GB1561107A - N-acetyl thienamycin - Google Patents

Abstract

The microorganism Streptomyces cattleya, preferably the strain NRRL 8057, is cultivated under submerged aerobic conditions in an aqueous nutrient medium containing assimilable C and N sources as well as inorganic salts. N-Acetylthienamycin is obtained. Non-toxic, pharmaceutically acceptable salts of N-acetylthienamycin can be obtained by reaction with inorganic or organic bases. N-acetylthienamycin and its salts show remarkable antibiotic properties and are active against a wide range of pathogens as well as against resistant strains of particular microorganisms. The antibiotic is active against Gram-positive and -negative bacteria and is suitable for use in human and veterinary medicine and as antibacterial agent for commercial and industrial purposes.

Description

(54) N-ACETYL THIENAMYCIN (71) We, MERCK & CO INC., a corporation duly organized and existing under the laws of the State of New Jersey, United States of America, of Rahway, New Jersey, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed to be particularly described in and by the following statement:- The discovery of the remarkable antibiotic properties of penicillin stimulated great interest in this field which has resulted in the finding of many other valuable antiobiotic substances such as other penicillins, cephalosporins, streptomycin, bacitracin, tetracylines, chloramphenicol and erythromycins. In general, each of these antiobiotics do not act against certain clinically important pathogenic bacteria.For example, some are principally active against only grm-positive types of bacteria. Furthermore, acquired resistance over the course of widespread use of existing antibiotics in the treatment of bacterial infection has caused a serious resistance problem to arise.

Accordingly, the deficiencies of the known antibiotics have stimulated further research to find other antibiotics which will be active against a wider range of pathogens as well as resistant strains of particular microorganisms.

This invention provides an antibiotic substance herein called N-acetyl thienamycin. The invention encompasses the antibiotic in dilute forms. as crude concentrates and in pure forms.

In accordance with the present invention, N-acetyl thienamycin is produced by growing under controlled conditions the microorganism Streptomyces cattleya.

Growth of Streptomyces cattleya in a nutrient media also results in the production of thienamycin. Acetylation of thienamycin provides a further process for the preparation of N-acetyl thienamycin. The preparation of thienamycin by fermentation of Streptomyces cattleya is set forth herein and also in the specification of our copending Application No. 48208/75 (1A98,087) Based upon extensive taxomic studies, Streptomyces catqeya. isolated from a soil sample, was identified as an actinomycete and has been designed MA-4297 in the culture collection of Merck & Co. Inc. Rahway, New Jersey.

A culture thereof has been placed on permanent deposit with the culture collection of the Northern Regional Research Laboratories, Northern Utilization Research and Development Division, Agricultural Research Service, U.S.

Department of Agriculture, Peoria, I11., and has been assigned accession No.

NRRL 8057.

The morphological and cultural characteristic of Streptomyces cattleya are set forth in Table 1.

TABLE 1 Morphology-Sporophores are compact spirals occurring as side and terminal branches on aerial mycelium. Spores are ellipsoidal to cylindrical in shape, 0.9 x1.2 in size, occurring in chains of more than 10.

Cultural Tomato paste-oatmeal agar Vegetative growth-Reverse-tan, flat, spreading; Aerial mycelium-Orchid (10 gc) mixed with white; Soluble pigment-None.

Czapek Dox agar (sucrose nitrate agar) Vegetative growth-Colorless, flat, spreading; Aerial mycelium-Sparse, pinkish white; Soluble pigment-None.

Egg albumin agar Vegetative growth-Tan with grayed-orchid cast, flat, spreading; Aerial mycelium-Orchid (10 gc) mixed with lighter shades of orchid and some white; Soluble pigment-None.

Glycerol asparagine agar Vegetative growth-Reverse-tan with gray-pink cast, flat, spreading; Aerial mycelium-Orchid (10 gc) mixed with some white; Soluble pigment-None.

Yeast extract-glucose+salt agar Vegetative growth-Tan with grayed pink cast; Aerial mycelium-Orchid (10 gc) mixed with pinkish-white; Soluble pigment-None.

Yeast extract-malt extract agar Vegetative growth-Tan; Aerial mycelium-Orchid (10 gc) mixed with pinkish-white; Soluble pigment-None.

Peptone-iron-yeast extract agar Vegetative growth-Tan; Aerial mycelium-none; Soluble pigment-Slight browning of medium; Melanin-Negative; H2S prodction-Negative.

Nutrient agar Vegetative growth-Light tan; Aerial mycelium-None; Soluble pigment-None.

Nutrient starch agar Vegetative growth-Cream to tan; Aerial mycelium-None; Soluble pigment-None; Hydrolysis of starch-Moderate.

Nutrient gelatin agar Vegetative growth-Cream-colored; Aerial mycelium-None; Soluble pigment-None; Liquefaction of gelatin-Moderate.

Gelatin stabs Vegetative growth-Tan; Aerial mycelium-None; Soluble pigment-None; Liquefaction of gelatin-Moderate.

Potato plug Vegetative growth-Moderate, tan; Aerial mycelium-Sparse, grayish-pinkish-white; Soluble pigment-None.

TABLE 1 (cont.) Loeffler's Blood serum Vegetative growth-Cream-colored; Aerial mycelium-None; Soluble pigment-None; Liquefaction-None.

Skim milk agar Vegetative growth-Tan; Aerial mycelium-Sparse, whitish; Soluble pigment-Slight browning of medium; Hydrolysis of casein-Positive.

Litmus milk Vegetative growth-Tan to brown; Aerial mycelium-None; Color-No soluble pigment, litmus indicator becoming bluish: Coagulation and/or peptonization-Partial peptonization, becoming alkaline.

Skim milk Vegetative growth-Tan; Aerial mycelium-None; Soluble pigment-None; Coagulation and/or peptonization-Partial peptonization, becoming alkaline.

Tyrosine agar Vegetative growth-Tan; Aerial mycelium-Mixture of orchid (10 gc) and white; Soluble pigment-None; Decomposition of Tyrosine--Positive.

All of the readings reported above were taken after three weeks incubation at 28"C. unless noted otherwise. The pH of the media used in these studies was approximately neutral, namely, pH 6.8-7.2. The color designations used in the description are in accordance with the definitions of the Color Harmony Manual. 4th Edition (1958), Container Corporation of America, Chicago, Illinois.

Streptomyces cattleya was also tested for its ability to utilize or assimilate various carbohydrates. For this purpose, the microorganism was grown on basal synthetic medium (Pridham and Gottlieb) containing 1% of the carbohydrate at 28 C. for three weeks. The pH of the media employed in the study was approximately neutral (6.8-7.2). Table 2 shows the utilization of these carbohydrate sources by Streptomyces cattleya: +indicating good growth, +poor growth, and -no growth on the particular carbohydrate.

TABLE 2 Glucose + Maltose + Arabinose - Mannitol + Cellulose - Mannose + Fructose + Raffinose Inositol - Rhamnose Lactose - Sucrose + Xylose + The amount of growth with change in temperature, the oxygen requirement and the effect on nitrate by the microorganism is as follows: Temperature range (Yeast extract-glucose+salts agar); 28"C.--Good 370C.-Moderate 500C.-No growth Oxygen requirement (Stab culture in yeast extract-glucose+salt agar); Aerobic Nitrate reduction-Positive.

It is to be understood that for the production of the new antibiotic of this invention, the present invention is not limited to the organism Streptomyces cattleya or to organisms fully answering the above growth and microscopic characteristics which are given for illustrative purposes. In fact, it is desired and intended to include the use of mutants produced from the described organism by various means, such as X-radiation, ultraviolet radiation, nitrogen mustard and phage exposure.

The novel antibiotic of the invention, N-acetyl thienamycin, is produced during the aerobic fermentation of suitable aqueous nutrient media under controlled conditions by inoculation with the organism, Streptomyces cattleva'.

Aqueous media, such as those employed for the production of other antibiotics are suitable for producing N-acetyl thienamycin. Such media contain sources of carbon, nitrogen and inorganic salts assimilable by the microorganism.

In general, carbohydrates such as sugars, for example, glucose, fructose, maltose, sucrose, xylose and mannitol and starches such as grains, for example, oats, rye, cornstarch and corn meal can be used either along or in combination as sources of assimilable carbon in the nutrient medium. The exact quantity of the carbohydrate source or sources utilized in the medium depend in part upon the other ingredients of the medium but, in general, the amount of carbohydrate usually varies between 10/, and 6 S by weight of the medium. These carbon sources can be used individually, or several such carbon sources may be combined in the medium. In general, many proteinaceous materials may be used as nitrogen sources in the fermentation process.Suitable nitrogen sources include, for example, yeast hydrolysates, primary yeast, soybean meal, cottonseed flour, hydrolysates of casein, corn steep liquor, distiller's solubles or tomato paste. The sources of nitrogen, either alone or in combination, are used in amounts ranging from 0.2?o to 6% by weight of the aqueous medium.

Among the nutrient inorganic salts which can be incorporated in the culture media are the customary salts capable of yielding such ions as sodium, potassium, ammonium, calcium, phosphate, sulfate, chloride and carbonate. Also included are trace metals such as cobalt, manganese, iron and magnesium.

It should be noted that the media described in the Examples are merely illustrative.

The fermentation is carried out at temperatures ranging from 200 C. to 370C.; however, for optimum results it is preferable to conduct the fermentation at temperatures of from 22"C. to 30"C. The pH of the nutrient media suitable for growing the Streptomyces cattleya culture and producing N-acetyl thienamycin can vary from about 6.0 to 8.0.

Although the novel antibiotic N-acetyl thienamycin is produced by both surface and submerged cultures, it is preferred to carry out the fermentation in the submerged state.

A small-scale fermentation of the antibiotic is conveniently carried out by inoculating a suitable nutrient medium with the antibiotic-producing culture and, after transfer to a production medium, permitting the fermentation to proceed at a constant temperature of about 24"C. on a shaker for several days.

