JP3718521B2 - Purified form of streptogramins - Google Patents

Description

  The present invention relates to a purified form of streptogramins consisting of group B components (minor components) of streptogramins.

  Among the known streptogramins, pristinamycin (RP 7293) was first isolated in 1955, a natural source antibacterial agent produced by Streptomyces pristinaesspiralis. The pristinamycins marketed under the name Pyostacine ™ mainly contain pristinamycin IA and pristinamycin IIA.

Another antibacterial agent of the streptogramin class, virginiamycin, was prepared from Streptomyces virginia, ATCC 13161 (see Non-Patent Document 1). Virginiamycin (Staphylomycine (TM)) mainly include factor S and factor _{M 1.}

An antibiotic constituting the antibiotic 899 belonging to streptogramins, ie, a pharmaceutical composition containing Factor S and Factor M ₁ has been disclosed (see Patent Document 1).

  In addition, the use of group A and group B components of streptogramins in the treatment of acne is disclosed (for example, see Patent Document 2).

  Antibacterial agents containing the natural source streptogramin species contain two groups of components, a mixture of Group B and Group A components, each group exhibiting its own antibacterial activity. Combinations composed of these two groups of components have been shown to produce a synergistic effect, resulting in enhanced bacteriostatic and bactericidal activity and broadening of the activity spectrum (Non-Patent Document 2, (See Non-Patent Document 3 and Non-Patent Document 4.) Moreover, the group A and B component of another streptogramin is described (refer nonpatent literature 5). Furthermore, natural pristinamycin as well as the different components that make it up are also described (see Non-Patent Document 6).

  Every attempt to produce a purified combination of streptogramins involves a group A major component [ie, pristinamycin IIA (PIIA)] that is consistently responsible for its activity and synergy. is there. The conclusions obtained in some studies further point out the importance of this component that also leads to better synergies (see patent document 3).

  However, these attempts have not been successful and partly because industrial manufacture is difficult, mainly because purified pristinamycin IIA is a crystalline product. However, it has been confirmed that the bioavailability is too low to be used as an active ingredient of a pharmaceutical.

  From the point of view of manufacturing the above products industrially, so far it has been possible to obtain a pre-scaled form that is sufficiently refined to satisfy the legal requirements of some countries regarding registration. It was not possible to produce batches with qualities that were unsatisfactory and showed sufficiently consistent reproducibility.

  For example, an industrial batch of natural pristinamycin contains an amount of impurities that can reach 20% even after purification. Attempts to carry out purification so far have been consistently unsuccessful, and very often one of these groups of components has been degraded, because they are unstable products. This is because many operations result in ring opening of the ring structure or dehydration of the group A component. As a result, for many years, it was considered impossible to achieve purity improvement. Even in 1988, purification was still considered difficult (see Non-Patent Document 7). In 1988, N.N. K. SHARMA and M.M. J. et al. O. ANTUNIS has announced that it is possible to separate and purify the virginiamycin component for analytical purposes, but because of the difficulties it faces, it is unlikely that these products will be produced industrially (Refer nonpatent literature 8.).

  Under these circumstances, commercialization of pristinamycin (Pyostacine ™) has been limited to certain countries, such as France and Belgium. The same applies to virginiamycin (Staphylomycine ™), which has been commercialized only in a limited number of countries as a human drug, as well as the commercialization of mikamycin (limited to Japan) Was also stopped. Thus, some groups have not been treated with efficacious efficacy in severe infections (especially infections caused by methicillin-resistant staphylococci) caused by gram-positive cocci or sexually transmitted diseases. .

  In the antibacterial field, it is well known to practitioners that allergies or tolerance can develop after administration of certain antibiotics (see Non-Patent Document 9). In particular, a large number of Staphylococcus aureus strains exhibiting resistance are known in hospital environments. For this reason, it is very effective for doctors to ensure a wide range of chemically different types of antibacterial agents in the hospital so that they can be adapted to the special case of patient treatment to be treated. The consequences of a particular type of marketed antibacterial agent failing can be very serious or even dramatic, because it can treat patients who do not fit other types of antibiotics. This is because it cannot be received.

