JPS5834115B2 - Antibiotics - Google Patents

Description

[Detailed description of the invention] Antibiotic thienamycin
is effective against both gram-positive and gram-negative bacteria.

This antibiotic is produced by growing a newly discovered, undescribed Streptomyces species in a suitable fermentation medium.

The discovery of the remarkable antibiotic activity of penicillin inspired great interest in this field and led to the discovery of many other valuable antibiotics such as other penicillins, streptomycin, bacitracin, tetracyclines, chloramphenicol, erythromycin, etc. .

Generally, the antibacterial activity of each of these antibiotics excludes certain clinically important pathogens.

For example, some are primarily effective only against Durham-positive bacteria.

The resistance acquired during the widespread use of existing antibiotics in the treatment of bacterial infections has created a serious resistance problem.

Therefore, deficiencies in known antibiotics are stimulating further research to discover antibiotics that are effective against a broader range of pathogens as well as resistant strains of certain microorganisms.

The present invention relates to novel antibiotics.

More specifically, the present invention relates to a novel antibiotic herein referred to as chenamycin.

The invention includes antibiotics in diluted form, crude concentrate and pure form.

It is an object of the present invention to provide new and useful antibiotics that are highly effective in inhibiting the growth of various Durham negative and positive bacteria.

Another objective is to provide a method for the preparation of the novel antibiotic by fermenting a nutrient medium with undescribed microorganisms.

Other objects will become apparent from the detailed description of the invention that follows.

The novel antibiotic of the present invention is derived from the hitherto undescribed microorganism Streptomyces C.
attleya) is grown and produced under controlled conditions.

Based on detailed taxonomic studies, Streptomyces cattleya (S.
s Cattleya) was identified as a previously undescribed actinomycete and was produced by Merck & Co., Rahway, New Chassis (MERKCo, Rahw, New York).
ay, N, J) collected strains (culture c
collection) and was named MA-4297.

This strain was obtained from the Northern Research Institute, Northern Utilization Research and Development Division, Agricultural Research Facility, United States Department of Agriculture, Peoria, Illinois (t
he NorthernBegional Re5e
aroh haboratories.

Northern Utilization Re5e
arch and Development Divi
sion, Agricultural Research
ch 5service, U, S, Departme
nt ofAgriculturelPeoria,
For the permanent deposit of Ill), accession number NRRL80
It was set as 57.

Streptomyces Cattleya MA-4297 Microorganism Accession No. 3309 Manual of Determinative Bacteriology (Bergey)
Determinative Bacteriology
y) 7th edition, 1957 and S. A. Waxman (
The Actinomycetes, Volume 2 (1961) by John Waksman
and E. B. Shirling and R. Gotlieb, “Collaborative Paper on the Type Culture of Streptomyces,” International Journal of Systematic Bacteriology.
of Systematic Bacteriology
y) 18.69-189 (1968) 18.279
-392 (1968), 19.391-512 (196
9) and Streptomyces found in 22.265394 (1972)
Classification key for the genus and Streptomyces (S tr
MA-
We looked for Streptomyces species with morphological and cultural characteristics similar to 4297.

These classical reference books do not describe the Streptomyces species whose aerial hyphae, which are characteristic of MA-4297, are pale purple in color and do not emit soluble pigments.

These inventions are based on new Streptomyces (S trept
omyces) justifies and requires the assignment of species.

Streptomyces Cattleya (S treptomyc
The following table shows the morphological and culture characteristics of esCattleya).

Morphology The sporophyte is helical, occurring as lateral and terminal branches on the aerial hyphae.

The spores are oval or cylindrical, and the size is 0.9μ x 1.2
μ, and consists of 10 or more chains.

Growth of vegetative mycelia on cultured tomato paste oatmeal agar - yellow-backed and colored, flattened, abundant (Spr
eading): Aerial mycelia - light purple mixed with white (10 gc): Soluble pigment - no Czapek Dox agar (
sucrose nitrate agar) Growth of vegetative hyphae - colorless, flattened, abundant: Aerial hyphae - sparse, pinkish white; Soluble pigments - non-ovalbumin agar) Growth of vegetative hyphae - grayish-pale yellowish color;
Squamous, abundant; aerial hyphae - pale purple with light shades of pale purple and some white (10gc); soluble pigments - no glycerin asparagine agar growth of vegetative hyphae - underside grayish pinkish yellow and color; Squamous, abundant; aerial hyphae - light purple with some white (10 gc): soluble pigment - yeast-free extract - glucose + salt agar vegetative hyphal growth - yellowish-brown with a hint of grayish pink; aerial hyphae - pink Pale purple with a hint of white (10gc
): Soluble pigment - yeast-free extract - malt extract agar Growth of vegetative mycelia - yellowish brown; Aerial mycelium - light purple with pinkish white (10gc)
; Soluble pigments - no peptone - iron - yeast extract Agar vegetative hyphal growth - yellow-brown; Aerial hyphae - no soluble pigment - makes the medium slightly brown; Melanin - negative H2S production - negative nutrient agar vegetative hyphal growth - light yellowish Color; Aerial hyphae - Non-soluble pigment - Non-nutrient starch agar Growth of vegetative hyphae - Cream to yellowish brown; Aerial hyphae -
Non-soluble dye-hydrolysis of starch-moderately trophic gelatin agar vegetative mycelia growth-cream color; aerial hyphae-insoluble dye-free gelatin liquefaction-moderate gelatin puncture vegetative mycelia growth-yellow-brown; aerial hyphae - No soluble pigments - no gelatin liquefaction - medium Potato Flag (Plug) Vegetative mycelial growth - medium, yellow-brown color; aerial hyphae - thin, greyish-pinkish white; soluble pigments - no Loefflers Blood
serum) Growth of vegetative hyphae - cream color; Aerial hyphae - non-soluble pigment - non-liquefied - non-skimmed milk agar Growth of vegetative hyphae - yellowish-brown; aerial hyphae - dilute, white soluble pigment - makes the medium slightly and colored hydrolysis of casein - positive litmus Lactotrophic hyphae growth - yellow and to brown; aerial hyphae - colorless - no soluble pigment produced, lidmus indicator turns blue; coagulation and/or peptonization; partially peptonization; Growth of skim milk vegetative hyphae that becomes alkaline - yellow-brown color: Aerial hyphae - no soluble pigment - no coagulation and/or peptonization - tyrosine agar that becomes partially peptonized and alkaline vegetative hyphae - yellow-brown color; aerial hyphae - pale Mixed purple (10 gc) and white; Soluble dye - Decomposition of no tyrosine - Positive All determinations reported above were made after 3 weeks of incubation at 28°C unless otherwise noted.

The pH of the medium used in these experiments was approximately neutral, ie, pH 6.8-7.2.

The color tone expression used in the description is Color Harmony Manual 4
(1958), Container Corporation of America.
on of America) according to the Chicago, Illinois definition.

Streptomyces Cattleya (S treptomyc
es Cattleya) was also tested for its ability to utilize or assimilate various carbohydrates.

For this purpose, the fungi were cultured for 3 weeks at 28°C on a basal synthetic medium (Pridum and Cottribe) containing 1% carbohydrate.

The pH of the culture medium used in the experiment was approximately neutral (6.8~
7.2).

Table 1 shows Streptomyces cattleya (Streptomyces cattleya).
yces Cattleya) shows the utilization of these carbohydrate sources by: + showing good growth on its carbohydrates, + poor growth, no growth.

The effects of temperature changes on bacterial growth, oxygen demand, and nitrate are as follows: Temperature range (yeast extract - glucose + salt agar); 28°C
~Good 37°C - Moderate 50°C - No growth and oxygen requirement (yeast extract - glucose + puncture culture in salt agar); Aerobic nitrate reducing - Positive For the production of the novel antibiotics of the invention, the present invention is Streptomyces cad'ya (S) presented for illustrative purposes.
It should be understood that the present invention is not limited to microorganisms that perfectly match the microscopic growth and microscopic characteristics.

In fact, it is desirable and contemplated to include the use of mutant strains made from the above-mentioned microorganisms by various methods such as X-ray irradiation, ultraviolet irradiation, nitrogen mustard, phage infection, etc.