The fermentation is initiated in a sterilized flask of medium in one or more stages of seed development. The nutrient medium for the seed stage may be any suitable combination of carbon and nitrogen sources. The seed flask is shaken in a constant temperature chamber at about 28"C. for one dr two days, or until growth is satisfactory, and some of the resulting growth is used to inoculate either a second stage seed or the production medium. Intermediate stage seed flasks, when used, are developed in essentially the same manner; that is, part of the contents of the flask from the last seed stage are used to unoculate the production medium. The inoculated flasks are shaken at a constant temperature for several days, and at the end of the incubation period the contents of the flasks are centrifuged or filtered.

For large-scale work, it is preferable to conduct the fermentation in suitable tanks provided with an agitator and a means of aerating the fermentation medium.

According to this method, the nutrient medium is made up in the tank and sterilized by heating at temperatures of up to about 1200C. Upon cooling, the sterilized medium is inoculated with a previously grown seed of the producing culture, and the fermentation is permitted to proceed for a period of time as, for example, from 3 to 5 days while agitating and/or aerating the nutrient medium and maintaining the temperature at about 24"C. This method of producing N-acetyl thienamycin is particularly suited for the preparation of large quantities of the antibiotic.

Physical and Chemical Properties of N-Acetyl Thienamycin An NMR spectrum at 100 MHz of N-acetyl thienamycin revealed the following peaks: S1.27, d, 3 H, 56.5; 61.98, S, 3 H; 62.94 m, 2H; 63.17, m, 2 H; S3.38; t, 2 H.

J=6.5; 83.38, m, 1 H; S4.20, m, 2 H.

The NMR spectrum of a combined solution of N-acetyl thienamycin obtained by fermentation and obtained by acetylating thienamycin is indistinguishable from those of the individual solutions. From the Amax dependence on pH, a PKa of 3.3t0.1 for a COOH group in N-acetyl thienamycin has been determined.

Upon paper electrophoresis in 0.1M potassium phosphate buffer, pH 7.

employing Schleicher and Schuell No. 2043-B paper at a voltage gradient of 50 v/m., both N-acetyl thienamycin obtained by fermentation and obtained by acetylating thienamycin migrate 2.7 cm. toward the anode over a 20-minute period at 10 C. The antibiotic is localized by bioautography on Vibrio percolans, ATCC 8461 (without intervening drying of the paper), and migration is measured from the point of application to the center of the zone of inhibition.

Thin-layer chromatography using cellulose-coated sheets and an ethanol: H2O, 70:30 (v/v) solvent system reveals an Rf of 0.7 for N-acetyl thienamycin obtained by fermentation and acetylation, as detected by bioautography on Vibrio percolans, ATCC 8461. The Rf value refers to the distance from the origin to the center of bioactivity divided by the distance from the origin to the center of bioactivity divided by the distance from the origin to the solvent front.

The IR spectrum of N-acetyl thienamycin is set forth in Figure 1.

N-Acetyl thienamycin is one of the compounds having the planar molecular structure

Other stereoisomers are claimed the specification of our copending Application No. 48233/76. Serial No. 1,561,108.

N-Acetyl thienamycin is further characterized by the following antibiotic spectrum profile. The test employs the Bauer-Kirby disc diffusion method modified only in respect to the 2 mm. agar depth employed here. The results, expressed in terms of the diameter in millimeters of the zone of inhibition are as set forth in Table 3. Table 3 sets forth the antibiotic spectrum profile of N-acetyl thienamycin and that of the material obtained by acetylating thienamycin. TABLE 3 Antibiotic N-Acetyl Thienamycina N-Acetyl Thienamycinb Test Organism Merck No. ATCC No.Resistance* 9.85 g/disc** 10.35 g/disc** Staphylococcus aureus MB2985 - - 37 37 MB2314 - P 37 37 Bacillus subtilis MB 964 6633 - 43 44.5 Escherichia coli MB2884 - - 31.5 31.5 MB2964 - P,C 28.5 29.5 MB2482 - P 28 28 Klebsiella pneumoniae MB2921 - P 28 28.5 MB2922 - P 27 30 Enterobacter cloacae MB2646 - P,C 26.5 27 MB2647 - - 29 29 Proteus mirabilis MB2830 - P,C 25 26 Proeus morganii MB2833 - P,C 25.5 25 Serratia MB2840 - P,C 27 27 Pseudomonas aeruginosa MB2824 - P,C 23 24 MB2835 - P,C 11 (hazy) 11 (hazy) MB3286 - P,C 0 0 *P=Penicillins as represented by ampicillin C=Cephalosporins as represented by cephalothin **Weight calculations based on an assumed E110/0cm., 301 nm=290 and a hydroxylamine extinguishability of 96% for pure material.

aPrepared by fermenting Streptomyces cattleya.

bPrepared by acetylating theinamycin.

N-acetyl thienamycin exhibits in vivo activity against gram-negative and grampositive organisms and-hence is useful in controlling bacterial infections in humans and animais. In determining the in vivo activity, N-acetyl thienamycin was dissolved in and diluted with 0.01 M sodium phosphate, pH 7.0 to provide five fourfold concentrations of drug for testing. Female white Swiss mice, averaging about 21 g.

in weight, were infected intraperitoneally with the test organism suspended in broth. The numbers of organisms injected were determined by standard platecount techniques. At the time of infection, and again 6 hours later, certain of the mice were treated intraperitoneally with the antibiotic. Five mice were used for each concentration of drug tested. Controls of five mice for each of several dilutions of the infecting culture were included in each test in order to calculate the numbers of organisms that were lethal to 50% of the infected, untreated mice (LD50). This calculation was made using survival data of the seventh day after infection, at which time the amount of drug that should protect 50% of the infected mice (EDso) also was calculated.

All animals receiving this challenge and not treated with antibiotic died within 48 hours of the infection. The efficacy of N-acetyl thienamycin is recorded in Table 4: TABLE 4 Efficacy Studies (mice)8 Route of treatment, Organism No. LD50 doses ED50mg./kg./dosec Staphaureus 13 i.p.x2b 0.050 2949 "CD-I, female mice, body wt. 21 g.

bIndicates treatment at the time of infecting dose and again 6 hours later.

CWt. of N-acetyl thienamycin based on estimated E10/0Cm 301 nm=290 and hydroxylamine extinguishability of 96% for pure material.

N-Acetyl thienamycin is a valuable antibiotic active agent against various gram-positive and gram-negative bacteria and, accordingly, finds utility in human and veterinary medicine. The compound of this invention can be used as an antibacterial drug for treating infections caused by gram-positive or gram-negative bacteria, for example against Staphylococcus aureus, Proteus mirabilis, Escherichia coli. Klebsiella pneumoniae, and Enterobacter cloacae. The antibacterial material of the invention may further be utilized as an additive to animal feeding-stuffs, for preserving foodstuffs and as a disinfectant.For example, it may be employed in aqueous compositions in concentrations ranging from 0.1 to 100 parts or preferably in concentrations ranging from I to 10 parts of antibiotic per million parts of solution in order to destroy and inhibit the growth of harmful bacteria on medical and dental equipment and as a bactericide in industrial applications, for example in water-based paints and in the white water of paper mills to inhibit the growth of deleterious bacteria.

The antibiotic of this invention may be used in any one of a variety of pharmaceutical preparations as the sole active ingredient or in combination either with one or more other antibiotics or with one or more pharmacologically active substances. As an example of the former, an aminocyclitol antibiotic such as gentamicin may be coadministered in order to broaden the antimicrobial spectrum and to minimize any change that resistanc organisms will emerge. As an example of the latter, diphenoxylate and atropine may be combined in dosage forms intended for the therapy of gastroenteritis. The antibiotic may be employed in capsule form or as tablets, powders, or liquid solutions or as suspensions or elixirs. It may be administered orally, topically, intravenously or intramuscularly.

Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example, lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; lubricants, for example, magnesium stearate, talc, polyethylene glycol, silica; disintegrants, for example, potato starch or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in the art. Oral liquid preparations may be in the form of aqueous or oily suspension, solution, emulsions, syrups or elixirs or may be presented as a dry product, for reconstitution with water or other suitable vehicles before use.Such liquid preparations may contain conventional additives such as suspensing agents, for example, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate or acacia; nonaqueous vehicles which may include edible oils, for example, almond oil, fractionated coconut oil, oily esters, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoates or sorbic acid. Suppositories will contain conventional suppository bases, e,g., cocoa butter or other glyceride.

Compositions for injection may be presented in unit dose form in ampoules, or in multidose containers with an added preservative. The compositions may take such forms as suspensions, solutions, emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspensing, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.

The compositions may also be prepared in suitable forms for absorption through the mucous membranes of the nose and throat or bronchial tissues and may conveniently take the form of powder or liquid sprays or inhalants, lozenges or throat paints. For medication of the eyes or ears, the preparations may be presented as individual capsules, in liquid or semi-solid form, or may be used as drops. Topical applications may be formulated in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints or powders.

Also, in addition to a carrier, the compositions may include other ingredients such as stabilizers, binders, antioxidants, preservatives, lubricators, suspending agents, viscosity agents or flavoring agents.

In veterinary medicine, such as in the treatment of chickens, cows, sheep or pigs, the composition may, for example, be formulated as an intramammary preparation in either long acting or quick-release bases.

The dosage schedule and route administration depends to a large extent upon the condition of the subject being treated, the weight of the host, the susceptibility of the infecting organism and the state of infection; the parenteral route being preferred for systemic infections and the oral route for intestinal infections.

In the treatment of bacterial infections in man, the compound of this invention is administered orally or parenterally, in accordance with conventional procedures for antibiotic administration, in an amount of from 2 to 600 mg./kg./day and preferably 5 to 100 mg./kg./day in preferably divided dosage, e.g. three or four times a day. They may be administered in dosage units containing, for example, 25, 250, 400, 800 or 1000 mg. of active ingredient with suitable physiologically acceptable carriers or excipients. The dosage units are in the form of liquid preparations such as solutions or suspensions or as solids in tablets or capsules.It will, of course, be understood that the optimum dose in any given instance will depend upon the type and severity of infection to be treated, and that smaller doses will be employed for pediatric use, all of such adjustments being within the skill of the practitioner in the field.