  Thus, attempts that have been made to purification have always been in the direction of removing minor components of streptogramins, which are considered not essential and are often considered impurities. I came.

Among the group components A of natural streptogramins, pristinamycin IIB (PIIB) is a minor component, its weight ratio within natural pristinamycin is less than 10%, and often within virginiamycin It is about 8% and even about 6%.
US Pat. No. 3,325,359 French Patent Application No. 2,619,008 See European Patent Application Publication No. 506,561 (page 2) Antibiotics and Chemotherapy, 5, 632 (1955) Streptogramine als Modelsysteme fuer den Kationentransport durch Membranen, Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Faccultaet der Georg-August Universitaet zu Goettingen, Goettingen 1979 Antibiotics III, 521 (1975) C. Cocito, “Antibiotics of the Virginiamycinae Family, Synergistic Inhibitors” Microbiological Reviews, 145-98 (1979) J. et al. Preud'Home, P.A. Tarridec and A.M. Belloc, Bull. Soc. Chim. Fr. 2, 585 (1968) J. et al. of Liq. Chromatography, 11 (11), 2367 (1988) Bull. Soc. Chim. Belg. 97 (3) 193 (1988) The New England Journal of Medicine, 324 (9), 601 (1991)

  The general formula in the form of a cocrystallize, a coprecipitate or a finely divided physical mixture:

[Where:
A ₁ is the general formula:

(here,
R ′ is a hydrogen atom or a hydroxyl group, and Y is a hydrogen atom, a methylamino group or a dimethylamino group)
A group represented by
When R is an ethyl group, or when R ′ represents a hydrogen atom, R may also represent a methyl group, and R ₁ and R ₂ each represent a hydrogen atom, or alternatively A ₁ represents the formula :

A group represented by
R is an isobutyl group and R ₁ is a hydroxyl group and R ₂ is a methyl group]
One or more group B components represented by the general formula:

[Where:
R ″ is a hydrogen atom or a methyl or ethyl group]
It has now been found that a combination comprising one or more Group A “minor” components represented by is particularly advantageous in view of its biological (ie antimicrobial) activity in vivo, which Forms the subject of the invention.

The present invention provides a material in a purified form of streptogramins belonging to Group A that has been conventionally discarded without being isolated as an impurity, which is convenient for making a combination as described above:
Purification form formula:

[Where:
R ″ is a hydrogen atom or an ethyl group]
Streptogramins of group A component represented by
<Further description of the invention>
The combination according to the invention exhibits a biological action in vivo, which is more than that of the corresponding natural product (eg natural virginiamycin or natural pristinamycin) and that of the combination with the Group A main component. It is also very big. In addition, novel combinations that exhibit satisfactory bioavailability are provided and are manufactured on a large scale.

  Thus, it is possible to use a purified, bioavailable form of the final product that exhibits a good level of activity and naturally contains less than 6% impurities.

  Product represented by general formula (II) wherein R ″ is an ethyl group [hereinafter referred to as pristinamycin IIF (PIIF)], and product represented by general formula (II) wherein R ″ is a hydrogen atom. [Hereinafter referred to as pristinamycin IIG (PIIG)] is a novel product, which is a very low streptogramin component and its proportion in the natural product batch is less than 0.5% by weight.

  Combinations according to the invention are advantageously prepared using these components in a ratio of 10:90 to 90:10 (weight), preferably in a ratio of 20:80 to 80:20. These may be in the form of a physical mixture of powders, or in accordance with one aspect of the invention, in co-precipitated form, or in accordance with further aspects of the invention, as described in detail below. It may be in crystalline form.

  The present invention is also a purified product comprising a combination of at least one group B component represented by general formula (I) and at least one group A component represented by general formula (II) co-crystallized. Provide different forms.

This co-crystallization is a constant stoichiometry consisting of 1 mole of component (s) represented by general formula (I) and 2 moles of group A component (s) represented by general formula (II). [This stoichiometry occurs when component A is a product of the general formula (I), where A ₁ is represented by structure (Ia)) about 43-44: 57-56 Is equivalent to the relative weight ratio].