The novel antibiotic of the present invention, chenamycin, is used in Streptomyces Cattleya (Streptomyces Cattleya).
leya) during aerobic fermentation under controlled conditions in a suitable aqueous nutrient medium.

Aqueous media such as those used for the production of other antibiotics are suitable for the production of chenamycin.

Such media contain sources of carbon, nitrogen and inorganic salts that can be assimilated by the microorganisms.

In general, carbohydrates such as sugars, such as glucose, fructose, maltose, sucrose, xylose, mannitol, etc., and starches, such as grains, such as oats, rye, cornstarch, cornseal, etc., can be used alone or in combination as sources of assimilable carbon in nutrient media. .

The exact amount of carbohydrate source in the medium will depend in part on the other components in the medium, but generally the amount of carbohydrate is usually between about 1% and 6% of the weight of the medium.

These carbon sources can be used individually or several such carbon sources can be combined in the culture medium.

In general, many proteinaceous substances can be used as a nitrogen source in the fermentation process.

Suitable nitrogen sources include, for example, yeast hydrolyzate, yeast powder, soybean flour, cottonseed flour, casein hydrolyzate, corn starch liquor, distiller's solution or tomato paste.

Nitrogen sources are used alone or in combination in amounts ranging from about 0.2% to 6% of the weight of the aqueous medium.

Nutrient inorganic salts that can be used in the culture medium include the common salts that can generate ions such as potassium, ammonium, calcium, phosphoric acid, sulfuric acid, hydrochloric acid, carbonic acid, etc.

Also included are trace metals such as cobalt, mankan, iron and magnesium.

It should be noted that the media mentioned in the examples are merely illustrative of the variety of media that can be used and are not meant to be limiting.

The fermentation is carried out at a temperature in the range of about 20°C to 37°C, although for best results it is preferred to carry out the fermentation at a temperature of about 22°C to 30°C.

Streptomyces Cattleya (S treptomyc
The pH of the nutrient medium suitable for the growth of E. es Cattleya and the production of chenamycin can vary from about 6.0 to 8.0.

Although the new antibiotic chenamycin is produced by both superficial and submerged culture, it is preferred to carry out the fermentation in submerged conditions.

Small-scale fermentation of antibiotics is carried out by inoculating antibiotic-producing bacteria into a suitable nutrient medium, transferring to the production medium, and fermenting on a shaker for several days at a temperature of about 24°C.

Fermentation begins in a sterile flask containing medium through one or more stages of seed expansion.

The nutrient medium for the seed stage may be any suitable combination of carbon and nitrogen sources.

The seed flask is shaken in a thermostatic chamber at about 28° C. for 2 days or until growth is sufficient, and a portion of the resulting culture is used to inoculate the second stage seed or production medium.

When using an intermembrane seed flask, it is expanded in essentially the same way: a portion of the contents of the flask from the previous seed stage is used to inoculate the production medium.

The inoculated flask is shaken at a constant temperature for several days, and after the incubation period the contents of the flask are centrifuged or filtered.

For large-scale fermentations, it is preferred to carry out the fermentation in suitable tanks equipped with a stirrer and ventilation.

According to this method, a nutrient medium is prepared in a tank and sterilized by raising the temperature to about 120°C.

After cooling, the sterilized medium is inoculated with pre-grown seeds of the production bacteria, the nutrient medium is agitated and/or aerated, and the temperature is brought to about 24°C.
Fermentation is carried out for a certain period of time, for example 3 to 5 days, while maintaining the temperature at .degree.

This production method of chenamycin is suitable for the preparation of large quantities of antibiotics.

Physicochemical Properties of Chenamycin Chenamycin is a white solid that is highly soluble in water and slightly soluble in methanol.

The apparent molecular weight in phosphate buffer was determined by analytical ultracentrifugation using short column sedimentation and ultraviolet absorption optics to detect a sedimentation zone of material with absorption at 300n71L and was approximately 282 Daltons. Calculated.

Field-desorption, mass spectrum shows a molecular weight of 2720.

From energy dispersive analysis of X-rays emitted by the antibiotic under the beam of a scanning electron microscope, a bond molecular weight of 290±20 was determined based on sulfur, the only element with an atomic number of 9 or higher, present in significant amounts.

Chenamycin has the empirical formula C1°H16N204S as deduced from high resolution mass spectrometry.

The calculated elemental composition corresponding to this empirical formula is 48.52% carbon.
, hydrogen 5.92%, nitrogen 10.29%, oxygen 23.5%
and 11.77% sulfur.

Infrared absorption spectrum of chenamycin in Matashor (
Figure 1) shows the following absorption peak: 1765crrL'
Sharp peaks at 1650-1550crIL-1 2800-2500C1rL-1 and broad peaks at 3500-3100crIL-1 Other absorption bands are 1290, 1245.1150-1130.10
65.995.970.945-935.885.80
5.785 and 720 (m').

Concentration of chenamycin lyophilized from heavy water 1.5 m9
10.4rnlD20, a small amount of internal reference material 2.2-
The 100 MHz NMR spectrum containing sodium dimethyl-2-silapentane-5-sulfonate shows the following peaks: δ 1.275, d, 3H, J = 6.8; δ 3.15
, m, 6H: δ3,39, dd , IH, J=6.0.2.9; δ4
．． 20. m, 2H Chenamycin has a specific optical rotation [α917°C + 82.7 (
C=0.1%, W/V, in 10mM phosphate buffer;
pH 6.95).

Optical rotational dispersion shows one positive Kotton effect with a peak at 31111 m and a trough at 242 Hm, circular dichroism is 28
It shows a positive maximum at 7.5 nm and a negative maximum at 216 nm.

The ultraviolet absorption spectrum of chenamycin in aqueous solutions in the pH range from 4 to 8 has a peak of 1% at 296.5 nm (E = 290, ε-7900) at 242 crr.
A valley (E = 88) is shown at L 1% nm.

The spectrum taken immediately after the preparation of the solution CrrL at pH 2 shows the thickest absorption at 309 nm.
Indicates red color movement to.

The spectrum taken immediately after adjusting the pH to 12 shows the absorption maximum at 3.
The red shift to 0.00.5 nm is shown.

Chenamycin is an internally neutralized amphoteric substance;
its acidic function
When measured using the pH-induced shift in the ultraviolet absorption spectrum in an acid buffer (30 mM'), the pk
It has al=3.08.

Ninhydrin, iodoplatinate and Foli
n) Shows a positive color reaction to the reagent.

Grain test is negative.

Chenamycin was subjected to thin layer chromatography on a cellulose plate containing a fluorescent reagent and mixed with ethanol:water (70:30).
) is detected at an Rf value of 0.45 to 0.50 by each of the following criteria used separately: (1) The test bacterium Staphylococcus aureus (S taphylo
coccus aureus) MB-108.

Staphylococcus aureus (Staphylococcus aureus)
Bioautography on an agar plate inoculated with ATCC6538P (2) Extinction of fluorescence excited by projected ultraviolet light, (3) Pink color reaction by ninhydrin spray and heating, (4) Platinum iodine Acid spray reagent yutsu (sushi bleaching).

The mobility of chenamycin in several other chromatography systems is shown below:
It is a trademark of ``at ion''.

The Rf value is calculated from the origin divided by the distance of the solvent front from the origin to the center of biological activity (Staphylococcus aureus).
) means the distance determined by ATCC6538P bioassay).

Model KLA-8 Hitashi-Derhin
Amino acid analysis of the acid hydrolyzate of chenamycin performed on an E1mer analyzer showed one major ninhydrin-reactive component, with a retention time intermediate between alanine and glycine, with an absorption at 440 nm of that at 540 nm. Gives ninhydrin reaction product that is 3 times as large.

A typical method used to convert antibiotics to thioethers and sulfhydryls to sulfones and sulfonic acids, respectively, is hydrolyzed by cold formic acid followed by reaction with embodied hydrogen; amino acid analysis yields slightly larger than aspartic acid. It shows one major ninhydrin-reactive component with a retention time of 540n77L and an absorbance of 42On7.
Gives ninhydrin reaction product which is 3.2 times that of 7L.