Included in this invention are the non-toxic pharmaceutically acceptable salts of N-acetyl thienamycin, for example, the pharmacologically acceptable salts formed with inorganic and organic bases; which include, for example, metal salts derived from alkali metal or alkaline-earth metal hydroxides, carbonates or bicarbonates such as those derived from sodium, potassium, ammonium and calcium and salts derived from primary, secondary or tertiary amines such as monoalkylamines, dialkylamines, trialkylamines, lower alkanolamines, di-(lower alkanol)amines, lower alkylenediamines, N,N-diaralkyl lower alkylenediamines, aralkylamines, amino-substituted lower alkanols, N,N-di-(lower alkyl)amino substituted lower alkanols, amino-, polyamino- and guanidino-substituted lower alkanolic acids and nitrogen-containing heterocyclic amines, where "lower" means "containing up to six carbon atoms." Representative examples include salts derived from sodium hydroxide, ammonium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium hydroxide, calcium carbonate, trimethylamine, triethylamine, piperidine, N-ethylpiperidine, morpholine, quinine, Iysine, protamine, arginine, procaine, ethanolamine, morphine, benzylamine, ethylenediamine, N,N'-dibenzylethylenediamine, diethanolamine, piperazine, dimethylaminoethanol, 2-amino-2-methyl- 1-propanol, theophylline and N-methylglucamine.

The salts of the compound of the present invention may be prepared by conventional methods. For example, the mono-salts such as monosodium salt obtained by treating one equivalent of sodium hydroxide with one equivalent of the product (I) in a suitable solvent. Also mixed salts with divalent cations may be prepared by combining one mole of a divalent base with one mole of the product (I) plus one equivalent of another acid. Alternatively, salts may be obtained by treating one equivalent of a base having a divalent cation, such as calcium hydroxide, with one equivalent of the product (I). The salts of this invention are pharmacologically acceptable non-toxic derivatives which can be used as the active ingredient in suitable unit-dosage pharmaceutical forms. Also, they may be combined with other drugs to provide compositions having a broad spectrum of activity.

The antibiotic containing fermentation broths produced in accordance with the procedures described herein have activities ranging from 0.1 to 4 pg. per ml.

Antibiotic preparations can be purified and the antibiotic recovered by a number of procedures. One such procedure comprises passing filtered broth containing Nacetyl thienamycin through a column of strong cation exchange resin. Illustrative of such resins are those of the sulfonate type having a styrenedivinylbenzene matrix, for example the polystyrene nuclear sulfonic acid resin Dowex 50x2 (manufactured by Dow Chemical Co., Midland, Michigan), on the sodium cycle.

Other representative members of the class of strong cation exchange resins include the following: Dowex 50x4, Dowex 50x8 (manufactured by Dow Chemical Co., Midland, Michigan), Amberlite IR120 (manufactured by Rohm & Haas Co., Philadelphia, Pennsylvania), Duolite C25D (manufactured by Chemical Process Co., Redwood City, California), Permutit Q (manufactured by Permutit Co., Birmingham, New Jersey), Ionac C-249 (manufactured by Ionac Chemical Co., Birmingham, New Jersey) and Amberlite 200. The words 'Dowex', 'Amberlite', 'Duolite', 'Permutit', and 'Ionac' are trade marks.

The spent material from the cation-exchange resin containing the antibiotic Nacetyl thienamycin can be further purified, if desired, by other purification procedures. It may be noted that thienamycin itself remains adsorbed on the strong cation-exchange resin. Accordingly, the procedure for separating these two compounds is clearly distinguishable.

One such procedure comprises adsorbing N-acetyl thienamycin on a strongly basic anion exchange resin. Illustrative of such strongly basic anion exchange resins are those having a styrene-divinylbenzene matrix, for example the polystyrene nuclear quaternary ammonium resin Dowex 1 x2, (manufactured bv Dow Chemical Co., Midland, Michigan), on the chloride cycle. Other representative members of this class of strongly basic exchange resins include the following: Duolite A-40, A- 42, A-101, A-102 and A-114 (manufactured by Chemical Process Co., Redwood City, California); Amberlite IRA-400, IRA-401 and IRA-410 (manufactured by Rohm and Haas, Washington Square, Philadelphia 5, Pennsylvania).The antibiotic N-acetyl thienamycin contained in the eluate can be further purified by passing it through a column packed with an acrylic ester polymer of intermediate polarity such as XAD-7 or 8 or through a non-polar hydrophobic polystyrene polymer, crosslinked by divinyl benzene, such as XAD-I, 2 and 4, preferably XAD-2. (XAD1, 2, 4, 7 and 8 are manufactured by Rohm and Haas, Washington Square, Philadelphia, 5, Pennsylvania).

A method of obtaining further purified N-acetyl thienamycin is by the use of gel filtration through polyacrylamide gel having a pore size which excludes molecules having a molecular weight greater than 1800 such as Bio-Gel P-2 (manufactured by Bio Rad, Richmond, California).

A preferred method for recovering purified N-acetyl thienamycin is to pass a solution of the antibiotic, such as the filtered fermenation broth, the pH of which has been adjusted between 4 to 5 through a column containing a strong cation exchange resin of the sulfonate type in the sodium cycle (Dowex 50x4). The collected spent may be further purified by a sequence of processes involving the following chromatographic media: anion exchange resins of the polystyrenetrimethylammonium type (e.g. Dowex 1 x2 in the chloride cycle), polymeric absorbents (e.g., XAD-2, a polystyrene resin), Dowex 1 x4 in the chloride cycle and gel permeation resins (e.g., Bio-Gel P-2, a polyacrylamide resin). The bioactivity of the eluates is measured by assaying the eluate using Staphylococcus aureus ATCC 6538P as the assay organism or as purity permits, by hydroxylamine-extinguished absorbance.A preferred method of obtaining N-acetyl thienamycin from fermentation broth is set forth in Table 5.

N-acetyl thienamycin, the antibiotic of the present invention, may also be prepared by acetylation of thienamycin. Acetylation is carried out by acetyl halide or preferably by acetic anydride. Thienamycin is dissolved in a suitable inert solvent such as DMF and treated with an excess of acetylating agent at a temperature from -l00C. to 250C. but preferably at about 00C. The reaction is complete in about 1 minute to about 1 hour. A reaction time of about 10 minutes at 0 C. is usually sufficient. The crude N-acetyl thienamycin obtained by acetylating thienamycin can be purified by the process described above for purifying filtered fermentation broths containing N-acetyl thienamycin.

A preferred method of purifying N-acetyl thienamycin obtained by acetylation is to adsorb the crude material on an anion exchange resin such as Dowex 1 x4 in the chloride cycle and elute with an aqueous salt solution. A suitable aqueous salt solution contains sodium chloride, ammonium chloride and ammonia preferably in the concentration of about 0.07 M, 0.005 M and 0.0001 M, respectively. The fractions containing the product are combined and concentrated. The concentrate may be further purified by gel filtration through a polyacrylamide gel. A preferred gel is Bio-Gel P-2 eluted with deionized water.

The Examples, which follow and in which the words "Difco", "Nujol", "Millipore", and "Selas" are trade marks, and mesh and capsule sizes are U.S.

Standards, illustrate the methods by which the products of this invention may be obtained.

TABLE 5 OUTLINE OF PURIFICATION PROCESS FOR ANTIBIOTIC N-ACETYL THIENAMYCIN

a ca, I" I 4: rlt e o am InL, h 9 < o n Q N mlnn E & S o W e c < n X as M - ~ H A0 3t ot ztt ur E , W fi Q trt o Fermented O P Broth of (1) | Filtered (1) applied on ;; 6 o add Broth Dowex-SO x ~ O s 1 , s Z! O ~ i CD a c In rrr EDTA mesh) o I hl N 00 LI C r chill tu 60C.

3t adjust N to 4.5 (1) 3t of spent v 1 G Um mE adjusLed to nd xw (r z e, vv v r3 3 R r( ,(2) adsorb to xAD-2 (salt-washed) (50-100 mesh) (2) adsorb on x 2 -3) (3) elute with 1120 Eluate (3) wash in t s3 ( D ~I JJ tJ : X elute with t3 NaCl, 25uM EDTA, l0m1 buffr, k 7 I adsorb to Dowex-l U U cf (minus x mesh) 3 vo :: o ~ X s3 ~ cs X o elute with 0.07M NaCI 3 0. 005M NH4Cl 7 > a Sa e m -4 tn < . > t en < ~ to ~* ~ ~ 4 3J concentrate Bio-Gel --- - \ v Hn , CFt F \ Eluate (2) adsorb to Bio-Cel W (1800 Daltons) Eluate r3 adsorb to XAD-2 < o u rrt ~ O o: lK (3) elute with .1: X tat E v I o a hlp IT LIZED I" sw m (1) | Second TUIENn'CIN o (2) lyophilize XAD-2 Eluate et xt r d' < ~ W srt 3s Z = = z tlt ttM < u-l N tlt ~{ SX 3 E O v < s ~ ~ { O 19 (tt N EJ tt v H . i t i + Oe C ~i ~ F z tJ to < vi 3j 1 C O thi r c O H Q tN Z h zi .

3iX z eD < N ,.j ,C X i Q h rlJ ~ ~ i3 0 > 3J t-i Li i=l h ai ,^j 3 . c tn : ~1 &verbar; 3 o O o X ~i \ , X t D a tJ i3 3J , e 3i qw w 3) i- 3J C t Li O h. E t-r 4 ^^ a 2; ' C Xi t3 C n D < s < n ai s rai cl o r i 3 ~~ ~ ri4 r E ;i (J 3Ji t~i t3 trs D i-l; rs si O 'i h 3J * 3i h I C n o . j ii H 2 i'~ iS Z Eti ~I tNi tej : ~ J] z . ~s v iN r > i tNi X ; Z H 7 ilJ 1 3J t.> l JJ X JJ I i3 Q t3 X t; > a iX Assay Procedures for Antibiotic N-Acetyl Thienamycin I. Bioassay Assays of antibacterial activity are run according to the following disc-diffusion method using either yibrio percolans ATCC 8461 or StaphlXlococczxs aureus ATCC 6538P as tester organism.