  The co-crystallized combination of the present invention can be used as a purified stable antibacterial agent that exhibits good bioavailability with improved in vivo activity, or as a minor component of the general formula (II). It can be used as a means of purifying streptogramins.

  Until now, it was impossible to purify the group A component represented by the general formula (II) by crystallization. As a result, until now, only chromatography has been known as a method for producing purified Group A products represented by general formula (II), and these products are isolated in large quantities. No other purification means suitable for was known.

  It has now been found that the group A component of general formula (II) is available in pure form by processing through the co-crystallization combination defined above. Ketones (eg, acetone, methyl ethyl ketone, methyl isobutyl ketone, etc.), esters (eg, ethyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, etc.), chloride solvents (eg, methylene chloride, chloroform, 1,2-dichloroethane, etc.), Or a crude mixture dissolved in an organic solvent such as a nitrile (eg acetonitrile) containing at least 30% Group A minor component corresponding to general formula (II), as defined by general formula (I) By adding the Group B component, a compound that co-crystallizes in the ratio defined above is obtained. The amount of the compound represented by the general formula (I) to be introduced is appropriately selected so that the residual concentration of this product (after co-crystallization) is less than the solubility it exhibits in the medium. Moreover, regarding the product represented by the general formula (II) and the product represented by the general formula (I), variations in the individual contents of the initial medium may cause modification of the obtained co-crystallized compound. It is understood that it does not bring. The co-crystallisation combination thus obtained dissolved in a solvent such as methyl isobutyl ketone or dichloroethane and treated in an acid medium (eg sulfuric acid, hydrochloric acid etc.) is combined with the organic phase with a solvent such as hexane. By processing, a purified group A minor component free from the group B component can be obtained.

  Furthermore, this co-crystallization combination offers the advantage of greatly enhanced stability and higher purity, most particularly the advantage of being easy to industrialize.

  This method can also be adapted for the preparation of co-crystals with modified derivatives of the natural group B components of streptogramins. These co-crystals are also within the scope of the present invention. Similarly, it is within the scope of the present invention to produce a minor component represented by the general formula (II) in purified form from the co-crystal.

Preferred embodiments of the present invention, the included impurities is less than 6%, preferably less than 3%, as defined pristinamycin IB [above, A ₁ is, Y is methyl amino group And R ′ represents a group represented by the general formula (Ia) which is a hydrogen atom, and when R is an ethyl group] or Virginiamycin S ₁ [as defined above, A ₁ represents Y and R ′ represents a group represented by the general formula (Ia) which is a hydrogen atom and R is an ethyl group] or a mixture of Virginiamycin S ₁ and Virginiamycin S ₄ [as defined above. Wherein A ₁ is defined in the same way as Virginiamycin S ₁ and R is a methyl group] and pristinamycin IIB [as defined above for the general formula (II) wherein R ″ is methyl. It is a combination with].

  Another preferred embodiment of the present invention is a group B of streptogramins as defined by general formula (I), which comprises a group B component and an A component in a relative molar ratio of about 1: 2. The combination of a component and the group A component represented by general formula (II) is included.

  According to a feature of the present invention, for example, by preparing a co-crystallized compound as defined above, at least one group B component of streptogramins represented by general formula (I) and general formula (II) Novel combinations with at least one group A component represented by are available. When it is desired to obtain a combination of different ratios, the compound thus co-crystallized and at least one of the components represented by the general formula (I) or the general formula (II) At least one group A component represented (these components are pre-purified), combined in an appropriate amount to obtain the desired ratio, or represented by general formula (II) A group A component (or a mixture of the above components) may be purified from the co-crystal and then mixed with one or more group B components represented by the general formula (I) in a desired ratio. In addition, the combination according to the present invention is obtained by isolating each of these components from the corresponding natural streptogramin after the group B component (s) and the group A component represented by the general formula (II) are purified, It can be prepared by mixing the purified components in the desired ratio as defined above.

