted with mastitis in order to prevent the non-afllicted cattle from receiving Gram-negative bacteria possessing resistance determinants to various antibiotics and chemical antibacterial agents, such as sulfa drugs.

EXAMPLE 12 Capsules One thousant two-piece hard gelatin capsules for oral use, each containing 250 mg. of clinimycin hydrochloride 12 are prepared from the following types and amounts of materials:

Gm. Clinimycin hydrochloride 250 Corn starch Talc 75 Magnesium stearate 25 The materials are thoroughly mixed and then encapsulated in the usual manner.

The foregoing capsules are useful for the inhibition of transfer of resistance determinants in adult humans by the oral administration of 1 capsule every 4 hours.

Using the procedure above, capsules are similarly prepared containing clinimycin hydrochloride in 25, 50, 125, 350 and 500 mg. amounts by substituting 25, 50, 125, 350 and 500 gm. of clinimycin hydrochloride for the 250 gm.

used above.

EXAMPLE 13 Capsules One thousand two-piece hard gelatin capsules for oral use, each containing 50 mg. of the 4-pentyl-l'-demethyl analog of clinimycin hydrochloride and 250 mg. of tetracycline hydrochloride, are prepared from the following types and amounts of ingredients:

4'-pentyl-1'-demethyl analog of clinimycin hydrochloride 50 Tetracycline hydrochloride 250 Talc 75 Magnesium stearate 25 The ingredients are thoroughly mixed and then encapsulated in the usual manner.

The foregoing capsules are useful for the inhibition of transfer of resistance determinants in adult humans by the oral administration of 1 capsule every 6 hours.

Using the procedure above, capsules are similarly prepared containing the 4'-penty1-1'-demethyl analog of clinimycin hydrochloride and each of the following antibiotics in place of tetracycline by substituting 250 gm. of such other antibiotic for tetracycline: penicillins, ampicillin, methicillin, nafcillin, oxacillin, phenecillin, chlortetracycline, oxytetracycline, demethylchlortetracycline, metacycline, rolitetracycline, doxycycline, erythromycin, oleandomycin, cephalothin, kanamycin, novobiocin, streptomycin, neomycin, cephaloglycin, cephaloridine, bacitracin, polymyxin, spectinomycin, colistin, paromomycin, dihydrostreptomycin, dihydronovobiocin, sulfonamides, nitrofuran compounds, chloramphenicol, fumagillin, tylosin and moenomycin. When a penicillin, such as potassium penicillin G, is to be used in place of tetracycline, 250,000 units per capsule is employed.

Such combination products are useful for the inhibition of transfer of resistance determinants in mixed infections in adult humans by the oral administration of 1 capsule every 6 hours.

EXAMPLE 14 Tablets One thousand tablets for oral use, each containing 500 mg. of clinimycin hydrochloride are prepared from the following types and amounts of materials:

Gm. Clinimycin hydrochloride 500 Lactose Corn starch 65 Magnesium stearate 25 Light liquid petrolatum 3 The ingredients are thoroughly mixed and slugged. The slugs are broken down by forcing through a number sixteen screen. The resulting granules are then compressed into tablets, each tablet containing 500 mg. of clinimycin hydrochloride.

13. The foregoing tablets are useful for the inhibition of transfer of resistance determinants in infections in adult humans by oral administration of 1 tablet every 4 hours.

Using the above procedure, except for reducing the' amount of clinimycin hydrochloride to 250 gm., tablets containing 250 mg. of clinimycin hydrochloride are prepared.

EXAMPLE 15 Tablets One thousand oral tablets, each containing 250 mg. of clinimycin hydrochloride and a total of 250 mg. (83.3 mg. each) of sulfadiazine, Sulfamerazine, and sulfamethazine, are prepared from the following types and amounts of materials:

The ingredients are thoroughly mixed and slugged. The slugs are broken down by forcing through a number sixteen screen. The resulting granules are then compressed into tablets, each containing 250 mg. of clinimycin hydrochloride and a total of 250 mg. (83.3 mg. each) of sulfadiazine, Sulfamerazine, and sulfamethazine.

The foregoing tablets are useful for the inhibition of transfer of resistance determinants in infections by the oral administration of 4 tablets first and then 1 every six hours.

For the inhibition of transfer of resistance determinants in urinary infections, the triple sulfas in the above formulation is advantageously replaced by 250 gm. of sulfamethylthiadiazole or 250 gm. of sulfacetamide.