Plates containing Vibrio percolans ATCC 8461 are prepared as follows: A lyophilized culture of Vibrio percolans ATCC 8461 is suspended in 15 ml. of a sterilized medium containing 8 g./1. of Difco Nutrient Broth and 2 g./l. of yeast extract in distilled water (hereinafter designated NBYE). The culture is incubated overnight on a rotary shaker at 280C. This culture is used to inoculate the surface of slants containing 1.5so agar in NBYE, and the inoculated slants are incubated overnight at 280C. and then stored in a refrigerator.

The refrigerated slants prepared from a single lyophilized culture are used for up to four weeks from their preparation, as follows: A loop of inoculum from the slant is dispersed in 50 ml. of NBYE contained in a 250 ml. Erlenmeyer flask. The culture is incubated overnight on a rotary shaker at 280C., and is then diluted to a density giving 50% transmittance at 660 nm. 33.2 ml. of this diluted culture is added to I liter of NBYE containing 15 g. of agar and maintained at 46"C. the inoculated agar-containing medium is poured into 100x15 mm. plastic petri dishes, 5 ml. per dish, chilled, and maintained at 2--4"C for up to 5 days before using.

Plates containing Staphylococcus aureus ATCC 6538P are prepared as follows: An overnight growth of the assay organism, Staphylocccus aureus ATCC 6538P, in nutrient broth plus 0.2% yeast extract is diluted with nutrient broth plus 0.20 yeast extract to a suspension having 55% transmittance at a wavelength of 660 nm.

This suspension is added to Difco nutrient agar supplemented with 2.0 g./l. Difco yeast extract at 470 C. to 48"C., to make a composition containing 33.2 ml. of the suspension per liter of agar. Five ml. of this suspension is poured into petri dishes of 85 mm. diameter, and these plates are chilled and held at 40C. until used (5 day maximum).

Samples of antibiotic to be assayed are diluted to an appropriate concentration in phosphate buffer at pH 7. Filter paper discs, 1/4 or 1/2 inch diameter, are dipped into the test solution and placed on the surface of the assay plate. The plates are incubated at 370 C. overnight, and the zone of inhibition is measured as mm.

diameter. The zone of inhibition measured in mm. determines relative potencies.

II. Hydroxylamine-extinguishable Absorbance The proportion of absorbance measured at 301 nm which can be attributed to the antibiotic content in impure samples is determined by the selective extinction of this absorbance (with concommitant inactivation of antibiotic activity) upon reaction with dilute hydroxylamine.

Samples containing antibiotic to be tested are prepared in 0.01 M potassium phosphate buffer at pH 7 to have an initial A30, between 0.1 and 1.0. Freshly prepared, neutralized hydroxylamine (NH2OH HCl plus NaOH to a final pH of 7) is added to a final concentration of 10 mM, and the reaction is allowed to progress at room temperature for at least 30 minutes. The resulting A301 when subtracted from the initial reading (after correction for dilution by added reagent) yields the hydroxylamine-extinguishable absorbance. Solutions of pure N-acetyl thienamycin show a hydroxylamine-extinguishable absorbance of 96.0%.

EXAMPLE 1 A tube of lyophilized culture of Streptomyces cattleya NRRL 8057 is opened aseptically and the contents suspended in a tube containing 0.7 ml. of sterile Davis salts having the following composition: Davis Salts Sodium citrate 0.5 g.

K2HPO4 7.0 g.

KH2PO4 3.0 g.

(NH4)2SO4 1.0 g.

MgSO4-7H2O 0.1 g.

Distilled H2O 1000 ml.

0.2 ml. of this suspension is used to inoculate a culture slant of Medium A (plus agar), which has the following composition: Medium A Yeast Autolysate (ardamine*) 10.0 g.

Glucose 10.0 g.

+Phosphate Buffer 2.0 ml.

MgSO4 7H2O 0.05 g.

Distilled H2O pH: adjust to 6.5 using NaOH 1000 ml.

*Ardamine: Yeast Products Corporation +Phosphate Buffer Solution KH2PO4 91.0 g.

Na2HPO4 95.0 g.

Distilled H2O 1000 ml.

For Slants: add agar-25.0 g.l.

The inoculated slant is incubated for 8 days at 280C. and then stored at 4"C.

A portion of the spores and aerial mycelia of this slant is used to inoculate a baffled 250-ml. Erlenmeyer seed flask containing 50 ml. of Medium A (without agar). This seed flask is shaken at 28"C. on a 220 rpm shaker (2" throw) for two days at which time the growth is satisfactory.

Fifteen 250 ml. Erlenmeyer flasks, each containing 40 ml. of Medium B, are inoculated with 1 ml. per flask of the growth from the seed flask. The Medium B has the following composition: Medium B Corn Meal 20.0 g.

Distiller's Solubles 10.0 g.

Soybean Meal 15.0 g.

Sodium Citrate 4.0 g.

CaCl2 # 2H2O 0.5 g.

MgSO4. 7H2O 0.1 g.

CoCI2. 6H20 0.01 g.

FeSO4. 71120 0.01 g.

**Polyglycol 2000 0.25% by Vol.

Distilled H2O 1000 ml.

pH: adjust to 6.5 using NaOH **Polyglycol 2000: Dow Chemical Co.

These 15 production flasks are shaken at 28"C. on a 220 rpm shaker (2" throw) for 53 hours. At harvest (53 hours age) the broth from the 15 flasks is pooled and a portion is centrifuged for assay. Prior to assay the pH of the centrifuged broth is adjusted to 6.5 from 5.9 with NaOH.

Assays are run on Staphylococcus aureus ATCC 6538P and Vibrio percolans ATCC 8461 assay plates using 1/2" discs dipped in the supernatant of centrifuged broth.

Assay results are as follows: ATCC 6538P Activity ATCC 8461 Activity (mm. zone) (mm zone) 39/44 SH 35/44 SH SH=slightly hazy 200 ml. of filtered broth is adjusted to pH 8.0 and adsorbed on 10 ml. of Dowex I x2 resin in the chloride cycle at 2 ml./min., collecting the spent effluent stream in 10x20 ml. fractions.

The adsorbate is eluted with 90% methanol: 10% water; 3% ammoniumchloride v/v/w, collecting the eluate in 10x5 ml. fractions. Eluate fractions I to 6 are combined and concentrated in vacuo to remove the methanol. The concentrate is assayed by the disc-diffusion procedure using a 1/2-inch diameter disc containing 100 pl. antibiotic solution against Staphvlococcus aureus MB-2985 and gives a zone size of 28 mm.

Assay plates of MB-2985 are prepared as follows: An overnight culture of MB-2985 grown in brain heart infusion medium at 37"C. with shaking, is diluted 20,000x and swabbed onto the surface of 10 ml.

brain-heart infusion agar contained in an 85 mm. diameter petri dish. Discs of 1/2 inch diameter containing 100 pal. antibiotic solution are placed on the plates which are then incubated 18 hours at 370C. Zones of inhibition are read in mm.

EXAMPLE 2 A tube of lyophilized culture of Streptomyces cattleya NRRL 8057 is opened aseptically and the contents is used to inoculate a baffled 250 ml. Erlenmeyer seed flask containing 50 ml. of Medium A (without agar), which has the composition set forth in Example 1.

*Ardamine: Yeast Products Corporation +Phosphate Buffer Solution KH2PO4 91.0 g.

Na2HPO4 95.0 g.

Distilled H2O 1000 ml.

This seed flask is shaken at 280C. on a 220 rpm shaker (2" throw) for two days at which time the growth is satisfactory.

Fifteen 250 ml. Erlenmeyer flasks, each containing 40 ml. of Medium B, are inoculated with 1 ml. per flask of the growth from the seed flask. The Medium B has the composition set forth in Example 1.

These flasks are shaken at 280C. on a 220 rpm shaker (2" throw) for three days with assays performed during the fermentation cycle. Assays are run on standard Staphylococcus aureus ATCC 6538P and Vibrio percolans ATCC 8461 assay plates using 1/2" discs dipped in the supernatant of centrifuged broth. The pH of this broth is adjusted prior to assay as shown in the following table. The results are as follows: Age (Hours) 48 53 72 ATCC 6538P Activity 33/39h 31/38h 21/27h ATCC 8461 Activity 35sh/46h 36sh/44h 33sh/38h pH, initial 5.2 5.1 4.8 pH, adjusted 6.1 6.2 6.9 sh=slightly hazy h=hazy At 53 hours age, the broths are pooled and filtered to yield 590 ml. of filtrate at pH 5.9. The pH of the filtrate is adjusted to pH 7.0 and 5.9 mg. of ethylenediamine tetraacetic acid (EDTA) is added.

A 580 ml. portion of the above filtrate, at pH 7.0, is adsorbed on 130 ml. of Dowex 1x2 resin in the chloride cycle collecting the spent stream in 2x290 ml.

fractions. The adsorbate is then washed with 130 ml. of deionized water. The washed adsorbate is stored in the coldroom overnight and then eluted with 5% NaCI solution collecting 6x50 ml. fractions.

The percent recovery of initial bioactivity, determined by the disc plate method, is tabulated below: Vibrio percolans Staph. aureus Fraction ATCC 8461 ATCC 6538P Eluate Fractions 1 two 5 26yen 1% Antibiotic N-acetyl thienamycin is found in fractions 1 to 5 of the NaCI eluate.

Fraction 4 is assayed by the disc-diffusion procedure using 1/2 inch diameter discs containing 100 pl. antibiotic solution against Staphylococcus aureus MB-2985 and gives a zone size of 29 mm. Plates containing MB-2985 are prepared according to the process set forth in Example 1.

EXAMPLE 3 A tube of lyophilized culture of Streptomyces cattleva NRRL 8057 is opened aseptically and the contents suspended in 0.8 ml. of sterile Davis salts having the composition defined in Example 1.

This suspension is used to inoculate four slants of Medium A (plus agar) which has the composition set forth in Example 1.

The inoculated slants are incubated for one week at 280 C. and then stored at 4 C.