  In addition, a solution of the component represented by the general formula (I) and the component represented by the general formula (II) in methyl isobutyl ketone or acetone or methylene chloride [or co-crystal And a solution containing a component represented by the general formula (I) or one of the components represented by the general formula (II)]. Alternatively, it may be poured into water to coprecipitate the combination according to the invention in the desired ratio.

  The production and separation of group A and B components is described in J. Org. Preud'homme et al., Bull. Soc. Chim. Fr. 2, 585 (1968), Antibiot. & Chemother. 5,632 (1955) or 7,606 (1957), Chromatog. Sym. 2 ° Brussels, 181 (1962), Antibiot. Ann. , 728 784 (1954-55), U.S. Pat. No. 3,299,047 or "Streptogramine als Modelsysteme fuer den Kationentransport durch Membranen, Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Faccultaet der Georg-August Universitaet zu Goettingen, Goettingen 1979" Isolating its constituents from this fermentation medium after fermentation, as described in or in accordance with the methods described in or in accordance with the methods described therein or as described in the examples below. Drunk is done. In particular, in the case of pristinamycin, the separation of the group A component and the B component is carried out by suspending crude streptogramin in an organic solvent such as acetate (eg, ethyl acetate), This can be done by filtration or centrifugation and extraction of the Group B components in an acidic aqueous medium followed by re-extraction in an ethylene chloride medium. Separation of Group A and B components can also be achieved by acid extraction of a solution of crude streptogramin in methyl isobutyl ketone followed by extraction of Group B component from this aqueous phase and It can also be done by isolating the group A component by precipitation from the organic phase.

  After separation, purification of the group B components of streptogramins can be done in alcohols such as ethanol, methanol or isopropanol, in acetate esters (such as isopropyl acetate or butyl acetate), or ketones (such as methyl ethyl ketone). ), Crystallized in acetonitrile, or chromatographed. Purification of the group A component represented by general formula (II) can be performed using chromatography and eluting with an acetonitrile / water mixture.

The production of the group A component and the B component represented by the general formulas (II) and (I), respectively, as described in French Patent Application No. 2,689,518, includes the following steps:
-First stage (optional), ie mutagenesis on non-selective microorganisms producing streptogramins, and-second stage, selection of selective microorganisms,
According to individual fermentation.

  These non-selective microorganisms are generally Actinomyces and fungi. Starting microorganisms that can be used in the present method are specifically selected from the group comprising pristinamycin, virginiamycin, micamycin, ostreoglycin, viridoglysein, vernamycin and etamycin. It is a microorganism that is a non-selective producer of streptogramin. As an example, some non-selective microorganisms that may be used are listed in the table below.

Microorganisms Antibacterial Bacteria, Micromonospora sp. Bernamycin Streptomyces sp. S. alborectus Virginiamycin S. griseeus (NRRL 2426) S. ravendulae Etamycin S. ravendulae S. loidensis
(ATCC 11415) Vernamycin S. S. mitakaensis
(ATCC 15297) Micamycin Ostreogriseus (S. ostreogriseus)
(ATCC 27455) Osteoglycine S. Pristina espiralis (S. pristinaesspiralis)
(ATCC 25486) Pristinamycin Virginia (ATCC 13161) Virginiamycin Actinomyces (ACTINOMYCES)
A. A. daghestanicus etamycin

More specifically, the production is carried out using a microorganism selected from Streptomyces alborexus, Streptomyces mitakaensis, Streptomyces pristina espiralis, Streptomyces osteoglyceus and Streptomyces virginia. .

The first step in this production is to modify these non-selective microorganisms to increase the antibiotic production capacity it exhibits and / or to synthesize only one of the two components of streptogramins. This may be due to genetic modification (mutation of a structural gene for an enzyme involved in the biosynthetic pathway, or, for example, a mutation of a sequence that allows expression of the structural gene) or biochemical modification ( Modification of post-translational mechanism, damage of feedback inhibition mechanism, etc.). Various mutagenesis means are used as follows:
-Physical agents: X-ray, UV, etc .; or-Chemical agents: alkylating agents such as ethyl methanesulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (Delic et al., Mutation Res. 9 (1970) 167-182) or 4-nitroquinoline 1-oxide (NQO), etc .; dialkylating agents; intercalating agents, etc.-or in DNA, especially in transposons, integration plasmids, phages or prophages Or any system that mutates into a protoplast fusion (Cohen, Nature 268 (1977) 171-174).