EXAMPLE 16 Oral syrup One thousand ml. of an aqueous suspension for oral use, containing in each 5-ml. dose, one-half gram of total sulfas and 250 mg. of clinimycin hydrochloride is prepared from the following types and amounts of ingredients:

Clinimycin hydrochloride gm 50 Sulfadiazine gm 33.3 Sulfamerazine gm 33.3 Sulfamethazine gm 33.3 Citric acid gm 2 Benzoic acid gm 1 Sucrose gm 700 Tragacanth gm 5 Lemon oil ml 2 Deionized water, q.s. 1000 ml.

The citric acid, benzoic acid, sucrose, tragacanth, and lemon oil are dispersed in suflicient Water to make 850 ml. of solution. The clinimycin hydrochloride and finely powdered sulfas are stirred into the syrup until uniformly distributed. Suflicient Water is added to make 1000' ml.

The composition so prepared is useful for the inhibition of transfer of resistance determinants at a dose of 1 teaspoonful (5 ml.) 4 times a day in adult humans being treated systemically for pneumonia.

EXAMPLE 1? By substituting 150 gm. of lincomycin hydrochloride for clinimycin hydrochloride in Example 16, an oral syrup containing one-half gram of total sulfas and 750 mg. of lincomycin hydrochloride in each S-ml. dose is prepared.

The composition so prepared is useful for the inhibition of transfer of resistance determinantsat a dose of 1 tea- 14 spoonful (5 ml.) 4 times a day in adult humans being treated systemically for pneumonia.

EXAMPLE l8 Parenteral solution A sterile aqueous solution for intramuscular use, containing in 1 ml. 200 mg. of clinimycin hydrochloride is prepared from the following types and amounts of material-s:

Gm. Clinimycin hydrochloride 200 Lidocaine hydrochloride 4 Methylparaben 2.5 Propylparaben 0.17

Water for injection, q.s. 1000 ml.

The ingredients are dissolved in the Water and the solution sterilized by filtration. The sterile solution is filled into vials and the vials sealed.

EXAMPLE 19 Parenteral preparation A sterile aqueous solution for intramuscular use, containing in 1 ml. 200 mg. of clinimycin hydrochloride and 400 mg. of spectinomycin sulfate, is prepared from the following types and amounts of ingredients:

Gm. Clinimycin hydrochloride 2 00 Spectinomycin sulfate 400 Lactose 50 Water for injection, q.s. 1000 ml.

EXAMPLE 20 Cream One thousand grams of a vaginal cream are prepared from the following types and amounts of ingredients:

Gm. Clinimycin hydrochloride 50 Tegacid Regular 150 Spermaceti Propylene glycol 50 Polysorbate 8O 5 Methylparaben 1 Deionized water, q.s. 1000 gm.

1 Self-emulsifying glyceryl monostearate from Goldschmidt Chemical Corporation, New York, N.Y.

The Tegacid and spermaceti are melted together at a temperature of 70-80" C. The methylparaben is dissolved in about 500 gm. of Water and the propylene glycol, Polysorbate 80, and clinimycin hydrochloride are added in turn, maintaining a temperature of 7580 C. The methylparaben mixture is added slowly to the Tegacid and Spermaceti melt, with constant stirring. The addition is continued for at least 30 minutes with continued stirring until the temperature has dropped to 40- 45 C. The pH of the final cream is adjusted to 3.5 by incorporating 2.5 gm. of citric acid and 0.2 gm. of dibasic sodium phosphate dissolved in about 50 gm. of water. Finally, sufficient Water is added to bring the final weight to 1000 gm. and the preparation stirred to maintain homogeneity until cooled and congealed.

The foregoing composition is useful for the inhibition of transfer of resistance determinants in conjunction with the treatment of vaginal infections in humans.

EXAMPLE 21 Ointment, ophthalmic One thousand grams of an ophthalmic ointment containing 0.5% clinimycin hydrochloride are prepared from the following types and amounts of ingredients:

Gm. Clinimycin hydrochloride 5 Bacitracin 12.2 Polymyxin B sulfate (10,000 units/mg.) 1 Light liquid petrolatum 250 W001 fat 200 White petrolatum, q.s. 1000 gm.

The solid ingredients are finely divided by means of an air micronizer and added to the light liquid petrolatum. The mixture is passed through a colloid mill to uniformly distribute the micronized particles. The wool fat and white petrolatum are melted together, strained, and the temperature adjusted to 45-50 C. The liquid petrolatum slurry is added and the ointment stirred until congealed. Suitably the ointment is packaged in one dram ophthalmic tubes.