Ten ml. of Medium A (without agar) is transferred aseptically to one of these slants, the spores and aerial mycelia scraped into suspension, and 1.2 ml. of this suspension used to inoculate three niter baffled Erlenmeyer flasks containing 500 ml. of Medium A (without agar). These seed flasks are shaken at 280C. on a 160 rpm shaker for 24 hours, at which time the growth is satisfactory.

The growth from these seed flasks is pooled and used to inoculate a 756 liter stainless steel fermentor containing 467 liters of Medium A (without agar). This tank is operated at 280C. using an agitation rate of 130 rpm and an airflow of 10 cu.

ft. per minute for 24 hours. Defoamer, Polyglycol 2000 (Dow Chemical Corp.), is used as required but does not exceed 0.1%. pH determinations are made as follows: Age, Hours 0 24 pH 6.3 6.4 454 liters of the growth in this seed tank is to incoulate a 5,670 liter stainless steel fermentor containing 4,082 liters of Medium E, which has the composition: Medium E Cerelose 25.0 g.

Corn Steep Liquor (wet basis) 15.0 g.

Distiller's Solubles 10.0 g.

Cottonseed Media (Pharmamedia) 5.0 g.

CoCI2 6H20 0.01 g.

CaCO3 (after pH adjustment) 3.0 g.

Polyglycol 2000 0.25% Tap water 1000 ml.

pH: adjust to 7.3 using NaOH This tank is run at 24 C. using an agitation rate of 70 rpm and an airflow of 54.3 cu.

ft. per minute for 138 hours. Additional defoamer, Polyglycol 2000, is added as required, but does not exceed 0.1% by weight. Antibacterial assays are run and the data are as follows: ATCC No. 6633 Age pH (3/8" disc.) (mm.) 0 6.9 0 24 6.3 0 36 6.0 0 48 5.9 0 60 6.0 23 72 5.9 84 6.0 21 96 6.2 108 6.5 35 120 6.6 36 132 6.7 41 138 6.7 39 The 4082 liters of fermentation broth is filtered using a 30 inch filter press and a filter aid admix to the extent of 4% w/v. 46 g. of ethylenediamine tetraacetic acid (EDTA) sodium salt is added to the filtrate. The filtrate is cooled to 60 C., adjusted to pH 4.5+0.2 and maintained at 6"C. The cold filtrate is applied to a 480-liter column of Dowex 50x4 Na+, 2050 mesh at about 48 I./minute. After a 1400 liter forerun has passed through, 18.9 liters of spent material is collected and its pH is adjusted to 7.08 with NaOH. It is stored at 5"C.

A 3.8 cm. diameter column packed with 300 ml. of Dowex 1 x2, 50100 mesh, resin in the chloride cycle is prepared and washed with 600 ml. of deionized water at 5"C. Four liters of the cold Dowex 50x4 spent material is passed through the column at the rate of 30 ml./minute. The column is washed with 300 ml. 25 pM EDTA. The antibiotic, N-acetyl thienamycin, is eluted at 50C. at the rate of 15 ml/minute with 900 ml. 5% NaCI solution containing 0.01 M potassium phosphate buffer, pH 7.0, and 25 pM EDTA. Fourteen fractions of 75 ml. are collected and assayed for biological activity by the disc-diffusion procedure. Eluate fractions 3 to 9, comprising 525 ml. are pooled and concentrated under vacuum to 115 ml.The concentrate contains 65 /n of the total bioactive material applied on the Dowex 1 x2 Cl column.

The Dowex 1x2 Cl- eluate concentrate is applied to 5"C. on a 3.8 cm.

diameter column packed with 450 ml. prewashed XAD-2. The XAD-2 is prewashed column wise successively with 4 column volumes: 1) 0.001 M EDTA, 2) NNaOH, 3) deionized H2O, 4)1 N HCI, 5) deionized H2O, 6) methanol, 7) acetone, 8) deionized water and, prior to use, with 2250 ml. 5% NaCI solution containing 25 pM EDTA.

After the sample has been applied to the column, it is followed by two 25 ml.

portions of 1120. The column is developed at 5"C. with deionized H2O at a flow rate of 10 ml./minute. The first fraction contains 400 ml. and 11 additional fractions of 75 ml. are collected. The pH of each fraction is adjusted to between 6.9 and 7.13 with 1 N NaOH or 1 N HCI. Fractions 3 to 9, containing 46% of the total bioactive material applied to the XAD-2 column, are combined and have a total volume of 490 ml. 45 ml. is removed for bioassays, by the standard disc-diffusion procedure against Staphylococcus aureus ATCC 6538P. The remaining 445 ml. is concentrated under vacuum to 50 ml.

The 50 ml. XAD-2 eluate concentrate is pumped at 50C. onto a 1.5 cm.

column packed with 40 ml. prewashed Dowex 1 x4 Cl-, minus 400, at 50C. and at a rate of 1 ml./min. The Dowex I x4 Cl-, minus 400 (defined by decanting from water) resin is washed column-wise prior to use with 240 ml. 0.2 M NaC1 containing 0.005 M NH4Cl and 0.1 mM NH40H at the rate of 1 ml./minute and then with 120 ml.

deionized water at the same rate.

After the sample has been applied to the column, it is followed by two 5 ml.

portions of deionized water. The column is developed at 50C. at the rate of 0.92 ml./minute with 0.07 M NaCI containing 0.005 M NH4Cl and 0.0001 M NH4OH.

Fractions of 8.6 to 9.3 ml. are collected. Fractions obtained after 600 ml. of eluate have been collected and ending with 710 ml. are pooled and contain 98% of the total bioactive material applied on the Dowex 1x4 column. This pool is concentrated under vacuum to 2 ml.

The Dowex 1 x4 concentrate is applied to a 2.2 cm. diameter column packed with 225 ml. Bio-Gel P-2, 200400 mesh, with an exclusion limit of 1800 Daltons (defined prior to use by decantation form distilled water).

The Bio-Gel P-2 column is washed prior to use with 225 ml,. 1 M NaCI followed by 100 ml. deionized water. The column is developed with 50C. deionized water at the rate of 1 ml./minute, and two ml. fractions are collected. Fractions from 104 ml.

to 128 ml. eluate, containing 83% of the bioactivity applied to the Bio-Gel P-2 column, are combined and concentrated to 1.58 ml.

A 50 ml. XAD-2 column (1.6 cm.x27 cm.) is prepared and prewashed columnwise with 200 ml. of 0.001 M EDTA, 1 N NaOH, deionized water, 1 N HCI, deionized water, methanol, acetone, and deionized water. The Bio-Gel P-2 concentrate containing 44.4 hydroxylamine-extinguishable optical density units is applied to the XAD-2 column at 5"C. and is followed by two 2 ml. portions of deionized water. The column is washed with deionized water at the rate of I ml./minute until the UV absorbance at 300 nm of the washings is reduced to 0.060.

The column is eluted with 50% methanol in deionized water at the rate of 1 ml./minute and 1 ml. fractions are collected. Fractions having absorbance at 300 nm over 0.1 are combined and concentrated under vacuum to give the product, Nacetyl thienamycin, containing 11.6 hydroxylamine-extinguishable O.D. units.

EXAMPLE 4 A tube of lyophilized culture of Streptomyces cattleya NRRL 8057 is opened aseptically and the contents suspended in 0.8 ml. of sterile Davis salts having the composition set forth in Example 1.

This suspension is used to inoculate four slants of Medium A (plus agar), which has the composition set forth in Example 1.

The inoculated slants are incubated for one week at 280 C. and then stored at 4"C.

Ten ml. of Medium A is transferred aseptically to one of these slants, the spores and aerial mycelia scraped into suspension, and 1.2 ml. of this suspension used to inoculate three 2 liter baffled Erlenmeyer flasks containing 500 ml. of Medium A (without agar). These seed flasks are shaken at 280C. on a 160 rpm shaker for 24 hours at which time the growth is satisfactory.

The growth from these seed flasks is pooled and used to inoculate a 756 liter stainless steel fermentor containing 467 liters of Medium A (without agar). This tank is operated at 280C. using an agitation rate of 130 rpm and an airflow of 10 cu.

ft. per minute for 24 hours. Defoamer, Polyglycol 2000 (Dow Chemical Corp.), is used as required but does not exceed 0.1%. pH determinations are made as follows: Age, Hours 0 24 pH 6.3 6.4 454 liters of the growth in this seed tank is used to inoculate a 5,670 liter stainless steel fermentor containing 4,082 liters of Medium E, which has the composition defined in Example 3.

This tank is run at 240 C. using an agitation rate of 70 rpm and an airflow of 54.3 cu. ft. per minute for 138 hours. Additional defoamer, Polyglycol 2000, is added as required, but does not exceed 0.10/,. Antibacterial assays are run and the data are as follows: ATCC No.6633 Age pH (3/8" disc.) (mm.) 0 6.9 0 24 6.3 0 36 6.0 0 48 5.9 0 60 6.0 23 72 5.9 84 6.0 21 96 6.2 108 6.5 35 120 6.6 36 132 6.7 41 138 6.7 39 The 4082 liters of fermentation broth is filtered using a 30-inch filter press and a filter aid admix to the extent of 4% w/v. 46 g. of ethylenediamine tetraacetic acid (EDTA) sodium salt is added to the filtrate. The filtrate is cooled to 6"C., adjusted to pH 4.5+0.2 and- maintained at 60C.The cold filtrate is applied to a 480 liter column of Dowex 50x4 Na+, 2050 mesh at about 48 I./minute.After a 1400-liter forerun has passed through, 18.9 liters of spent material is collected, the pH of the spent material is adjusted to 7.08 with NaOH, and it is stored at 50C.

A 3.8 cm. diameter column packed with 300 ml. of Dowex 1 x2 50100 mesh resin in the chloride cycle is prepared and washed with 300 ml. of deionized water at 5"C. Four liters of the cold Dowex 50x4 spent is passed through the column at the rate of 30 ml./minute. The column is washed with 350 ml. 25 pM EDTA and then eluted at 50C. with 900 ml. of 5% NaCI solution containing 0.01 M Tris HCI, pH 7, and 25 pM EDTA at the rate of 15 ml./minute. Fractions of 75 ml. are collected and assayed by the disc-diffusion procedure against Staphylococcus aureus ATCC 6538P. Fractions 4 to 10 containing 47% of the bioactivity applied are added to 42.5 ml. of the sample removed for bioassay from the first XAD-2 pool described in Example 3.These combined fractions are concentrated under vacuum to 100 ml., and the pH is adjusted to 6.32 with HCI.

