US2843526A - Radioactive antibiotic - Google Patents

Description

carbon.

United States Patent RADIOACTIVE ANTIBIOTIC Junius F. Snell, Paterson, N. J., assignor to Chas. Pfizer & Co., Inc., New York, N. Y., a corporation of Delaware No Drawing. Application July 22, 1955 Serial No. 523,946

3 Claims. (Cl. 195-80) This invention is concerned with radioactive antibiotic substances and processes for producing said substances. This invention is also concerned with the metal and acid addition salts of these radioactive antibiotics. More particularly, it is concerned with the tetracycline antibiotics containing organically bound radioactive carbon, the metal salts of said antibiotics, and the acid addition salts of said antibiotics.

The tetracycline antibiotics are well known groups of antibiotics with a broad range of antimicrobial activity. Specifically the term tetracyline antibiotics applies to tetracycline, oxytetracycline, chlortetracycline, and the metal and the acid addition salts of said substances. The tetracycline antibiotics have the following structure wherein R and R'=I-I in tetracycline; R=H, and R'=OH in oxytetracycline; and R=Cl, and R=H in chlortetracycline.

R cm 011 R These substances are amphoteric and form both metal and acid addition salts. The non-radioactive tetracycline antibiotics and their production are more particularly described in the following patents and articles:

The valuable products of the present invention are identical with the hitherto known tetracycline antibiotics except that they contain organically bound radioactive Therefore, these valuable products have much higher radioactive disintegration rates than the hitherto known substances. The term organically bound is intended to apply to those atoms which are incorporated into the fundamental ring structure and its appendages of said antibiotics by means of one or more covalent bonds. Therefore, an organically bound atom is an integral component of the molecule and it is not merely present in the anion. of an acid addition salt of said substance.

In addition to being useful therapeutic agents in themselves, these substances are very useful materials for research purposes by virtue of their high radioactive disintegration rates. They have made possible certain types of investigations that otherwise could not have been undertaken. Therein lies the great value of these radioactive products. For some time, the need has existed for radioactive tetracycline antibiotics for research purposes. The object of this invention therefore, is to provide the radio- 2,843,526 Patented July 15, 1958 active tetracycline antibiotics to fulfill the above need. A further object is to develop a sufficiently economical process so that adequate quantities of these unique materials may be produced for research and therapeutic purposes.

The most immediate needs for research employing a radioactive tetracycline antibiotic lie in the fields of pharmacy and pharmacology. Radioactive materials such as these make possible the study of dosage forms on an exact and quantitative basis. By means of such materials the site and rate of absorption of the antibiotics can be ascertained and their anatomical distribution pattern determined. The effect of various pharmaceutical carriers on the rate of absorption and the distribution of the antibiotics in the animal body can be determined to assist in developing improved dosage forms. Thus a rational and scientific approach to the study of tetracycline antibiotic dosage forms in a manner that has not been available hitherto is now made possible.

Other questions that may yield to pharmacological and biochemical research based on the radioactive antibiotics of this invention include explaining observed differences between the in vivo and in vitro activity of the antibiotics to improve the performance of the antibiotics in the animal. In the bacteriological and biochemical fields of research investigations based on these radioactive antibiotics are concerned with studies on the mechanism of antimicrobial action and microbial resistance, and biosynthetic pathways by which the antibiotics are produced.

A fermentation process for producing radioactive tetracycline antibiotics has now been invented. In essence, the process of this invention consists of carrying out the fermentation using a suitable selected species of Streptomyces on a medium containing a minimal carbon content and then adding the radioactive carbon source at an appropriate time during the fermentation. By a suitable species of Streptomyces is meant any of the known producers of the various tetracycline antibiotics. A number of species are known producers of these antibiotics and are either described in the literature or generally known in the art. It is not intended to limit the process of this invention to any particular species since this is not a critical feature of the invention. The critical features are the composition of the fermentation medium employed and the nature .and time of addition of the radioactive carbon precursor. It is important to restrict the nonradioactive carbon content of the medium in order to obtain a product of useful specific activity. In addition radioactive tetracycline has also been produced by the hydrogenation process of the Conover patent cited above by using the radioactive chlortetracycline of the instant invention as a starting material.