  These means (alone or in combination) can be applied to spores or non-selective microorganisms in the form of spores after or during germination, or can be applied to hyphae. In this production, an operation (randomly or directly) that makes it possible to obtain from a non-selective microorganism a microorganism that can selectively produce one component of streptogramins can be used.

  This second stage of production concerns the identification and isolation of selective microorganisms. This step can be carried out using means for testing the selectivity, especially with respect to microorganisms. There are various microorganisms that exhibit specific susceptibility to group A or group B components of streptogramins, such as Bacillus subtilis (ATCC 6633), Bacillus circulans, particularly sensitive to group B components. (Bacillus circulans), Bacillus cereus (Watabebe, J. Antibio. Ser. A XIII (1) (1960) 62) or C.I. Streptococcus agalactiae B96 (Antimicrob. Agents Chemother. 10 (5) (1976) 7 Micrococcus luteus (Prikrylova, cited above) or Sarcina lutea (ATCC 341) is present. It is specifically sensitive to one component of streptogramins by inserting a gene suitable for resistance to one of these two components into a microorganism sensitive to both components of streptogramins It is also possible to prepare artificial microorganisms. Some of these genes have been cloned (Le Goffic et al., J. Antibio.XXX (8), 665 (1977); Le Goffic et al., Ann. Microbiol. Inst. Pasteur 128B, 471 (1977); Solh Path. Biol. 32 (5), 362 (1984)), introducing the gene into different microorganisms using standard molecular biology techniques. This selection step can also be performed using an ELISA test with an antibody specific for component A or B, or alternatively using analytical techniques such as chromatography (liquid chromatography, thin layer chromatography, etc.). When testing selectivity for microorganisms, it is further preferred to confirm the selectivity using a chromatographic assay.

  Thus, according to the present invention, it is now possible to obtain a new purified form of streptogramin on an industrial scale, where the impurity level, clarity and uniformity of this composition are in accordance with the legal requirements for registration. And, further, it exhibits improved in vivo activity and bioavailability and is less toxic. Thus, this novel combination can compensate for the lack of acceptable treatment with this type of antimicrobial agent in many countries.

  The novel combination of the group B component of the streptogramins represented by general formula (I) and the group A component of the streptogramins represented by general formula (II) is particularly advantageous against gram-positive microorganisms. Shows in vivo activity. In vivo, mice were shown to be active against Staphylococcus aureus IP 8203 when orally administered to mice at a dose of 30 to 50 mg / kg.

As an example, the oral CD ₅₀ exhibited by several combinations of components represented by general formulas (I) and (II) in experimental Staphylococcus aureus IP 8203 infection in mice is shown below.

  The test combinations in Table I below are prepared by co-precipitation in hexane using a solution of the components represented by general formulas (I) and (II) in methyl isobutyl ketone or acetone. .

Table I
Product (I) / Product (II) combination CD ₅₀ (mg / kg) p. o.
PI (Example 1) PIIB (Example 18)
10:90 44
20:80 32
30:70 30
70:30 30
80:20 30
90:10 50

The combinations shown in Table II below are co-crystallized products prepared as described in the Examples.

Table II
Co-crystallized product (I) / product (II) CD ₅₀ (mg / kg) p. o.
Combination PI / PIIB (Example 9) 38
PIA / PIIB (Example 11) 28
PIB / PIIB (Example 12) 32
PIC / PIIB (Example 13) 36
PID / PIIB (Example 14) 50
Factor S / PIIB (Example 15) 32
Factor S ₁ / PIIB (Example 16) 50
Factor S / PIIF (Example 17) 50

The combinations described in Table III below are prepared in the form of a physical mixture of powders.