A 3.8 cm. diameter column packed with 450 ml. XAD-2 resin is prewashed column-wise successively with 4 column volumes: 1) 0.001 M EDTA, 2)1 NNaOH, 3) deionized H20, 4)1 N HCI, 5) deionized H20, 6) methanol, 7) acetone, and 8) deionized water, and washed prior to use with 2250 ml. 5% NaCI solution containing 25,up EDTA. The above concentrate is applied to the XAD-2 column and is followed by two 5 ml. portions of deionized water. The column is developed at 5"C. at the rate of 10 ml./minute with deionized water. The first fraction contains 40 ml., and subsequent fractions of 75 ml. are collected and assayed by the discdiffusion process. Fractions 9 to 15, which contain 22% of the bioactivity applied to the XAD-2 column are pooled and concentrated under vacuum to 56 ml.

A 21 cmx 1.7 cm. column packed with 40 ml. of Dowex 1x4 Cl-, minus 400 mesh, (defined by decanting from water) is washed column-wise prior to use with 240 ml. 0.2 M NaCI containing 0.005 M NH4CI and 0.0001 M NH40H at the rate of I ml./minute and then with 120 ml. deionized water at the same rate.

The XAD-2 concentrate is applied on the column and is followed by two 2 ml.

portions of deionized water and then by two 2 ml portions of eluting buffer. The column is eluted at 50C. with a solution of 0.07 M NaCI containing 0.005 M NH4Cl and 0.0001 M NH40H at the rate of 1 ml./minute. Fractions of 10 ml. are collected and assayed by the disc diffusion method. Eluate fractions from 544 ml. to 647 ml.

containing an apparent 100% of the applied bioactivity are combined and concentrated under vacuum to 2.3 ml. The concentrate contains 36.4 hydroxylamine-extinguishable optical density units.

A 2.2 cm.x62 cm. column packed with 225 ml. Bio-Gel P-2 200--400 mesh resin with an exclusion limit of 1800 Daltons is washed prior to use with 225 ml. 1 M NaCI followed by 100 ml. deionized water. The Dowex 1 x4 concentrate is applied to the column and is followed by two 2 ml. portions of deionized water. The column is developed at 50C. with deionized water at the rate of 1 ml./minute and 2 ml.

fractions are collected and assayed by the disc-diffusion procedure. Fractions from 124 ml. to 129 ml., which contain 7.04 hydroxylamine-extinguishable O.D. units, are combined and concentrated under vacuum to 2 ml. to give an aqueous solution of the product, N-acetyl thienamycin. Fractions from 117 to 123 ml. and 130 to 139 ml. are combined to give a solution of N-acetyl thienamycin containing 13.7 hydroxylamine-extinguishable O.D. units.

EXAMPLE 5 Preparation of Thienamycin A tube of lyophilized culture of Streptomyces cattleya NRRL 8057 is opened aseptically and the contents suspended in 50 ml. of sterile Medium A contained in a 250 ml. baffled Erlenmeyer flask. Medium A has the composition set forth in Example I, without the agar.

The inoculated flask is shaken at 280C. on a 220 rpm shaker (2" throw) for 48 hours. 40 ml. of the 48 hour broth is removed aseptically and mixed with 40 ml. of sterile 20% (v/v) aqueous glycerol. Two ml. quantities of the resulting mixture are pipetted into sterile I dram vials, which are then frozen and stored in the vapor phase of a liquid nitrogen freezer.

Frozen vial contents are used to inoculate a 250 ml. baffled Erlenmeyer flask containing 50 ml. of Medium A. This seed flask is shaken at 280C. on a 160 rpm shaker at 28"C. for 24 hours.

Ten ml. portions from this seed flask are used to inoculate a 2 liter baffled Erlenmeyer flasks containing 500 ml. of Medium A. These seed flasks are shaken on a 160 rpm shaker at 280C. for 24 hours.

A 1000 ml. portion of the pooled contents of these seed flasks is used to inoculate a 756 liter stainless steel fermentor containing 467 liters of Medium A.

This tank is operated at 280C. using an agitation rate of 130 rpm and an airflow of 10 cu. ft. per minute for 24 hours. Polyglycol 2000 (Dow Chemical Corp.) is used as required as a defoamer but not to exceed 0.1%. Measurements of pH and dextrose are made and are as follows: Age (Hours) 0 12 24 pH 6.4 6.4 6.6 Dextrosemg./ml. 8.1 8.1 8.1 453 liters of this growth is used to inoculate a 5670 liter stainless steel fermentor containing 4082 liters of Medium E, which has the composition set forth in Example 3.

This tank is operated at 24"C. using an agitation rate of 70 rpm and an airflow of 54.3 cu. ft. per minute for 144 hours. Defoamer, Polyglycol 2000, is added as required but does not exceed 0.1 /O. Centrifuged broth is assayed against Staphylococcus aureus ATTC 6538P by the standard disc-diffusion procedure. The results are tabulated in the table below under the heading "Antibiotic Activity vs ATCC 6538P". Assays are also run by the disc-diffusion procedure using 3/8 inch filter-paper discs and 10 ml. assay plates and the results tabulated in the table below under the heading "Antibiotic Activity (10 ml. plates)".

The 10 ml. assay plates are prepared as follows: An overnight growth of the assay organism, Staphyloccous aureus ATCC 6538P, in nutrient broth plus 0.2% yeast extract is diluted with nutrient broth plus 0.2% yeast extract to a suspension having 40% transmittance at a wavelength of 660 mp. This suspension is added to Difco nutrient agar supplemented with 2.0 g./l. Difco yeast extract, at 470 C. to 48"C., to make a composition containing 33.2 ml. of the suspension per liter of agar. Ten ml. of this suspension is poured into petri plates of 85 mm. diameter, and the plates are chilled and held at 4"C. until used (5 day maximum).

Antibiotic Antibiotic Activity vs Activity ATCC 6538P (10 ml. plates) Age pH Dextrose mg./ml. (mm.) (mm.) 0 6.6 22.2 12 6.3 20.2 24 5.8 18.0 0 36 6.0 13.2 21.5 48 6.0 8.6 21.5 60 5.7 6.4 26.5 72 5.8 2.7 25.5 84 6.2 0.3 27.5 96 6.4 0.2 36.0 108 6.4 0 35.0 120 6.3 41.5 37.0 132 5.8 37.5 144 5.9 43.0 37.5 The 4,082 liters of fermentation broth is filtered using a 30 inch filter press and a filter aid admix to the extent of 4% w/v. 12 g. of ethylenediamine tetraacetic acid sodium salt is added to the filtrate. The filtrate is cooled to 6"C., adjusted to pH 4.5+0.2 and maintained at 6"C. The cold filtrate is adsorbed on 480 1. of Dowex 50x4 Na+, 2050 mesh at about 48 I./min.The adsorbate is washed with 480 1. of deionized water and then eluted with 2% aqueous pyridine at 24 I./min. and three fractions of 300 1., 520 1. and 240 1. are collected and assayed at pH 7.0. The assays indicate that the eluate fractions contain 4%, 16% and. 6% respectively of the bioactivity applied on the Dowex 50x4 Na+ column. Eluate fraction two is concentrated to 48 1. and adjusted to pH 7.

The 48 1. concentrate is adjusted to pH 7.3 and adsorbed on 76 1. of Dowex lox2, 50 to 100 mesh, chloride cycle resin at 7.6 I./min. The resin is eluted with deionized water at the same rate. Four fractions are collected, two of 48 1., one of 70 1. and one of 48 1. The fractions are adjusted to pH 7 as collected. Assays indicate that 68% of the starting bioactivity is in the 70 1. fraction. This fraction is concentrated to 18 1. at pH 7.0 and filtered using a 0.45 micron Millipore Filter.

The filtrate is tray freeze-dried to yield 99 grams of product having a potency of 310 units/mg. where one unit is defined as the amount calculated to produce the same inhibition against Staphylococcus aureus ATCC 6538P using the disc-diffusion procedure as 1 ,ug. of cephalothin/ml., that zone of inhibition being between 16 and 21 mm. diameter.

Ten g. of the freeze-dried solids is taken up in 0.1 M 2,6-lutidine acetate buffer, pH 6.3. The solution, 125 ml. readjusted to pH 6.3 with acetic acid, is applied to a column of Dowex 50x8 (200400 mesh) in the 2,6-lutidine cycle, 7.6x142 cm., which had previously been equilibrated with buffer, and developed with 0.1 M buffer at 25 ml./min. A 31. fore-cut is collected followed by 200 fractions of 20 ml.

of each. Every fourth fraction from 36 to 192 is assayed at a dilution of 1:200. The bioactivity is contained in fractions 56 to 192, reaching a maximum in fractions 92 to 96. Fractions 80 to 136 are combined and 590 ml. of deionized water added to give 1760 ml. The pooled diluted solution which contains 62% of the starting bioactivity applied on the Dowex 50x8 column, is freeze-dried.

The freeze-dried solids are dissolved in 0.1 M 2,6-lutidine acetate pH 7.0 buffer. The solution, 27 ml., is applied to a column of Bio-Gel P-2 (200400 mesh) 5x112 cm. which had previously been equilibrated with 0.1 M buffer. The gel is then developed with the same buffer at 10 ml./min.

The effluent stream is monitored with a Meccomatic recording differential refractometer. The development is continued until 105 fractions, 20 ml. each, are collected. Every fraction, 70 to 93, is assayed at a dilution of 1:300. The bio-activity is found in fractions 73 to 82, reaching a maximum in fractions 77 and 78. Fractions 75 to 80 are freeze-dried to obtain 90 mg. an antibiotic with an average potency of 10,000 units/mg.