This component was found to be the same as taurine (2-aminoethanesulfonic acid) by comparison with the elution time of a standard substance subjected to similar oxidation and hydrolysis.

The proportion of taurine recovered from this comparison was determined to be 0.8 μmol per chenamycin reacted.

Chenamycin has a p
It exhibits the best stability in aqueous solution when kept in the presence of a phosphate buffer keeping H between 6 and 7.

For example, a 11n9/1rLl solution in water (pH 7,0) is 0
Half-life of 20 days when kept at ℃
while at the same concentration in 10 mM potassium phosphate (pH 7.0) the half-life increases to 37 days.

However, 10mM of antibiotic! When a 12μ/ml solution in J acid buffer is kept at 10°C, it is only 3%
Indicates the half-life in days.

The solution is initially yellowish-white but takes on a deep amber color after 48 hours of storage.

The half-life of chenamycin at room temperature in the acidic pH range is 1346 minutes, pH 4.0; 30 minutes, pH 3.0; 6.7.
min, pH 2.0, and in each case 30 mM phosphate buffer is used for hydrogen ion adjustment.

Half-life at 30°C under alkaline conditions is 110 minutes, pH 8
, 2; 5.7 min, pH 8,8 (borate buffer) and 2,1 min, pH 12 (0,01M sodium hydroxide)
It is.

When kept at 20°C at pH 7.0 in a reaction mixture containing 0.05 μmol of antibiotic and 4 μmol of hydroxylamine per rul, the antibiotic activity and UV absorbance measured at 296.5 μm were determined with an accurate half-life of 5.7 min. Decrease in percentage.

The reagent is 4 μmol cysteine per rrLl, pH 7.0.
When kept at 20°C, a half-life of 2.5 minutes is observed.

Finally, the antibiotic activity and absorbance disappear upon treatment with β-lactamase obtained by sonication of an Enterobacter cloacoe strain.

Chenamycin is thought to have the following molecular structure: Therefore, the present invention provides a compound represented by the general formula (chenamycin) and a bacterium containing the compound represented by the general formula (chenamycin) as a main component. Concerning agents for treating infectious diseases.

More specifically, the bacterial infection therapeutic agent has a content of the compound represented by the general formula (chenamycin) of at least 50 units/rIll, and the content of the compound represented by the general formula (chenamycin) is At least 2 units/■(
The present invention relates to the above-mentioned bacterial infection therapeutic agent which is solid).

Chenamycin is further characterized by the following antibacterial spectrum:

The test was conducted by Bauer-Kilby (Bauer-K 1rb).
Using the disk diffusion method of y), only the thickness of the agar used in the two cases used here was corrected.

Table 1 shows the results expressed in millimeters of direct continuation of the blocking circle.

Further evaluation of sufficiently pure antibiotics used the agar dilution method.

0.002ml containing 2X10' to lXl0'' bacteria
A drop of 0.01 to 73tt? Mueller-Hinton agar surfaces containing serial 2-fold dilutions of antibiotics ranging from /rIll are inoculated onto Mueller-Hinton agar surfaces.

The results (Table 2) are reported as the minimum inhibitory concentration μ9/rul, which is the lowest antibiotic concentration at which growth of the test bacteria is inhibited over 18 hours at 37°C.

Chenamycin exhibits in vivo activity against Gram-negative and Gram-positive bacteria and is therefore effective in controlling bacterial infections in host animals.

To measure in vivo activity, chenamycin was added to 0
．． 15M NaCl, 0.01M sodium phosphate,
Dissolve and dilute at pH 7.0 to obtain 5 concentrations of 4-fold dilutions of drug for testing.

Female White Swiss mice with an average body weight of 211 were infected intraperitoneally and subcutaneously with the test bacteria suspended in culture medium.

The number of bacteria injected was determined by standard plate counting method.

Mice were treated intraperitoneally, subcutaneously and orally with certain antibiotics at the time of infection and again 6 hours later.

Five mice were used for each concentration of drug tested.

Furthermore, two uninfected mice were treated with antibiotics to determine whether the amount of the injected drug was toxic.

Five mice were used as controls for each dilution of infected bacteria to calculate the number of bacteria that would kill 50% of the infected and untreated mice (LD 50 ).

This calculation was performed using the survival number 7 days after infection, and the drug dose (ED50) that would make 50% of the infected mice survive was calculated.

All animals that underwent this challenge but were not treated with antibiotics died within 48 hours of infection.

The potency of chenamycin at a concentration of 930,000 units is reported in Table 3: Low toxicity to mammals is confirmed as no adverse effects were observed for 7 days after intravenous administration of 500 η/kg of mouse body weight of the antibiotic. Ta.

Chenamycin is a valuable antibiotic that is effective against a variety of Durham-positive and -negative bacteria and therefore can be used in human and animal medicine.

The antibiotic of the present invention can be used as an antibacterial agent for the treatment of infections caused by Durham-positive or -negative bacteria, such as Staphylococcus aureus.
s), Proteus mirabilis (Proteus m1
labilis), Escheritsxakoli (Escher
ichia coli), Klebsiella Pneumoniae,
Pseudomonas aeruginosa
saeruginosa) and Enterobacter cloacae
Can be used for

The antimicrobial substances of the invention can furthermore be used as additives in animal feed, for food preservation and as disinfectants.

For example, this antibiotic contains 0.1 to 10 parts per million in solution.
As an aqueous component with a concentration in the range of 0 parts, for destroying and inhibiting the growth of toxic bacteria on medical and dental instruments, and in the industrial field, for example in white water in the water-based paint and paper industries. It can be used as a disinfectant.

The antibiotics of the invention may be used as sole active ingredients or in combination with one or more other antibiotics or in combination with one or more pharmaceutically active substances in any of a variety of pharmaceutical preparations. can also be used.

An example of the former is gentamicin.
) can be administered together to minimize the chance of developing resistant bacteria.

As an example of the latter, ginenoxylate and atropine can be combined in a dosage form for the treatment of gastroenteritis.

Antibiotics can be used in capsules or as tablets, powders or solutions or as suspensions or elixirs.

It can be administered orally, topically, intraperitoneally or intramuscularly.

Tablets and capsules for oral administration may be in unit dosage form, with binders such as syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers such as lactose, sugar, corn starch, calcium phosphate, sorbitol. or glycine: lubricants such as magnesium stearate,
It can contain the usual excipients such as: talc, polyethylene glycol, silica: deliquescent agents, acceptable wetting agents such as potato starch or sodium lauryl sulfate.

The tablets can be coated in a conventional manner.

Oral liquid preparations include water or oil suspensions, solutions, emulsions, syrups,
It can be made into an elixir or the like, or it can be presented as a dry product for reconstitution with water or other suitable carrier before use.

Such liquid preparations may contain suspending agents such as sorbitol, syrup, methylcellulose, crucose/sugar syrup,
gelatin, hydroxymethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats; emulsifiers such as lecithin, sorbitan monooleate or gum arabic; edible oils such as almond oil, fractionated coconut oil, oily esters, propylene glycol, or ethyl Conventional additives may be included, such as a non-aqueous carrier including an alcohol; a preservative such as methyl or propyl P-hydroxybenzoate or sorbic acid.

Herbal medicines include the usual herbal bases such as cocoa butter or other glycerides.

Compositions for injection may be presented in unit dosage form in ampoules or in multi-dose containers with a preservative.

The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous carriers and can contain forming agents such as suspending, stabilizing, and/or dispersing agents.

Alternatively, the active ingredient may be in powder form for reconstitution with a suitable carrier, eg, sterile pyrogiene-free water, before use.

The compositions may also be prepared in a suitable form for absorption through the mucous membranes of the nose and throat or the bronchial tissues, and may conveniently take the form of powder or liquid sprays or inhalants, lozenges, throat liniments, and the like.

For administration to the eyes and ears, the preparations can be presented as individual capsules, liquid or semi-solid, and can be used as drops.

Topical preparations can be formulated in hydrophobic or hydrophilic bases such as ointments, cream lotions, paints, powders, and the like.