In the fermentation process of this valuable invention, it is undesirable to include the radioactive carbon source in the fermentation medium at the outset of the fermentation since substantial radioactivity then appears in the mycelium. Rather it is preferred to permit the cycelium to develop on the minimal nonradioactive carbon source supplied and to add the radioactive source to the mixture containing the mature mycelium at a stage when the antibiotic potency is rapidly increasing. It has been found that the radioactive precursor is most advantageously added after from 25 to hours of incubation. A total incubation period of 96 to hours is preferred.

It is also preferred to employ as radioactive carbon sources those containing C in the valuable process of this invention. However, other radio-isotopes may be employed. Reasons for this are many-fold. A number of suitable C labeled materials are commercially available; this isotope has a long half-life thus making it convenient to use in biological experiments; the low energy beta radiation derived from it is easily shielded thus rein the expired CO ducing the hazard to both operators and to experimental animals; and it is harmless to experimental animals when used in small amounts. With respect to the latter point, it is frequently convenient to dilute the radioactive tetracycline antibiotics of this process with non-radioactive material to yield a composition with a level of radiation suitable for the particular experiment at hand. In general, compositions with specific activities greater than about 0.05 microcurie per millimole are useful.

The selection of the radioactive carbon source while critical may be made from a substantial latitude of materials. A wide variety of assimilable carbon sources have been found suitable for the process of this invention. These include C labeled amino acids, carbohydrates, and fatty acids. Simpler materials have also been employed including sodium acetate, glucose, carbonate, and bicarbonate labeled with C Simple materials of the latter types are commercially available as are some of the more complex precursors. More complex precursors which are commercially available include a C labeled yeast hydrolysate manufactured by Schwarz Laboratories of Mount Vernon, New York and a C labeled leaf preparation derived from plants of the genus Canna available from Atom Laboratories of New York, New York. Both of the above materials are complex mixtures containing C labeled amino acids and other substances.

The novel radioactive teracycline antibiotics produced by the valuable process of this invention are shown to be labeled generally throughout the molecule by known degradative techniques. General labeling occurs regardless of whether a simple carbon source such as acetate or bicarbonate is used or a more complicated one such as glucose or yeast hydrolysate is employed. It was found that radioactive carbon is incorporated into each of the four rings and into the dimethylamino group of the tetracycline antibiotics. This property makes these unique products particularly desirable for the research purposes outlined above since radioactivity is retained even by the small fragments remaining after extensive degradation of the antibiotic. The general type of labeling achieved in the valuable product of this invention adds to the novelty and usefulness of this already valuable product. Previous attempts to make radioactive antibiotics have not resulted in generally labeled products of this type.

Ordinary known techniques for producing the tetracycline antibiotics by fermentation under submerged, aerated, and agitated conditions with the incorporation of but a few refinements may be employed to produce the valuable products of this invention. Some means must be provided for adding the radioactive carbon precursor during the course of the fermentation without disturbing the sterility of the batch. Second, for reasons of economy and safety the expired CO should be scrubbed from the exit gases, for instance by means of aqueous alkali, since 70-80% of the C charged appears Although the C carbonate recovered in this fashion is of relatively low specific activity, some overall efiiciency can be gained by recycling it to future batches.

One method for carrying out the fermentation is to use shaker flasks incorporating a small open compartment Within the flask which serves as a reservoir for an aqueous alkaline solution to absorb the CO expired during the fermentation. Erlenmeyer flasks containing a short piece of glass tubing sealed in a vertical position to the inside of the bottom of the flask with the upper end of the tube open have proven suitable. The tube serves as a reservoir for an aqueous potassium hydroxide solution and the annulus of the flask as a container for the fermentation medium. The flask to which the aqueous alkali and fermentation medium were charged were then sealed with tape and sterilized in an autoclave. Inoculation of the sterile medium with an inoculum containing a suitable selected tetracycline antibiotic producing species of Streptomyces was accomplished by means of a hypodermic needle inserted through the tape closure. A similar aseptic technique was used to introduce the radioactive precursors at suitable times during the fermentation.

A minimal carbon medium consisting of the following ingredients in 1 1. of water was found to give satisfactory potencies in the preparation of radioactive oxytetracycline using Streptomyces rimosus NRRL 2234 described in the Sobin patent.