The 90 mg. of freeze-dried solid is taken up into 4 ml. of 0.01 M potassium phosphate buffer, pH 7. This solution, containing 596 hydroxylamine-extinguishable optical density units (this measure of the thienamycin content being described in the section headed "Assay") is applied on a 1.7 cm. diameter column packed with 90 ml. prewashed XAD-2 and equilibrated prior to use with 180 ml. of 0.01 M potassium phosphate buffer, pH 7, at 50C. The XAD-2 is washed prior to use successively with 1) 5 volumes of 1 N NaOH followed by deionized H2O until effluent is neutral; 2) 5 volumes 1 N HCI followed by deionized H2O until the effluent is neutral; 3) 5 volumes each of methanol, acetone and 0.001 M EDTA tetrasodium, and finally distilled 1120. Vacuum is applied to all solvents before use.

After the sample is applied on the column it is followed by two 2 ml. portions of the phosphate buffer. The column is developed at 50C. with the buffer at a flow rate of 2 ml./min. Four ml. fractions of eluate are collected. Fractions obtained after 100 ml. of eluate has been collected and ending with 253 ml. are combined and concentrated on a rotary evaporator under vacuum and below 100C. to a volume of 6 ml.

This solution, containing 436 hydroxylamine-extinguishable optical density units, is applied on a 1.7 cm. diameter column packed with 90 ml. XAD-2 prewashed as above and equilibrated at 50C. with distilled water. The sample is followed by two 2 ml. portions of distilled water. The column is developed with distilled water at the rate of 2 ml./min. Four ml. fractions of eluate are collected.

Fractions obtained after 100 ml. of eluate has been collected and ending with 151 ml. are pooled and concentrated on a rotary evaporator to a volume of 2.73 ml. and the solution is lyophilized to yield 6.49 mg. of thienamycin. Fractions obtained between 152 ml. and 345 ml. are pooled and concentrated on a rotary evaporator to a volume of 3.34 ml. and lyophilized to yield 11.53 mg. of thienamycin. These fractions contain a total of 369 hydroxylamine-extinguishable optical density units.

This represents a 3.1 fold purification over the material applied to the first XAD-2 column and yields a calculated potency of 31,000 units/mg. Spectrophotometric analysis of a sample of this product shows an E1o'com=253 when measured in phsophate buffer, -pH 7, at 297 nm.

10 g. of the 99 g. freeze-dried solids obtained by the Dowex 1x2 purification above is taken up in 0.1 M2,6-lutidine acetate buffer, pH 6.3. The solution, 125 ml., readjusted to pH 6.3 with acetic acid, is applied to a 7.6x 142 cm. column of Dowex 50x8 in the 2,6-lutidine cycle, which has previously been equilibrated with buffer.

The column is developed with 0.1 M buffer at 35 ml./min. A 3.6 1. fore-cut is collected, followed by 200 fractions of 20 ml. each. Every forth fraction from 6 to 194 is assayed at a dilution of 1:200. The bioactivity is contained in fractions 18 to 178, reaching a maximum in fractions 62 to 82. Fractions 42 to 102 are combined and 640 ml. of deionized water is added to give 1920 ml. The pooled diluted solution containing 63% of the bio-activity applied on the Dowex 50x8 column, is freeze-dried.

The freeze-dried solids are dissolved in 0.1 M 2,6-lutidine acetate, pH 7.0 buffer. The solution, 25 ml., is applied to a 5x 112 cm. column of Bio-Gel P-2 (200- 400 mesh), which had previously been equilibrated with 0.1 M buffer. The gel is then developed with the same buffer at 10 ml./min.

The effluent stream is monitored with a Meccomatic recording differential refractometer. The development is continued until 125 fractions of 20 ml. each have been collected. Every fraction from 70 to 89, is assayed at a dilution of 1:300, The bio-activity is found in fractions 72 to 81, reaching a maximum in fraction 77.

Fractions 75 to 79 are freeze-dried to obtain 100.5 mg. of antibiotic with a potency of 8,320 units/mg.

The 100.5 mg. of freeze-dried solid is taken up into 4 ml. of 0.01 M potassium phosphate buffer, pH 7. This solution, containing 692 hydroxylamineextinguishable optical units is applied on a 1.7 cm. diameter column packed with 90 ml. prewashed XAD-2 and equilibrated prior to use with 180 ml. of 0.01 M potassium phosphate buffer, pH 7, at 50C. The XAD-2 is washed prior to use successively with 1) 5 volumes of 1 N NaOH followed by deionized H2O until effluent is neutral; 2) 5 volumes 1 N HCI followed by deionized H2O until the effluent is neutral; 3) 5 volumes each of methanol, acetone and 0.001 M EDTA tetrasodium, and finally distilled H2O. Vacuum is applied to all solvents before use.

After the sample is applied on the column it is followed by two 2 ml. portions of the phosphate buffer. The column is developed at 50C. with the buffer at a flow rate of 2 ml/min. Four ml. fractions of eluate are collected. Fractions obtained after 109 ml. of eluate has been collected and ending with the 309th ml. are combined.

To this combined eluate is added the 11.53 mg. sample of XAD-2 purified antibiotic obtained above comprising 186 hydroxylamine-extinguishable optical density units. The combined eluate together with the added antibiotic is concentrated in vacuo on a rotary evaporator at a temperature below 10 C. to a volume of 7 ml.

This solution, containing 720 hydroxylamine-extinguishable optical density units is applied on a 1.7 cm. diameter column packed with 90 ml. XAD-2 prewashed as above and equilibrated at 5 C. prior to use with distilled water. The sample is followed by two 2 ml. portions of distilled water. The column is developed with distilled water at the rate of 2 ml./min. Four ml. fractions of eluate are collected. Fractions obtained after 109 ml. of eluate have been colllected and ending with the 301st ml. are pooled and concentrated on a rotary evaporator to a volume of 10.3 ml. This solution, containing 589 hydroxylamine-extinguishable optical density units, is lyophilized to yield 23.6 mg. of antibiotic with a calculated potency of 30,140 units/mg.

The antibiotic thienamycin thus prepared is a white, amorphous solid with a fibrous consistency, a sample of which on exposure in a glass capillary tube to temperatures elevated at a rate of 3C degrees per minute, underwent decomposition without the invervention of a liquid phase in the following stages: softening occurred at 130 to 1400C. with a contraction in volume of the solid continuing until 170 to 174 C. in which range the material yellowed; sintering and a progressive intensification of color to reddish-brown being observed in the range 180 to 200 C. and finally carbonization of residual traces of solid being found at 250 C.

A further sample of this material on spectrophotometric analysis shows an absorbance peak at 296.5 nm with an E1 cm1-@@=268.2. Elemental analysis yields the following results: 1) a 5.67 ,; weight loss upon drying at room temperature for 4 hour under vacuum, and 2) the composition 47.68% carbon, 6.22% hydrogen, 11.48% nitrogen.These results are consistent with the empirical formula C11H16N2O4S#(NH3)0.28, the calculated elemental composition corresponding to this empirical formula being C=47.68%; H=6.13%, N=11.52%, S=11.57% and 0=23.1%. Polarimetric analysis of a 1 mg./ml. solution of this sample in 0.01 M potassium phosphate buffer showed a specific optical rotation []D7tC+80. The infrared spectrum of a Nujol mull of this sample revealed characteristic absorption peaks at 1765 cm.-1, 1650-1550 cm.-1, 2800-2500 cm.-1, and 3500-3100 cm.-1.

An NMR spectrum at 100 MHz of a sample of this product dissolved in D,O reveals a doublet at # 1.275, a pair of doublets at a3.39 and multiplets at 33.15 and #4.20, these peaks being characteristic of thienamycin.

EXAMPLE 6 Acetylation of Thienamycin Thienamycin, 10.9 mg. is stirred for 10 minutes at OOC. in 1 ml. dry DMF plus 2 ml. freshly prepared acetic anhydride. The DMF and acetic anhydride are removed by washing repeatedly (5 to 6 times) with 25--40 ml. portion of hexane and one last portion of hexane after the addition of 1 ml. dry ethyl ether. The crude sample of N-acetyl thienamycin is dissolved in 20 ml. of deionized water which contains 100 moles of Tris base [tris(hydroxymethyl)aminomethane] and 35 poles of HCI. The pH, after dissolution of the sample, is 7.9. The solution contains 244 absorbance units at 298 nm and a 1/2-inch assay disc containing 0.1 ml. of a 1000 fold dilution, produces a 23 mm. zone of inhibition when incubated on ATCC 8461 plates at 250C.

This sample is applied on a column (1.3 cm.xl4 cm. bed dimensions) of Dowex-lx4 (Cl-) minus 400 mesh. The column is washed with 10 ml. of deionized water and the antibiotic, N-acetyl thienamycin, is eluted with 0.07 M NaCl+0.005 M NH4Cl+0.0001 M NH3 in deionized water, 6.1 ml. fractions are collected at a flow rate of 0.7 ml. per minute. The main peak of UB absorbance at 298 nm appears in fractions 36 to 50, with a maximum at fraction 40. Fractions 38 to 46 are combined, containing a total of 107 absorbance units at 298 nm. The combined fractions are rotary evaporated under reduced pressure to 2 ml. and 5 pl of 1 M NaOH is added.

This concentrate is applied on a column (2.2x80 cm. bed dimensions) of Bio Gel P-2, 200400 mesh. The sample is washed in with two 1 ml. portions of deionized water and eluted with deionized water at a flow rate of 0.6 ml. per minute. 3.04 ml. fractions are collected.

The main peak of UB absorbance at 300 nm appears in fractions 58 to 64, with a maximum at fraction 60. Fractions 59 to 62 containing 83.7 A300 units are pooled.

A portion equivalent to 2.2 A300 units is removed for reference, and the remainder is concentrated to 1.5 ml. and lyophilized in a 14 ml. glass vial to give 3.9 mg. of Nacetyl thienamycin.

Amax 301 nm, Eax/Emin=4.45, E%301 in deionized water=208.

EXAMPLE 7 Acetylation of Thienamycin Step A) Nine mg. thienamycin is stirred for 10 minutes at 0 C. in 1 ml. dry DMF plus 2 ml. freshly prepared acetic anhdride. The DMF and acetic anhdride are removed by washing repeatedly (5 to 6 times) with 25-40 ml. portions of hexane and one last portion of hexane after the addition of 1 ml. dry ethyl ether. Ten ml. cold distilled water containing 50 pi I M Tris/base, and 50 L1 1 M Tris . HCI, pH 8, is added. The pH of this solution is 7.8 and it contains a total of 102 hydroxylamineextinguishable O.D. units. Ten ml. cold distilled water is added and the solution held at 0 C.