In addition to carriers, the present invention also provides stabilizers, binders, antioxidants, 1. Other ingredients such as preservatives, lubricants, suspending agents, thickening agents or flavoring agents may be included.

In veterinary medicine, such as in the treatment of chickens, cattle, sheep, pigs, etc., the compositions can be formulated, for example, as intramammary preparations in long-acting or rapidly deliquescent bases.

The dosage to be administered depends on the symptoms to be treated, the host's weight,
Parenteral administration is preferred for common infections and oral administration is preferred for enteric infections, although this largely depends on the type of infection, route and frequency of administration.

In the treatment of bacterial infections in humans, compounds of the invention may be administered at doses of about 2 to 600 my/k according to conventional methods of antibiotic administration.
It is administered orally or parenterally, preferably in an amount of about 5 to 100 g/ky/day, preferably in divided doses, eg 3 to 4 times a day.

They can be administered as a dosage unit containing, for example, 25.250.500 or 1000 μ of active ingredient together with suitable physiologically acceptable carriers or excipients.

Dosage units can be in the form of liquid preparations, such as solutions or suspensions, or solid, such as tablets or capsules.

Of course, it should be understood that the optimal dosage in a given case will depend on the type and severity of the infection to be treated, with lower doses being used for children, and all these adjustments should be made by experts in the field. is clear.

The fermentation solution containing antibiotics prepared according to the method described herein is a fermentation solution containing Staphylococcus aureus (5taphylococcus aureus).
ococcus aureus) ATCC6538
It has an activity of about 50 to 400 units per 1'll when assayed by disk diffusion method using P.

Antibiotic preparations having at least 2 units per square can be purified and the antibiotic recovered in a number of ways.

One such method is characterized by the adsorption of chenamycin onto a strong cation exchange resin.

Examples of such resins include those of the sulfonic acid type with styrene divinylbenzene crosslinks, such as the polystyrene core sulfonic acid resin Dowex 5Q
X41 Dow Chemical Company, Midland, Michigan (D
ow chemical Co1M1dland Mi
There is a sodium form of chigan).

Other representative examples of strong cation exchange resins include: DOWEX 50X4, DOWEX 50X8 (Dow Chemical Company, Midland, Mich.), Amberlite IR120 (Rohm and Haas Company). , Philadelphia, Pennsylvania (Ror
m&Haas Co, Ph1ladelphia P
enn5ylvania)), Duo Light (Duo
lite) C25D C Chemical Process, Inc. Redwood City, California (Chemical
Process (:0.Redwood C1ty
.

Ca1ifornia) ), palm tree (Pe
rmutit ) Q [Permutit Co., Hermingham, New Chassis (Permutit Co., B
irmingham.

New Jersey ), Ionac
) C249 (manufactured by Ionac Chemical, Birmingham, New Chassis)
al Co.

Birmingham 1New Jersey ))
and Amberlite) 20
0.

A further method of obtaining purified chenamycin is the use of gel filtration through a polyacrylamide gel with a pore size that excludes molecules having a molecular weight greater than 1800.

A preferred gel is Biogel (Bi.-Gel) P
-2 (Bio-Rad, Richmond, California).

California)).

Adsorbed antibiotics are easily eluted from the cation exchange resin with aqueous solutions of ammonia or organic bases such as pyridine, picoline, lutidine, collidine and alkyl amines.

The eluate thus obtained can be further purified by other purification methods if desired.

The eluate is thus passed through a polyester polymer such as XAD-7 or 8, or XAD-1, 2 and 4;
Preferably, it can be purified through a polystyrene hydrophobic crosslinked divinylbenzene polymer such as XAD-2.

A preferred method of recovering purified chenamycin is to prepare a solution containing the antibiotic, such as the fermentation filtrate, to a pH of between 4 and 5 and transfer it to a column containing a strong cation exchange resin of the sulfonic acid type (Dowex 50X2). It is passed through sodium type.

The resulting adsorbate is then eluted with a suitable eluent such as a 2% aqueous pyridine solution.

The eluate is fractionated, and the amount of fractionation depends on the size of the column used.

Further purification is obtained by a series of methods involving the following chromatographic solvents: anion exchange resins of the polystyrene triethylammonium type (e.g. the hydrochloric acid form of DOWEX-I), cation exchange resins of the polystyrene-sulfonic acid type (e.g. 2,6-lutidinium type of DOWEX-50),
Gel pore resins (e.g. Pyogel P-2, polyacrylamide resins) and polymeric adsorbents (e.g. XAD
-2 polystyrene resin) eluate was determined by comparing the eluate with Staphylococcus aureus (5taphylococcus
ccus aurous) ATCC6538P as a test bacterium.

The following example describes how to obtain the products of the invention.

However, it will be apparent to those skilled in the art that the examples are for illustrative purposes only and that the invention includes functionally equivalent products and methods of their preparation.

Therefore, the same product is produced. Modifications to the methods described herein should be construed as constituting analogous methods.

The described method is susceptible to occasional variations and modifications and minor deviations or extensions are within the knowledge of the expert and are considered to be within the scope of the invention.

Assay Antibacterial activity is assayed according to the following disk diffusion method, unless otherwise indicated.

Prepare the assay plate as follows.

Test bacteria, Staphylococcus aureus in nutrient medium + 0.2% yeast extract
Add overnight culture of ATCC6538P to nutrient medium + 0.2%
Dilute with yeast extract to a suspension with 55% transmission at a wavelength of 660 mμ.

This suspension also contains Difco yeast extract 2.
0? /l of Difco nutrient agar at 47°C to 48°C to obtain 33% suspension per liter of agar.
．． Obtain a composition containing 2 ml.

5TrLl of this suspension is poured into a Petri dish with a diameter of 857IL1L, and the plate is cooled and kept at 4°C until use (up to 5 days).

The antibiotic sample to be assayed is diluted to the appropriate concentration in pH 7 phosphate buffer.

A 12.7 mm diameter mouthpiece disc is immersed in the test solution and placed on the surface of the assay grate, usually two discs for each sample are placed on either side of the plate.

Plates are incubated overnight at 37°C and the zone of inhibition is measured between diameters.

The zone of inhibition measured in terms of diameter determines the relative concentration or unit/unit antibiotic concentration when compared to a purified control standard such as cephalothin.

The activity units reported in Examples 4 to 7 are 8.4.2 and 1μ? /rul based on cephalothin standard solution.

Is 1 unit 1μ of cephalothin? /rrdl is calculated to exhibit the same blocking, ie the blocking circle is defined as the amount between 16 and 21 diameters.

Example 1 Streptomyces Cattleya (S treptomyc
The lyophilized tube of esCattleya was opened aseptically and the contents were poured into a sterile Davis tube with the following composition:
s) Suspend in a test tube containing 0.7 ml of salt; Davis (I) avis) Salt-inoculated slants are incubated at 28°C for 8 days and then stored at 4°C.

The spores and aerial mycelial portion of this slant are used to inoculate a 250 ml pleated Erlenmeyer type 1 flask containing 50 ml of medium A (no agar).

Shaking this seed flask at 28° C. on a 22 Orpm shaker (2 inch amplitude) for 2 days is sufficient for growth.

15 250 ml tubes each containing medium B40rILl
Inoculate Rehnmeyer flasks with the culture from the seed flask, 1 ml per flask.

Medium B has the following composition: Medium B These 15 production flasks were incubated at 28° C. for 22 Orp.
Shake on a m shaker (2 inch amplitude) for up to 3 days and assay during the fermentation period.

The assay is performed using centrifuged culture fluid.

Before the assay, adjust the pH of the culture medium as shown in the table below.

At 53 hours of age, cultures from 13 flasks are collected.

Centrifuge a portion and assay. Pass the remaining culture solution through the mouth, and
Adjust to H7 and freeze-dry 500 ml to obtain a solid 10.
Get 7 fl.

1.5i of this solid was mixed with n-butyl alcohol:water (1
:99) Dissolve in 25 ml.

The pH of the solution is 7.0. This solution is preliminarily
Butyl alcohol: Biogel equilibrated with water (Bio
-Gel) P-2 (200-400 mesh) 5 x 118 CrfL column.