Sodium nitrate g 8.0 Corn meal g 15.0 Beet molasses g 4.0 Soybean oil ml 7.0

This medium, containing 7.28 g. of carbon per liter, consistently produced 600-1000 meg/ml. of oxytetracycline under the fermentation conditions employed. Other media, of course, can be employed that afford a favorable ratio of antibiotic potency to carbon content, so long as the carbon content is restricted to an extent that makes possible the incorporation of the C from the radioactive precursor with reasonable efficiency. In fact the composition of the minimal carbon medium is preferably tailored to the particular strain of organism employed and the antibiotic to be produced. It has been found that the non-radioactive carbon content of the medium should be less than about 10 g./l. in order to obtain satisfactory conversions of radioactivity. Further, it was found that radio-carbon precursor should preferably be added at an equivalent specific activity of at least about 20 uC/S ml. of fermentation medium in order to obtain a useful level of radioactivity in the product.

As was indicated above a number of radioactive precursors have been found useful. Of those tested uniforrnly labeled C glucose and methyl (i. e. 2-C) labeled C acetate gave the highest incorporation of radioactivity into the antibiotic. The best efficiency obtained was about 6% using methyl labeled C acetate in a 96 hour fermentation using S. rimosus NRRL 2234 for the preparation of radioactive oxytetracycline wherein the radioactive acetate was added substantially between 50 and hrs. after the batch was inoculated. About 7080% of the radioactive carbon charged appeared in the expired carbon dioxide. Very little appeared in the mycelium and the remainder appeared to be transferred to other products.

In small scale work, paper chromatography was employed in isolating the radioactive antibiotic either from the filtered fermentation broth or from concentrates obtained by solvent extraction. Useful solvent systems for the isolation of radioactive oxytetracycline and chlortetracycline by the papergram technique included: n-butanol saturated with 2 N hydrochloric acid; 1: 1:1 ethyl acetateglacial acetic acid-water: 1:1:1 ethyl acetate-pyridine water; and 1:1:1 ethanol-butanol-water. In the production of radioactive tetracycline, mixtures of tetracycline and chlortetracycline were generally obtained. For the isolation and separation of these materials by paper chromatography the method described by H. L. Bird, IL, and C. T. Pugh, Antibiotics and Chemotherapy, IV, 750 (1954) using the solvent system ethyl acetate saturated with water with filter paper that had been treated with a pH 3.0 phosphate buffer was found suitable. The papergrams were then assayed by two methods; one to detect radioactivity and the other to detect biological activity. The radioactive and bio-active zones on the papergrams coincided thus indicating that the radioactive antibiotic had been produced.

A variety of instruments and methods can be used to assay the papergrams for radioactivity. One useful apparatus that was used was made up of a rotating drum contained within a methane flow counter and shielded from the anode of the counter by a partition with a slit in it. The papergram was placed on the rotating drum and appropriate measuring devices were used to measure the fluctuations in the current resulting from the ionization of the gas in the vicinity of the anode caused by the radioactive emissions emanating from the papergram in front of the slit. With proper calibration of this apparatus, it was possible to determine accurately the R values of the radioactive zones and to determine semiquantitatively their C analyses.

Assay of the papergrams for zones of biological activity was done by a standard bio-autograph technique employing a B. subtilis plate assay. Sterile agar plates were prepared and inoculated with B. subtilis. The papergrams were placed on the plates to transfer the antibiotics to the medium, and the plates were incubated. The zones of inhibition then indicated the location of the antibiotics on the papergrams. As indicated previously, a correlation was observed between the zones located by the two techniques. A quantitative bio-assay of the chromatographed material permitted a calculation of the approximate specific activities of various samples of the radioactive tetracycline antibiotics produced by this technique.