The foregoing ointment is usefully applied to the eye for the inhibition of transfer of resistance determinants in humans and other animals.

Advantageously the foregoing composition can contain 5 gm. (0.5%) of methylprednisolone for the treatment of inflammation, and, alternatively, the bacitracin and polymyxin B sulfate can be omitted.

EXAMPLE 22 Eye-ear drops One thousand cc. of a sterile aqueous solution for eye or ear use containing mg. of clinimycin hydrochloride and 5 mg. of methylprednisolone phosphate sodium in each ml. is prepared from the following types and amounts of ingredients:

Clinimycin hydrochloride 10 Methylprednisolone phosphate sodium 5 Sodium citrate 4.5 Sodium bisulfite l Polyethylene glycol 4000 120 0.2

Myristyl-y-picolinium chloride Polyvinylpyrrolidone 1 Deionized water, q.s. 1000 ml.

bacteria to mating Gram-negative recipient bacteria which comprises contacting said donor and recipient bacteria with an effective amount of a lincomycin antibiotic of the formula:

R2 IL CH3 a H 51 (Ham. 1 1', AczO o OAB1 OAcs wherein R is methyl or ethyl; R is cis or trans alkyl of 2 to 8 carbon atoms, inclusive; R is hydrogen or alkyl of 1 to 8 carbon atoms, inclusive; X is hydroxy, chlorine, bromine, or iodine, each in the (R) or (S) configuration; Ac and A0 are hydrogen or acyl, selected independently, wherein acyl is the acyl of a hydrocarbon carboxylic acid of not more than 18 carbon atoms, or a halo-, nitro-, hydroxy-, amino-, cyano-, thiocyan0-, or loweralkoxy hydrocarbon carboxylic acid of a total of up to 18 carbon atoms; and Ac is of the same group as AC1 and Ac or phosphate, or acid-addition salts thereof.

2. A process, according to claim 1, wherein the lincomycin antibiotic is lincomycin free base or an acid addition salt thereof having the following structure:

3. A process, according to claim 1, wherein the lincomycin antibiotic is a chlorinated lincomycin antibiotic.

4. A process, according to claim 1, wherein the lincomycin antibiotic is 7(S)-chloro-7-deoxylincomycin.

5. A process, according to claim 1, wherein the lincomycin antibiotic is 7(S)-chloro-7-deoxy-1-demethyllincomycin.

6. A process, according to claim 1, wherein the lincomycin antibiotic is 7 (S)-chloro-7-deoxy-4'-depropy1- 4-pentyll '-demethyllincomycin.

7. A process, according to claim 1, wherein said efiective amount of a lincomycin antibiotic is in the range of from about 0.5 to about ng./ml.

References Cited UNITED STATES PATENTS 3,086,912 4/1963 Bergy et a1. 424-181 3,380,992 4/1968 Argoudelis et a1. 260-210 OTHER REFERENCES Kawakami et al., Biochemical and Biophysical -Re search Communications, vol. 18, Nos. 5-6, 1.965, pp. 716724.

ALBERT T. MEYERS, Primary Examiner D. M. STEPHENS, Assistant Examiner UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3, 574,850 Dated Apr i l 13, 1971 Inventofls) Joanne Roese" It is certified that error appears in the above-identified patem and that said Letters Patent are hereby corrected as shown below:

Col umn 1, i ine 52, for ant ib iotic, defined, read ant i biotic, as here in defined i ine 69, for "of" read or Coi umn 5, i ine 28, for "1 .2" read 1,2 Col umn 6, i ine 5, for "(25 9 ./mi read (25 gg/mi i ine 22, for "deteminant' read determinant i ine +1, for "roi 1 read cel i i ine 65, for "d imethyl i incomyc read demethyl i incomyc in i ines 73-74, for tetracyi ine" read tetracyci ine Col umn 7, I ine 57, for i "1 .0 read 1 .0 Col umn 8, I ine 1, for "chloro-deox i'T' read chioro-T-deoxy-4' Coi umn 9, I ine 52, for "corn-q.s read corn meal q.s Column 10,

i ine 37, For "200 mg. read 2000 mg Coi umn 11, i ine 7 L, for "thousant" read thousand Signed and sealed this 31st day of August 1971 (SEAL) Attest:

EDWARD M.FLETCHER, JR. WILLIAM E. SCHUYLER, JR. Attesting Officer Commissioner of Patents