The gel was developed with the same solution at 10 ml/min and 650 TLl
After pre-development, collect 75 fractions of 20ml each.

The eluate was measured using a Mecomatic recording differential refractometer (Mecco-m).
aticrecoding differentia
Monitor with lrefractometer).

Each fraction is assayed for antibacterial activity.

Chenamycin is found in fractions 34 to 40 with a maximum in fraction 37.

1Qml of fraction 37 was freeze-dried to obtain 2.0ml of solid matter.

The resulting solid is dissolved in 5 mIC water for assay.

Incubate the assay plate overnight at 28°C.

The results are shown in the table below:
The lyophilized tube of sCattleya was opened aseptically and the contents were poured into a sterile Davis tube with the following composition:
) Suspend in 0.8 ml of salt: Davies salt This suspension is used to inoculate 4 slants of medium A (additional agar) with the following composition: Medium A For slants: agar 25 .0? The inoculated slant to which 1/l is added is incubated at 28°C for one week and then stored at 4°C.

The spores and aerial mycelium of one slant were placed in medium A3011.
250TLl Erlenmeyer (Er) with pleats including 11
lenmeyer) is used to inoculate the seed flask.

Incubation of this seed flask for 2 days at 28° C. on a 220 rprn shaker (2 inch amplitude → top) will provide sufficient growth.

15 pieces each containing medium B4Om1) 250rrtl A-
Reimeyer flasks are inoculated with 1=l of culture from the seed flask per flask.

Medium B has the following composition: These flasks were placed on a 22 Orpm shaker (
2 inch amplitude) for 3 days and assayed during the fermentation period.

The assay is performed using the supernatant of the centrifuged culture.

The results are as follows: At 53 hours of age, cultures from 13 flasks are collected and sifted.

The pH of 400a parts of the oral fluid is adjusted to 4.8 with dilute hydrochloric acid, and this is adsorbed onto DOWEX 50×2Na+ 140rnlE at a rate of 14rrLl/min.

Wash the adsorbate with 200 rrlls of deionized water and collect 6×70 ml fractions by elution with 2% pyridine in water.

Adjust the pH of the fraction to 7.0.

When assaying all fractions, the inhibition circle diameters are as follows: These assays indicate that 45% of the activity is contained in the eluate.

Elution fraction #2 was lyophilized to give a solid substance of 117 rrU? get.

117% solids were dissolved in n-butyl alcohol:water (1:9
9) Dissolve in 1.5III.

1.4 of Bio-Gel P-2 whose solution was equilibrated with n-butyl alcohol:water in advance.
Transfer to a X81.5 CIII column.

The gel is developed with n-butyl alcohol:water at 1 rnl for 1 minute and 2 ml fractions are collected.

The elution flow is monitored with a Mecomatic recording refractometer.

The antibacterial activity of the fractions is assayed at a 1:20 dilution.

The biological activity peak is observed in fractions 44-53.

Combine fractions 46 to 49 and half of fraction 500.

The Lrul sample of the combined fractions is removed for reassay and the remainder is lyophilized to yield 4.2 Ing of partially purified antibiotic.

Example 3 Streptomyces Cattleya
sCattleya) is aseptically opened and the contents suspended in 0.8 ml of sterile Divis salt having the following composition:

This suspension is used to inoculate four slants of medium A (plus agar) with the following composition: Medium A The inoculated slants are incubated overnight at 28°C and stored at 4°C.

The spores and aerial mycelium of one slant were each placed on medium A30.
Used to inoculate three pleated 250 ml Erlenmeyer Type 1 flasks containing ml.

These seed flasks were placed on a 220 rpm shaker (
2 inch amplitude) for one day is sufficient for growth.

Twelve 200 ml Erlenmeyer flasks, each containing medium C2C25O, are inoculated with 7 ml of a suspension aseptically collected from the contents of three seed flasks.

Medium C has the following composition: Shake the flasks at 28°C on a 220 rpm shaker (2 inch amplitude) for 72 hours.

The culture fluid is collected and aliquots are centrifuged for assay.

The collected culture solution has a pH of 7.4, and the antibacterial activity using the centrifuged supernatant is 431rL71L.

Pass the culture solution through the mouth, adjust the pH of both solutions to 4.0 with diluted HCI, and add 3000ml to DOWEX 50 x 2Na + 300ml.
1 at a speed of 30 ml and 11 minutes.

The adsorbate is washed with 300 ml of deionized water and eluted with 2% pyridine to collect 8 x 150 I7I1 fractions.

Adjust the pH of the fraction to 7.0.

Elution fractions #2 and #3.300 ml were collected and contain 48% of the total bioactive material loaded onto the Dowex 50 x 2 Na+ column.

One 280 ml portion of the collected fractions of the DOWEX 50 x 2 Na eluate at pH 7.0 obtained above was injected into DOWEX 1X.
Pass through a 40rnl column of 2C1-type resin.

Wash the resin with 160 ml of deionized water. Collect the flow-through to obtain 440 ml of solution.

This solution was adjusted to pH 8.2 with dilute sodium hydroxide, concentrated under reduced pressure to 300 ml, adjusted to pH 7.0 with dilute HCl, and lyophilized to obtain 189 ml of solid material.

189rv of the lyophilized solid obtained above is dissolved in n-phthyl alcohol:water (1:99).

Biogel (Blo-Ge1) P-2 (2
00-400 mesh) column, heat to 5X108α.

The gel is then developed with n-butyl alcohol:water at 6.7 ml/min.

The elution flow is monitored with a Mecomatic recording differential refractometer.

After taking 500 ml of pre-eluate, fractionate into 20 ml portions.

The antibacterial activity of each fraction is assayed at a 1:25 dilution.

Biological activity is seen in fractions 37 to 42, with maximum in fraction 39.
It is.

Fractions 38 to 41 with a total volume of 80 ml are combined.

The solution is concentrated to 10 ml, pH 7.0 and lyophilized.

The freeze-dried solid was 13.57/lp of Staphylococcus aureus.
(us) has a relative activity approximately 6 times that of the sample applied to a Biogel P-2 column, based on a comparison by disk diffusion assay.

Example 4 Streptomyces Cattleya
sCattleya) and the contents are suspended in 0.8 ml of sterile Davis salts having the following composition: Use to inoculate book slants: Inoculate slants at 28°C for 1 week, then store at 4°C.

Aseptically transfer 10 ml of medium A to one of these slants, scrape and suspend the spores and aerial mycelia, and inoculate 3.3 ml of this suspension into a 2t pleated Erlenmeyer Blask containing 300 ml of medium A. used for.

Shaking this seed flask at 28° C. on a 16° rpm shaker (2 inch amplitude) for 48 hours is sufficient for growth.

The culture solution from this seed flask was mixed with medium A160t.
Used to inoculate 901 stainless steel type tanks.

The tank is operated for 24 hours at 28° C. with an agitation number of 15 Orpm and an aeration rate of 85 t/min.

Add as much antifoaming agent, Polyglycol 2000 (Dow Chemical Company) as necessary, but not more than 0.1%.

The results of pH measurement are as follows: 43 tons of the culture solution in this type of tank was mixed with medium E with the following composition.
Used to inoculate a 756t stainless steel fermentor containing 467t: Medium E The tank is operated at 24°C for 120 hours using an agitation rate of 95 rpm and an aeration rate of 283, ff/min.

In addition, an antifoaming agent, polyglycol 2000, is added as much as necessary, but not in an amount exceeding 0.1%.

The data from the antibacterial assay are as follows: I say something.

(ethylene-2-nitrile)tetraacetic acid sodium salt 1.2
Add y' to both solutions.

Both solutions were cooled to 6°C and adjusted to pH 4.0 ± 0.2.
Keep the cold liquid at 6℃ and add DOWEX 50X4Na+ (2
0-50 mesh) 38. /- at 47/min.

The adsorbate was eluted with a 2% aqueous pyridine solution, and 19t' of 3
Collect and assay two fractions.

In the antibacterial assay, elution fractions 2 and 3 were charged with 22
Indicates that % is included.