The above technique is useful for determining opti mum fermentation conditions. Fermentations were also carried out from which the radioactive tetracycline antibiotics were recovered. Essentially the same type of shake-flask fermentation technique was used as was employed where paper chromatography was used to isolate the product. However, a number of these small fermentation batches were combined to increase the scale of the recovery operation. In the case of radioactive oxytetracycline, the radioactive product was recovered by the procedure described by P. P. Regna et al., I. Am. Chem. Soc. 73, 4211 (1951), for the isolation of the non-radioactive oxytetracycline. This procedure involves acidification of the broth to pH 2.0-2.5 and filtration of the mycelium; precipitation of the oxytetracycline as the barium'magnesium double salt; decomposition of the double salt; with sulfuric acid at pH 1.5; and adjustment of the filtrate after removal of the inorganic sulfates to pH 7.0 which results in the precipitation of the oxytetracycline as the crude base. The crude base was then converted to the hydrochloride salt and recrystallized from methanol. In the instant radioactive fermentation a useful adjunct, which increased the recovery efficiency of the radioactive antibiotic produced, involved adding a quantity of non-radioactive oxytetra-cycline to the broth prior to recovery to increase the total antibiotic concentration present and thus simplify the mechanical manipulations of recovery. This, of course, resulted in dilution of the radioactive antibiotic with non-radioactive material, but the so reduced specific activity of the product obtained was still of such magnitude as to be useful for the applications outlined above.

The valuable radioactive tetracycline antibiotics obtained by the unique process of this invention were found to be identical in physical and chemical properties with authentic samples of the non-radioactive antibiotics. They differ only in the radioactive disintegration rates which, of course, are much higher than those of the nonradioactive types., The ultra violet absorption spectra, bio-potency, and paper chromatographic behavior were identical with authentic samples of the non-radioactive materials. The valuable radioactive products of this invention have been routinely produced with specific activities of 50 to 200 microcuries per millimole. Some batch to batch variations were, of course, observed.

For most purposes it is desirable to blend a radioactive antibiotic of this invention with the corresponding nonradioactive material to obtain a composition of lower specific activity. In general, it is most economical in research studies to Work with the lowest specific activity possible. For instance, for chemical degradation studies, in which little dilution normally is necessary, specific activities of 1/10 to l/ 100 of the above may be used provided efficient counting techniques are available. Indeed specific activities as low as 0.05 microcurie per millimole are useful. Somewhat higher specific activities are required for biological experiments than are required for chemical investigation.

For the degradation studies with radioactive oxytetracycline, a small sample of the above antibiotic prepared from a methyl C acetate precursor was diluted 95 fold with non-radioactive oxytetracycline and submitted to known degradative techniques. The following degradation products were isolated. All were radioactive. The average specific activity per carbon atom in each of these degradation products was roughly equal to that of the starting radio-oxytetracycline except for the dimethylamine which was lower. This indicates that the valuable product of this invention is generally labeled throughout the carbon structure except for the dimethylamine group which is relatively lower in C content.

Terracinoic acid Succinic acid Succinic anhydride Dimethylamine 7-hydroxy-3-methylphthalide Known degradative techniques were similarly applied to tetracycline and chlortetracycline.

For determination of the C content of the radioactive tetracycline and its degradation products which were isolated in pure form, the combustion method of Van Slykeet al., J. Biol. Chem. 192, 769 (1951) was employed. The radioactive content of the CO produced was measured in a BernsteinBallentine tube.

The following examples were given by way of illustration and are not to be considered as limiting the scope of this invention. In fact, my varying embodiments may be employed without departing from the spirit and scope thereof. My invention is to be limited only by the specific wording of the appended claims.

Example I A fermentation medium was prepared containing the following materials in one liter of water.

Sodium nitrate grams 8 Corn meal grams 15 Beet molasses grams 4 Soybean oil milliliters 7 This mixture was then adjusted to pH 6.8 and a 5 ml. aliquot of it was placed in the annulus of a ml. modified Erlenmeyer flask to the bottom of which was sealed a 27 mm. length of glass tubing whose outside diameter was 8 mm. Four hundred microliters of 20% potassium hydroxide solution, was then placed in the well formed by the piece of glass tubing. The flask was then sealed with a piece of tape and sterilized in an autoclave at 15 p. s. i'. g. for 15 minutes. The inoculum had been previously prepared by transferring under sterile conditions spores of S. rimosus NRRL 2234 to a solution containing 1% NZ amine B (this material is an enzymatic digest of casein which was obtained from the Sheffield Chemical Company, New York, New York), 1% glucose, 0.5% baseamine Busch (this material is a yeast hydrolysate which was obtained from Anheuser-Busch of St. Louis, Missouri), 0.1% calcium carbonate and incubating at 28 for 48 to 72 hours. The sterile fermentation medium was then inoculated with 0.5 ml. of the above inoculum by means of a hypodermic needle and the mixture incubated with continuous shaking at 27 for from 27 to 50 hours. The flask was then removed, and a solution of the radioactive precursor at an equivalent total activity of 20 C was then added aseptically to the fermentation mixture by means of a hypodermic needle. Listed below are some of the radioactive precursors and their sources that were successfully used in this procedure.