A 1.7 cm. diameter column packed with 30 ml. Dowex 1x4 Cl- (minus 400 mesh) resin is prepared and washed with 90 ml. of each of the following: 1) 0.2 M HC1, 2) 0.5 N NaCI solution containing 0.01 M HCI, 3) deionized water.

The 20 ml. sample is applied to the Dowex 1x4 column, followed by two 2 ml.

portions of deionized water and then two 1 ml. portions of eluting buffer. The column is eluted with 0.07 M NaCl containing 0.005 M NH4Cl and 0.0001 M NH4OH. The rate is 4.3 ml./5 minutes, and 4.3 ml. fractions are collected. Fractions from 555 ml. to 594 ml. are combined and stored at 50C.

Step B) Thienamycin, 27 mg., is stirred for 10 minutes at OOC. in 2 ml. dry DMF plus 4 ml. freshly prepared acetic anhydride. The DMF and acetic anhydride are removed by washing 6 times with 2540 ml. portions of hexane and one last portion of hexane after addition of 2 ml. dry ethyl ether. Ten ml. cold distilled water containing 50 l 1 M Tris-base and 50 l 1 M Tris#HCl, pH 8, is added and the resulting pH is 6.85. The solution contains 148 hydroxylamine-extinguishable O.D. units.

The top cm. of the Dowex 1 x4 (minus 400 mesh column) described in Step A) is removed and the column washed with 90 ml. of each of the following: 1) 0.2 M HC1, 2) 0.5 N NaC1 solution containing 0.01 M HCI, 3) deionized water.

The 10 ml. mixture is added to 10 ml. 0 C. distilled water and is applied to the Dowex 1 x4 Cl- column and is followed by two 2 ml. portions of deionized water and then two 2 ml. portions of eluting buffer. The column is eluted with a 0.07 M NaCI solution containing 0.005 M NH4Cl and 0.0001 M NH40H. The rate is 4.3 ml./5 minutes, and 4.3 ml. fractions are collected. Fractions from 497 to 571 ml. are pooled and added to the pooled fractions from the Dowex 1 x4 column described in Step A. Together these fractions contain 117.5 hydroxylamine-extinguishable O.D.

units.

A 2.2 cmx62 cm. column packed with 225 ml. Bio-Gel P-2 (200-400 mesh) resin with an exclusion limit of 2600 Daltons (defined prior to use by decantation from distilled water) is prepared and washed with 50 ml. 1 M NaCI, followed by 225 ml. deionized 1130. The combined Dowex 1 x4 eluate pools are concentrated under vacuum to 2.66 ml. and applied to the Bio-Gel P-2 column, followed by two 2 ml.

portions of deionized water. The column is developed with deionized water at the rate of 1 ml./minute, and 2 ml. fractions are collected. Fractions from 190 ml. to 204 ml. are combined and concentrated to 2.5 ml.

A 2.2 cmx62 cm. column packed with 225 ml. Bio-Gel P-2 200400 mesh resin with an exclusion limit of 1800 Daltons (defined prior to use by decantation from distilled water) is washed prior to use with 225 ml. 1 M NaCl followed by 100 ml. deionized water.

The above 2.5 ml. concentrate, which contain 109.5 hydroxylamineextinguishable O.D. units, is applied to the column followed by two 2 ml. portions of deionized water. The column is developed with deionized water at the rate of 1 ml./minute and 2 ml. fractions are collected. Fractions from 104 ml. to 132 ml. are combined and contain 97 hydroxylamine-extinguishable O.D. units. A 2.4 ml.

portion is stored at OOC. for in vitro assays and the remainder is lyophilized to give N-acetyl thienamycin.

Compositions containing the antibiotic N-acetyl thienamycin may be administered in several unit dosage forms as, for example, in solid or liquid orally ingestible dosage form. The compositions per unit dosage, whether liquid or solid may contain from 0.1% to 99% of active material, the preferred range being from 10 to 60%. The composition will generally contain from 25 mg. to 1000 mg. by weight of the active ingredient based upon the total weight of the composition; however, in general, it is preferable to employ a dosage in the range of from about 250 mg. to 1000 mg., preferably 400 to 800 mg. for parenteral administration the unit dosage is usually the pure compound in a slightly acidified sterile aqueous solution or in the form of a soluble powder intended for solution.Representative formulations can be prepared by the following procedures: Capsules Per Capsule N-Acetyl thienamycin Lactose, U.S.P., a sufficient quantity to fill No. 0 Capsules, approx. 475 mg. each 400 ing.

In this formation the active compound and the diluent are mixed to produce a uniform blend, which is then filled into No. 0 hard gelatin capsules, by hand or on a suitable machine, as required. The mixing and filling is preferably done where the relative humidity is less than 40%.

Tablets Per Tablet N-Acetyl thienamycin 300 mg.

Calcium phosphate 192 mg.

Lactose, U.S.P. 190 mg.

Cornstarch 80 mg.

Magnesium stearate 8 mg.

800 mg.

In this formulation, the active component is blended with the calcium phosphate, lactose and about half of the cornstarch. The mixture is granulated with a 15% by weight cornstarch paste and rough-screened and screened again through No. 16 screens. The balance of the cornstarch and the magnesium stearate is added and the mixture is compressed into tablets, approximately 1/2" in diameter, each weighing 800 mg.

Alternatively, the active component is blended with the calcium phosphate, lactose and one-half the cornstarch. The mixture is "slugged" on a heavy-duty press to produce compacted tablet-like masses. These are broken down to a No. 16 mesh granule. The balance of the cornstarch and the magnesium stearate are added and the mixture is compressed into tablets approximately 1/2" in diameter, each weighing 800 mg.

Lyo Form (For Injection) Per Vial N-Acetyl thienamycin 25 mg.

Water-for-Injection, U.S.P. to make 5 ml.

In this formulation the active component is dissolved in sufficient water-forinjection in the ratio shown. The solution is filtered through Selas candles or Millipore membrane filters to sterilize it. The solution is subdivided into sterile vials. The vials and contents are frozen, and the water is aseptically removed by lyophilization. The vials containing the sterile dry solid are aseptically sealed.

To restore for parenteral administration, 5 ml. of sterile water for iniection is added to the contents of a vial.

Oral Liquid Forms Per 1000 ml.

N-Acetyl thienamycin 1.0 gm.

Sucrose 600.0 gm.

Glucose 250.0 gm.

Sodium Benzoate 1.0 gm.

Concentrated Orange Oil 0.2 ml.

Purified water U.S.P. to make 1000.0 ml.

The sucrose and glucose are dissolved in about 400 ml. of water using heat to aid solution This solution is cooled and the sodium benzoate is added, followed by the concentrated orange oil. The solution is brought to about 900 ml. volume with water and the antibiotic is added. The solution is clarified by filtration through a coarse filter.

WHAT WE CLAIM IS:- 1. N-Acetyl thienamycin and pharmaceutically acceptable salts thereof.

2. A composition comprising an antibacterially effective amount of a compound as claimed in Claim 1 and a non-toxic pharmaceutically acceptable carrier.

3. An aqueous solution suitable for disinfecting medical or dental equipment containing 1 to 10 parts per million by weight of a compound as claimed in Claim 1.

4. A water-based paint or white water in a paper mill containing as antibacterial agent a compound as claimed in Claim 1.

5. A composition as claimed in Claim 2 in the form of a tablet, capsule, powder, suspension or elixir.

6. A composition as claimed in Claim 2 in the form of a suppository.

7. A composition as claimed in Claim 2 enclosed in an ampule.

8. A composition as claimed in Claim 2 in the form of a spray, inhalent, lozenge or throat paint.

9. A composition as claimed in Claim 2 in the form of an ointment, cream, lotion, powder or paint suitable for topical application.

10. A composition as claimed in Claim 2 in the form of an optically or aurally administrable material.

11. A composition as claimed in Claim 2 in the form of an intramammary preparation suitable for veterinary use.

12. A composition as claimed in any one of Claims 2 and 5 to 11 also containing an aminocyclitol antibiotic.

13. A composition as claimed in any one of Claims 2 and 5 to 11 also containing a diphenoxylate and atropine.

14. A method of producing a compound as claimed in Claim 1 substantially as hereinbefore described in any one of the Examples.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (14)

**WARNING** start of CLMS field may overlap end of DESC **. water and the antibiotic is added. The solution is clarified by filtration through a coarse filter. WHAT WE CLAIM IS:-

1. N-Acetyl thienamycin and pharmaceutically acceptable salts thereof.

2. A composition comprising an antibacterially effective amount of a compound as claimed in Claim 1 and a non-toxic pharmaceutically acceptable carrier.

3. An aqueous solution suitable for disinfecting medical or dental equipment containing 1 to 10 parts per million by weight of a compound as claimed in Claim 1.

4. A water-based paint or white water in a paper mill containing as antibacterial agent a compound as claimed in Claim 1.

5. A composition as claimed in Claim 2 in the form of a tablet, capsule, powder, suspension or elixir.

6. A composition as claimed in Claim 2 in the form of a suppository.

7. A composition as claimed in Claim 2 enclosed in an ampule.

8. A composition as claimed in Claim 2 in the form of a spray, inhalent, lozenge or throat paint.

9. A composition as claimed in Claim 2 in the form of an ointment, cream, lotion, powder or paint suitable for topical application.

10. A composition as claimed in Claim 2 in the form of an optically or aurally administrable material.

11. A composition as claimed in Claim 2 in the form of an intramammary preparation suitable for veterinary use.

12. A composition as claimed in any one of Claims 2 and 5 to 11 also containing an aminocyclitol antibiotic.

13. A composition as claimed in any one of Claims 2 and 5 to 11 also containing a diphenoxylate and atropine.

14. A method of producing a compound as claimed in Claim 1 substantially as hereinbefore described in any one of the Examples.