These fractions are combined and concentrated to 3.8t and the pH adjusted to 7.

3.8 tons of the concentrate obtained above was mixed with Dowex 1 x 2.50 to 100 mesh, and 2.5 tons of hydrochloric acid type resin was added to 20 o'r.
Adsorb in 1 min.

Elute the resin with deionized water at the same rate.

Collect the 10 fractions and adjust each fraction to pH 7.0 if necessary.

The assay shows that 75% of the biological activity is in fractions 5 to 8.

A total of 0.45 micron millipores (Mi
lliPore).

Three parts of both solutions were freeze-dried to obtain 16.61 solids having an activity of 70 units/1n9.

The lyophilized solid 41 obtained above is dissolved in 5Qm of 0.1M 2.6 lutidine acetate buffer.

A solution of pH 6.3 was prepared as follows: DOWEX 5
Convert the 2.51 DOWEX 50X8 (200400 mesh) hydrogen type resin to the 2,6-lutidine type by applying it to a 0x8 column.

Equilibrate the resin with 6 column volumes of 0.1 M 2.6-lutidine acetic acid, pH 6-3, buffer.

The size of the equilibrated resin column is 5 x 114 cm.

The column is developed with 0.1 M buffer at 14 TLl/min.

The elution flow is monitored with a mekomatic recording differential refractometer.

The development is carried out until 220 fractions are collected in 20ml increments.

Every other fraction from 44 to 142 is assayed at a 1:50 dilution.

Biological activity was found in fractions 78 to 136;
It reaches its maximum at 94.

Select fractions 82 to 116 to obtain 700 ml of combined solution.

Lyophilize the solution in two 350ml portions.

One portion of the lyophilized solid is dissolved in 0.1 M 2.6-lutidine acetic acid, pH 7.0, buffer.

Bio-Gel P- in which solution 27+711 was equilibrated with 0.1M buffer in advance
Transfer to 2 (200,400 mesh) 5X108 cIIL column.

The gel is then developed with the same buffer at 10 ml/min.

The elution flow is monitored with a Mecomatic recording differential refractometer.

Continue development until 105 fractions have been collected in 20ml increments.

Each fraction from 51 to 90 is assayed at a 1:5o dilution.

The assay showed activity peaks in fractions 67 to 75, and fraction 70.
It has a maximum from 71 to 71.

Fractions 68 to 75 are combined to obtain 1621rLl.

3ml of 360mM, pH 7.0 sodium phosphate buffer was added to the combined fractions 1451rL, and the solution was diluted to 9ru.
Concentrate to l.

78 of the total biologically active substances exposed to the Biogel P-2 column
This concentrate containing % is lyophilized to obtain 1851n9.

The remaining 177711 aliquots of fractions 68 to 75 were lyophilized to give 1 solid with an activity of 1050 units/m9.
．． Get 9■.

Example 5 Streptomyces Cattleya
Aseptically open the freeze-dried tube of sCattleya and empty the contents into 0.8 m of sterile Taviis salt having the following composition:
This suspension is used to inoculate four slants of medium A (additional agar) with the following composition: The inoculated slants are incubated for one week at 28°C and then at 4°C. Save with .

Aseptically transfer 10 ml of medium A to one of these slants, scrape and suspend the spores and aerial mycelia, and
m1ta: Used to inoculate a 2e fluted Erlenmeyer flask containing 500 rnl of medium A.

The seed flask is shaken at 28°C on a 16 Orpm shaker (2 inch amplitude) for 48 hours.

The culture medium from this species flask was mixed with medium A160J.
Used to inoculate 90J stainless steel type tanks.

The tank is operated for 24 hours at 28° C. using an agitation number of 15 Orpm and an air flow rate of 851/min.

Antifoaming agent, polyglycol 2000 (Dow Chemical Company)
Use as much as necessary, but not more than 0.1%.

The results of pH measurement are as follows: 35 J of the culture solution in this species tank was mixed with medium E having the following composition.
Used to inoculate a 7561 stainless steel fermenter containing 467J: This tank is operated for 12020 hours at 24°C using an agitation rate of 95 rprrl and an aeration rate of 2831/min.

Add as much antifoaming agent, Polyglycol 2000, as necessary, but not more than 0.1%.

The data obtained from the antibacterial assay are as follows: 400 J of the total culture solution is passed through the mouth using a squeeze filter and a mouth filter aid of 40% W/V.

(ethylene-2-nitrile)4acetic acid sodium salt 1,2
? Add to both solutions.

Both solutions were cooled to 6°C and adjusted to pH 4.0 ± 0.2.
Keep at ℃.

The cold liquid is adsorbed onto DOWEX 50x4Na'', (20-50 mesh) 381 at 41/min.

The adsorbed matter is eluted with a 2% aqueous pyridine solution, and three fractions of 197 each are collected and assayed.

The assay shows that 27% of the dim biological activity is contained in fractions 2 and 3.

Elution fractions 2 and 3 are combined, concentrated to 3.71, and adjusted to pH 7.

Eluate concentrate 3.71 was adjusted to pH 7.4 and treated with Dowex 1X2cl-type resin (50-100 mesh) 2.5
Adsorb 200rnl in J for 1 minute.

The resin is then eluted with deionized water at the same rate.

2x21, lx800m111x4. J and 1X21 fractions are collected and the pH adjusted to 7.0 if necessary.

Fraction 4 (4, J) containing 50% of the activity in the concentrate was added to 0.
455 Millipore) Sip using a mouthpiece.

Freeze-dry both clear liquids to create sculpture 1 with an activity of 270 units/■
2.4? I got it.

Lyophilized solid 41 is dissolved in O,1M 2.6-lutidine acetate buffer pH 6.3.

Add acetic acid to 50rrL of solution to adjust pH to 6.3,
Dowex 50 x 8.2-6-lutidine type resin (200-400
Mesh) column, 5X114crfL.

The resin was then washed with the same OlIM pH 6.3 buffer at 147.
It develops at 711/min.

The elution flow is monitored with a mekomatic recording differential refractometer.

The development is carried out until 150 fractions of 20 rILl have been collected.

Every other fraction from 72 to 150 is assayed at a 1:50 dilution.

One bioactive peak in fractions 76 to 148 and in fraction 9.
2 to 102 with a maximum.

Fractions 86 to 113 were combined and applied to a Dowex 50x8 column. A solution containing 71% of the biological activity was 580 rI'Ll.
get.

This solution is then lyophilized.

The resulting lyophilized solid was dissolved in 0.1 M 2.6-lutidine acetic acid pH 7.0 buffer to give 25+711.

Bio-Gel P-2 (20
0-400 mesh) column, 5X warm to 108 temperature.

The gel is then developed with the same buffer at 9ru13/min.

The elution flow is monitored with a mecomatic recording refractometer.

Development is carried out until 105 fractions have been collected in 20ml increments.

Each fraction from 65 to 80 is assayed at a 1:200 dilution.

The bioactive peak is seen in fractions 67-76.

Fractions 70 to 72 are combined and lyophilized to yield 16.0 ml with an activity of 13,800 units/s.

Fractions 69, 73 and 74 were combined and lyophilized to 5200
We obtain 20.2779 with an activity of units/■.

16 μm of the freeze-dried solid was dissolved in 0.1 M 2,6-lutidine acetic acid pH 7.0 buffer to make 10 μm.

Biogel P-2 (200-400 mesh) column, 5X10, where the solution was pre-equilibrated with the same buffer.
Apply to 8cIrL.

Develop the gel with buffer at 9rrLl/min.

The elution flow is monitored with a mekomatic recording differential refractometer.

Continue development until 105 fractions have been collected in 20ml increments.

All fractions from 65 to 80 are assayed at a 1:200 dilution.

The bioactive peak is seen in fractions 67-75.

Fractions 68 to 72 were combined and lyophilized to yield 15,000 units/
9-1m9 with the activity of (2) is obtained.

Example 6 Streptomyces Cattleya (S treptomyc
The lyophilized tube of esCattleya) is aseptically opened and the contents suspended in 30 rul of sterile medium A contained in a 250 TLl pleated Erlenmeyer flask.