Sodium 1-C acetate-Tracer Laboratories, Boston,

Mass.

Sodium 2-C acetateTracer Laboratories, Boston,

Mass

Sodium C bicarbonate-Tracer Laboratories, Boston,

Mass.

C canna leavesAtom Laboratories, New York, New

York

Following addition of the radioactive precursor, the fermentation mixture was incubated as above until a total incubation period of 96 hours had elapsed. The flask was then removed from the incubator, the broth aseptically filtered, and the filtrate chromatographed on strips of acid washed Whatman No. 1 paper. Four different solvent systems were found useful with solvent rises of 30 'cm. The solvent systems employed and the R; values observed are listed below.

1 Butanoi saturated with 2 N HCl 0.40 12111 ethyl acetate-glacial acetic acid-water 0.80 1:1:1 ethyl acetate-pyridine-water 0.55 1:1:1 ethanol-butanoi-water 0.50

Following chromatography, the solvent free chromatogram was wound on the aluminum cylinder of the counting apparatus described above and placed inside of the methane flow counter. The cylinder rotated the paper strip slowly in front of a slit which opened to the sensitive counting volume and count obtained was recorded on an Esterline-Angus recorder (The Esterline-Angus Co., Inc., Indianapolis, Ind.) which was connected in series with a count rate meter, and a sealer. The strip was then removed and bioautographed as described above against B. subtilis. The zones of biological activity coincided with the positions of radio-activity observed above.

Example II An experiment was carried out as described in Example I using a fermentation medium containing the following materials in 1 l. of water and 2C acetate as the radio-active precursor.

Glutamic acid g 15 Glucose g Sodium ammonium phosphate g 2.25 KH PO g 1.5 Magnesium sulfate g 0.3 Sodium citrate g 4.5 FeSO .7H O g 0.05 ZHSO4.7H2O g Calcium chloride g 0.10 Soybean oil ml 7.0

Similar results were obtained.

Example III Three 5 ml. fermentations as described in Example I were carried out concurrently. Sodium 2C -acetate (Tracer Laboratories, Inc., Boston, Massachusetts) was used as the radioactive carbon source. At the completion of the incubation period the broths were combined and bioassayed by a standard turidimetric technique employing Klebsiella pneumoniae as the assay organism. Nonradioactive oxytetracycline was then added to the broth to effect a fold increase in the concentration of that substance prior to recovery. The radioactive oxytetracycline was then isolated as a composition with the nonradioactive material according to the procedure of P. P. Regna et al., J. Am. Chem. Soc. 73, 421i (1951). This procedure is outlined schematically below. The specific activity of the radioactive oxytetracycline composition obtained in this fashion varied somewhat from batch to batch in the range 5-20 C/miilimole. The crude radioactive oxytetracycline base so obtained was converted to the hydrochloride salt and purified by recrystallization from methanol. A small sample of the crude radioactive oxytetracycline base was also converted to the sodium salt again by the procedure of P. P. Regna et al. (loc. cit.).

Fermentation broth (2) Adjust to pH 2.0 (3) Stir with a diatomaceous earth and filter Fiitrate Cake (1) Add barium chloride Wash and combine and magnesium chloride washes with filtrate. (2) Adjust to pH 8.5 (3) Collect Ba-Mg double salt Filtratc Cake (1) Adjust to pll 3.0 Discard (2) Seed with oxytetracyclinc (3) Adjust to pH 5 and then to pH 7 (4) Collect the crude radioactive oxytetracycline base Filtrato Cake (1) Convert to HCl salt (2) Collect and recrystallize from methanol Radioactive oxytetracycllne.HCl

Example IV Radioactive chlortetracycline was prepared by a procedure analogous to that of Example I using Streptomyces allreofaciens NRRL 2209 as the fermentation organism and sodium 2-C acetate as the radioactive precursor. The chlortetracycline base so obtained had a specific activity of about 5 C/millimole but was otherwise identical with non-radioactive chlortetracycline. It was converted to the hydrochloride salt by suspending a sample of the base in water and acidifying with hydrochloric acid to a pH of 2.0 to 3.0 and then drying the solution from the frozen state. The sodium salt was similarly prepared by drying from the frozen state a solution containing equivalent quantities of chlortetracycline base and sodium hydroxide.