Medium A has the following composition: Shake the inoculated flask at 28°C on a 22 Orpm shaker (2 inch amplitude) for 48 hours.

Aseptically remove 401rLl of the 48-hour culture solution and sterilize it for 20 hours.
% (v/v) glycerin 40rnl.

2a volumes of the resulting mixture are pipetted into sterile 3,9rfLl vials (1 dramvials) and then frozen in the gas phase of a liquid nitrogen freezer and stored.

The contents of the frozen glass bottle were transferred to 25% containing medium A30m7.
Used to inoculate 0TrLl fluted Erlenmeyer flasks.

This seed flask was incubated at 28°C on a 16 Orpm shaker for 24 hours.
Shake for an hour.

10 rul from this seed flask was added to medium A500171.
Used to inoculate 21 fluted Erlenmeyer flasks containing 1.

Shake these seed flasks for 24 hours at 28°C on a 160 rpm shaker.

1000rrtl of the collected contents of these seed flasks
is used to inoculate a 75.6J stainless steel fermentor containing medium A4671.

The tank is operated for 24 hours at 28° C. with an agitation rate of 13 Orpm and an air flow rate of 2831/min.

Polyglycol 2000 (Dow Chemical Company) is used as an antifoaming agent as necessary, but not in an amount exceeding 0.1%.

The results of pH and dextrose measurements are as follows: The tank was stirred at 24°C with 7 orpm and aeration volume of 1.
Run for 144 hours using 5381/min.

Add as much antifoaming agent, Polyglycol 2000, as necessary, but not more than 0.1%.

The assay is performed using the centrifugation supernatant.

The results are shown in the table below under the heading "Antimicrobial activity against ATCC6538p".

Assays were also performed by the disk diffusion method using 9.5 crrL mouthpiece disks and 10 ml assay plates, and the results are shown in the table below under the heading "Antimicrobial Activity (10 mA'Plates)".

A 1-ornl assay plate is prepared as follows: Assay bacteria, Staphylococcus aureus (5 taptylococcus aureus)
An overnight culture of ATCC6538P in nutrient medium + 0.2% yeast extract is diluted with nutrient medium + 0.2% yeast extract to obtain a suspension with 40% transmittance at a wavelength of 660 mμ.

This suspension was mixed with Difco yeast extract 2.
0? /l of Difco Nutrient Agar from 47°C to 48°C.
to obtain a composition containing 33.2 TLl of suspension per liter of agar.

Pour 10rrLl of this suspension into a Petri dish with a diameter of 85mm, cool the plate and keep at 4°C until use (max.
days).

Fermented liquid 4082J was squeezed into a 76cIfL squeezer and 4%W
Pass through the mouth using a mouthwash aid of /V.

(ethylene-2-nitrile)tetraacetic acid sodium salt 122
Add the same amount to both liquids.

Keep both solutions at 6°C. Dowex 50X4Na cold liquid
+ (20-50 meshes) 4801 at a rate of about 481/min.

The adsorbate was washed with deionized water 4801, then eluted with 2% pyridine aqueous solution 24 l 7 min, 300. J, 520.
Three fractions of g and 2401 are collected and assayed at pH 7.0.

The assay shows that the eluates contain 4%, 16% and 6%, respectively, of the biological activity transferred to the DOWEX 50X4Na+ column.

Elution fraction 2 is concentrated to 48 J and adjusted to pH 7.

Adjust 48J of concentrate to pH 7.3 and add DOWEX IX
2.50X100 mesh, 7.6 to hydrochloric acid type resin 761
Let it absorb in 17 minutes.

Elute the resin with deionized water at the same rate.

Collect four fractions 2X487, 1X707 and 1X481.

The collected fractions are adjusted to pH 7.

The assay shows that 68% of the starting bioactivity is in the 7O1 fraction.

This fraction was concentrated to 18 J at pH 7.0 and passed through a 0.455 Millipore filter.

Freeze-drying both solutions yields product 9 with an activity of 310 units/■
Get 9g.

Dissolve the lyophilized solid log in 0.1 M 2.6-lutidine acetate buffer, pH 6.3.

Readjust 1251 nl of the solution to pH 6.3 with acetic acid, and add DOWEX 50 x 8 (
200-400 mesh) column 2, 6-lutidine type,
25 with 0.1M buffer over 7.6X142Crn
Develop at TLl/min.

Collect pre-eluate 31 followed by 200 fractions of 20 ml each.

Every third fraction from 36 to 192 is assayed at a 1:200 dilution.

Biological activity is contained in fractions 56-192 and reaches a maximum in fractions 92-96.

Fractions 80 to 136 are combined and 59 TILl of deionized water are added to yield 1760 ml.

6 of the starting bioactivity was applied to a Dowex 50×8 column.
The collected diluted solution containing 2% is lyophilized.

The lyophilized solid was dissolved in 0.1 M 2,6-lutidine acetic acid p
Dissolve in H7.0 buffer.

27 rnl of the solution was placed on a 5 x 112 cm Biogel P-2 (200-400 mesh) column equilibrated in advance with 0.1 M buffer.

The gel is then developed with the same buffer at 101 rLl/min.

The elution flow is monitored with a Meccomatic recording differential refractometer.

Continue development until 105 fractions have been collected in 20ml increments.

Fractions 70 to 93 are assayed at a 1:300 dilution.

Biological activity was seen in fractions 73 to 82 and fractions 77 to 7.
The 0 fractions 75 to 80, which reach the maximum at 8, are lyophilized to obtain 90 μ of antibiotic with an average activity of 10,000 units/μ.

90cm of freeze-dried solid matter was reduced to 0. OIMIJ potassium acid buffer 7' (pH 7) 4rILlK bath lees.

This solution containing 596 hydroxylmosine concentration is described at the end of this example) was loaded with pre-washed
Transfer to a 1.7 diameter CrIL column equilibrated with

Before use, the XAD was treated with (1) 5 volumes of NaOH, followed by deionized water until the flowthrough was neutral (2) 5 volumes of lNHCl, followed by deionization until the flowthrough was neutral. water(
3) 5 times volume each of methanol, acetone, 0.001M
Wash successively with tetrasodium EDTA and finally with distilled water.

All solvents are vacuum degassed before use.

After applying the sample to the column, add 2 ml of phosphate buffer twice.

The column is developed with buffer at 5°C at a flow rate of 2 rul/min.

Collect the eluate 41rl1 fraction. The fractions from 100 ml of the eluate to 253 TLl are combined and concentrated in vacuo using a rotary evacuation aporator at 10° C. or lower to a total volume of 6 ml.

436 hydroxylmo XAD-2, prewashed with this solution as above, 90 ml
A diameter of 1.7 CrrL was filled and equilibrated with distilled water at 50°C.
Cover the column.

Two times 2 TrLl of distilled water are added to the sample.

The column is developed with distilled water at a flow rate of 27711/min. Collect the eluate 4TrL1 fraction.

Fractions from 100 to 151 ml of the eluate were collected and concentrated to 2.7 ml using a rotary evaporator, and the solution was lyophilized to obtain chenamycin 6.491.

Fractions between 152 ml and 345 ml were collected, concentrated in a rotary evaporator to 3.34 ml, and lyophilized to yield 11.53 ml of antibiotic.

These fractions contained a total of 369 citroxyl moieties, representing a 3.1-fold purification relative to the material present on the initial XAD-2 column, and a calculated activity of 31.000 units/m2.
I? give.

Spectrophotometric analysis of θ samples of this product shows E = 253 at 297 nm 1% when determined by IUIJ in phosphate buffer.

crrL Hydroxylamine Disappearance Absorption The proportion of the absorption at 297 nm of an impure sample that is due to the antibiotic is determined by the selective disappearance of this absorption by reaction with dilute hydroxylamine (with inactivation of the antibiotic activity).

The sample was 0.01M potassium phosphate buffer pH 7.0
The sample is prepared to have an initial A297 of between 0.05 and 20.

Freshly prepared neutral 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 proceed at room temperature for at least 30 minutes.

Subtracting the resulting A297 from the initial reading (after correcting for dilution by added reagent) gives the hydroxylamine loss absorption.

Pure chenamycin solution shows hydroxylamine loss absorption of 94.5%.

Example 7 The IOP portion of 99 g of the lyophilized solid obtained by the Dowex IX2 purification of Example 6 was reduced to 0. I IVI 2.6 Lutidine Dissolve in acetate buffer, pH 6.3.

Readjust 125 ml of the solution to pH 6.3 with acetic acid and add 700ml of Dowex 50x8, pre-equilibrated with buffer.
．． Transfer to 6 x 142 cII column 2.6-lutidine type.

The column is developed with 0.1 M buffer at 35 rILl/min.

Collect 200 fractions of 2 or ILl following pre-elution 3.61.

Every third fraction from 6 to 194 is assayed at a 1:200 dilution.

The bioactive peak is contained in fractions 18 to 178, and fraction 6
It peaks at 2 to 82.

Fractions 42 to 102 are combined and 640 rnl of deionized water is added to give 19201711.

63% of the bioactivity lost to the DOWEX 50x8 column
Lyophilize the collected diluted solution containing.

The lyophilized solid was dissolved in 0.1 M 2,6-lutidine acetic acid,
Dissolve in pH 7.0 buffer.

Biogel P-2 (200-400 mesh) 5X with 25ml of solution equilibrated in advance with 0.1M buffer
Transfer to a 112Cm column.

The gel is then developed with the same buffer in 10 ml for 11 minutes.

The eluate is monitored with a mekomatic recording differential refractometer.

Continue development until 125 fractions have been collected in 20ml increments.

All fractions from 70 to 89 are assayed at l:300 dilution.

Biological activity is seen in fractions 72 to 81, reaching a maximum in fraction 77.

Fractions 75 to 79 were lyophilized and the activity was 8320 units/In.
9 antibiotic 100.5Tn9 is obtained.

Dissolve 100.5 μl of the lyophilized solid in 0.01 MIJ potassium acid buffer, pH 7.

692 Hydroxylumo-washed XAD-2,90rILl before use
．． Transfer to a 1.7 CrrL diameter column equilibrated with 01M potassium phosphate buffer, pH 7, at 5°C.

Before using the XAD-2 column, (1) dilute it with 5 volumes of lN.
NaOH followed by deionized water (2
) 5 volumes of 1N HCl followed by deionized water until the flow through is neutral (3) 5 volumes each of methanol, acetone, 0
．． Wash successively with 001 MEDTA 4-sodium and finally with distilled water.

All solvents are vacuum degassed before use. After applying the sample to the column, add 2 ml of phosphate buffer twice.

The column is developed at 5° C. with a buffer arch flow rate of 2 ml 11 min.

Collect the eluate 4fn1 fraction. After 109ml of eluate 3
Combine fractions up to 0.09ml.

To this combined solution is added 11.53 m9 of the 186 hydroxylmosubstance sample obtained from Example 6.

The combined solution was concentrated in vacuo on a rotary evaporator at a temperature below 10°C with the addition of antibiotics to 7TLl.
shall be.

This solution with 720 hydroxylamine vanishing photometric units was prewashed as described above.
diameter 1.0 mm, filled with
7 to column 771.

Two times 2 rul of distilled water is added to the sample.

The column is developed with distilled water at a flow rate of 2 ml and 11 minutes.

Collect 4 ml fractions of eluate. Eluate 109-after 301
Concentrate to 10.31 rLl using a rotary evaporator.

This solution with 589 hydroxyl molecules was lyophilized to give a calculated activity of 30.140 units/TrL9 of 23.67 antibiotics.
Get n9.

The antibiotic prepared in this way is a white fibrous amorphous solid which, when exposed to increasing temperatures at a rate of 3°C/min in a glass capillary, decomposes in the following steps without passing through the liquid state: softening. A contraction of the solid volume occurs between 130 and 140°C and continues up to 170 and 174°C, in this range the material is yellow: sintering
and a gradual change in color tone to reddish brown at 180 to 200℃
Finally, at 205° C., carbonization and a slight residue of solid matter are observed.

A sample of this substance was found to be 296.5 HmK in spectrophotometric analysis.
It shows an absorption peak of E"%-268.2.

Elemental Lcm analysis gives the following results: (1) 5.67% weight loss upon vacuum drying at room temperature for 4 hours, and (2) 47% carbon.
68%, hydrogen 6.22%, nitrogen 11.48% composition.

These results are based on the empirical formula % Formula % The calculated elemental composition corresponding to the empirical formula is C = 47.68%,
H=6.13%, N=11.52%, 5=11.57%
and 0=23.1%.

1/rn of this substance in 10mM phosphate buffer
Optical rotation analysis of the 1 solution showed a specific optical rotation [-α] of 37°C +80.

Infrared absorption spectrum of this substance in Mediyo (Figure 1)
is 1765 to 771', 1650-1550crr
t'2800-2500C1rL', and 3500
It showed a characteristic absorption peak at 3100c1'rL'.

The NMR spectrum at 100 MHz of a sample of this substance dissolved in D20 shows a doublet at δ1.275 and a doublet at δ3.3.
A pair of doublets at δ 3.15 and δ 4.2° are shown, and these peaks are characteristic of chenamycin.

Compositions containing the antibiotic can be administered in a number of unit dosage forms, such as solid or liquid oral dosage forms.

The composition per unit dose is either liquid or solid.
Contains from 1% to 99% active material, with a preferred range of about 1
It is 0 to 60%.

The composition generally ranges from about 25 cm to about 10 cm based on the total weight of the composition.
000 active ingredients, but generally about 250
It is preferred to use dosages in the range from 1 to 1000 .

For parenteral administration, the unit dosage form is usually the pure compound in the form of a soluble powder in slightly acidic sterile water or for solution.

A typical formulation is prepared by the following method: In the above example, the active substance and diluent are mixed to obtain a homogeneous mixture, which is then poured into NO,0 hard gelatin capsules by hand or in a suitable machine. Fill it up as much as possible.

Mixing and filling are preferably carried out in air at a relative humidity of 40% or less.

In the above example, the active ingredient is mixed with calcium phosphate, lactose and about half the cornstarch.

The mixture is granulated with 15% cornstarch paste, passed through a coarse sieve, then sieved through an N016 sieve, the remainder of the cornstarch and magnesium stearate are added and the mixture is compressed into tablets of diameter approximately 1+-α and weight SOO■.

Alternatively, the active ingredient is mixed with calcium phosphate, lactose and half the cornstarch.

The mixture is pressed in a heavy duty press.
press) and compress it into a tablet-like mass.

These are broken into No. 16 mesh particles.

The remainder of the cornstarch and magnesium stearate are added and the mixture is compressed into tablets approximately L3 cIrL in diameter and 800 m9 in weight.

In the above example, the active ingredients are dissolved in a sufficient amount of water for injection in the proportions indicated.

Pour the solution into 5elas candles
) or Millipore membrane to sterilize.

Aliquot the solution into sterile glass bottles.

The vial and contents are frozen and the water is removed aseptically by lyophilization.

Aseptically seal the vial containing the sterile dry solids.

To reconstitute for injection administration, add 5 ml of sterile water for injection to the contents of the vial.

Heat and dissolve sucrose and glycose in approximately 400 ml of water to obtain an auxiliary solution.

Cool the solution and add sodium benzoate followed by concentrated orange oil.

Add water to the solution to approximately 900 rnl and add antibiotic.

Clear the solution by passing it through a coarse sieve.

[Brief explanation of drawings]

FIG. 1 is an infrared absorption spectrum diagram of chenamycin of the present invention.

Claims (1)

[Claims] 1. A compound represented by the general formula (chenamycin). 2 A therapeutic agent for bacterial infections whose main ingredient is a compound represented by the general formula (chenamycin). 3. The bacterial infection therapeutic agent according to claim 2, wherein the content of the compound represented by the general formula (chenamycin) is at least 50 units/ml. 4. The therapeutic agent for bacterial infections according to claim 2, wherein the content of the compound represented by the general formula (chenamycin) is at least 2 units/solid.