Example V A fermentation was carried out in the same fashion as described in Example I except that 5.0 g. of sodium bromide was added to each liter of the fermentation medium. Spore of Streplomyces aureofaciens NRRL 2209 Were used in preparing the inoculum. This resulted in the formation of a mixture of. radioactive tetracycline and radioactive chlortetracycline which was isolated from the broth and separated by the paper chromatographic technique of H. L. Bird, Jr., et al., Antibiotics and Chemotherapy, vol. iV, p. 750 (1954). This separation involved developing of the crude mixture on Whatman No. l filter paper that had been previously treated with pH 3.0 phosphate buffer using the solvent system ethyl acetate saturated with water. Control runs in the B. subtilis plate assay of the papergrams confirmed the presence of both tetracycline and chlortetracycline and scanning of the papergrams with the methane flow counter apparatus described above demonstrated'that the two tetracycline antibiotics so obtained were of a radioactive type.

What is claimed is:

l. A process for producing a radioactive tetracycline antibiotic selected from the group consisting of tetracycline, oxytetracycline and chlortetracycline, which comprises cultivating a tetracycline antibiotic producing species of Streptomyces on an aqueous nutrient medium containing less than about 10 g./l. of non-radioactive carbon, under submerged aerobic conditions for substantially between 25 and 75 hours, thereafter adding a radioactive assimilable carbon source selected from the group consisting of a C14-labelled carbohydrate, a C14-labelled fatty acid, a C14-1abelled a-amino acid, a C14-labelled carbonate salt, and a C14-labelled bicarbonate salt to said medium and continuing said fermentation until substantial antibiotic activity is obtained.

2. A process as claimed in claim 1 wherein the radioactive tetracycline antibiotic is recovered from the medium at the conclusion of the fermentation.

3. In a process for producing a tetracycline antibiotic selected from the group consisting of tetracycline, oxytetracycline and chlortetracycline, by submerged aerobic fermentation the step which comprises adding to the medium after antibiotic production has begun a radio- References Cited in the file of this patent UNITED STATES PATENTS 2.482.055 Duggar Sept. 13, 1949 2,516,081) Sobin et al. July 18, 1950 2,653,931 lsbell Sept. 29, 1953 2,699,054 Conover Jan. 11, 1955 2,712,517 Gourevitch et al. July 5, 1955 OTHER REFERENCES Chemical Abstracts, vol. 46 (1952), 8329. Gordon et al.: Science, vol. 118 (1953), p. 43. Chem. and Eng. News, vol. 33, Jan. 3, 1955, p. 5.

Claims (1)

1. A PROCESS FOR PRODUCING A RADIOACTIVE TETRACYCLINE ANTIBIOTIC SELECTED FROM THE GROUP CONSISTING OF TETRACYCLINE, OXYTETRACYCLINE AND CHLORTETRACYCLINE, WHICH COMPRISES CULTIVATING A TETRACYCLINE ANTIBIOTIC PRODUCING SPECIES OF STREPTOMYCES ON AN AQUEOUS NUTRIENT MEDIUM CONTAINING LESS THAN ABOUT 10 G./1. OF NON-RADIOACTIVE CARBON, UNDER SUBMERGED AEROBIC CONDITIONS FOR SUBSTANTIALLY BETWEEN 25 AND 75 HOURS, THEREAFTER ADDING A RADIOACTIVE ASSIMILABLE CARBON SOURCE SELECTED FROM THE GROUP CONSISTING OF A C14-LABELLED CARBOHYDRATE, A C14-LABELLED FATTY ACID, A C14-LABELLED A-AMINO ACID, A C14-LABELLED CARBONATE SALT, AND A C14-LABELLED BICARBONATE SALT TO SAID MEDIUM AND CONTINUING SAID FERMENTATION UNTIL SUBSTANTIAL ANTIBIOTIC ACTIVITY IS OBTAINED.