Table III
Product (I) / Product (II) combination CD ₅₀ (mg / kg) p. o.
PIA (Example 1) / PIIB (Example 18)
30:70 36
PIA (Example 1) / PIIB (Example 18)
50:50 40
Factor S (Example 5) / PIIB (Example 18)
30:70 44

Furthermore, this novel combination is not toxic, i.e., when administered orally to mice at a dose of 150 mg / kg (2 doses), no signs of toxicity appear themselves.

  In accordance with another aspect of the present invention, the co-crystallized compound contained in a ketone (eg, methyl isobutyl ketone, etc.) using the co-crystallized combination as a means of purifying the component represented by general formula (II) The component represented by the general formula (II) can be purified by extracting the group A component by acid extraction and then isolating it by precipitation from the organic phase.

  The following examples illustrate the invention. The assay values in these examples are given in wt%.

Separation and purification of Group B components

In 210 liters of ethyl acetate, 30 kg of crude pristinamycin [pristinamycin IA (PIA): 20.7%, pristinamycin IB (PIB): 3.9%, pristinamycin IC (PIC): 0.6%, pristinamycin ID (PID): 0.3%, pristinamycin IIB (PIIB): 8%, pristinamycin IIA (PIIA): 45%, pristinamycin IIF (PIIF): <0 .5% (not analyzed), pristinamycin IIG (PIIG): 0.5% (not analyzed)] is suspended and stirred at room temperature for 15 hours. The suspension is filtered and the ethyl acetate filtrate is collected and extracted with 20 liters of 1N sulfuric acid and then with 20 liters of distilled water. The aqueous phases are combined and washed 6 times with 15 liters of ethyl acetate, then the pH is adjusted to 7 by adding 30 liters of 10% sodium bicarbonate solution and 3 with 30 liters of methylene chloride. Extract once. The methylene chloride phases are combined and then washed with 10 liters of distilled water. The methylene chloride is then distilled off and replaced with 50 liters of ethanol. The mixture is then treated under reflux with 0.8 kg of L3S charcoal for 30 minutes. After filtration and washing twice with 5 liters of ethanol, the mixture is cooled to 10 ° C. over 15 hours. After holding for 1 hour at 10 ° C., the suspension is filtered and washed three times with 7 liters of ethanol. After drying this solid at 40 ° C. under reduced pressure, 5.7 kg of purified pristinamycin I (hereinafter referred to as PI) is obtained.
Assay value: 96.8% (PIA: 81.1%, PIB: 12%, PIC: 2.6%, PID: 1.1%);
Yield on PIA: 74%.

  After taking up 1500 g of purified PI in 9 liters of 1,2-dichloroethane, 1.5 equivalents of succinic anhydride and 0.015 equivalents of dimethylaminopyridine are added. This solution is kept at 20 ° C. for 1 week, and then placed on a column [column height: 1 m; diameter: 20 cm] containing 10 kg of silica (20-45 μm). Elution is carried out by flowing a mixture of 1,2-dichloroethane / methanol at a flow rate of 18 liters / hour for 6 hours, and during this chromatography the methanol (water content 5%) percentage is changed from 0 to 4%. To rise. 47 2.4 liter fractions were collected.

  Fractions 5 to 15 are pooled and the 1,2-dichloroethane is evaporated off and replaced with 5 liters of ethanol. After crystallization, 365 g of PIA is obtained with an assay value of 99.8%.

Fractions 36 to 39 obtained from the chromatography experiment described in Example 1 are pooled and the 1,2-dichloroethane is evaporated off to give 210 g of solid. 40 g of this solid is taken up in 8 liters of water with 8 cm ^{3 of} 10N hydrochloric acid. After 3 hours at 90 ° C., the solution is neutralized to pH 6.5 with sodium bicarbonate. The solution is extracted three times with 1 liter of ethyl acetate and the extract is washed twice with 0.2 liter of water. After treatment with charcoal, the ethyl acetate is evaporated off and replaced with 600 cm ³ of ethanol. After recrystallization, 20 g of PIB are obtained with an assay value of 97%.

Fractions 22 to 26 obtained from the chromatography experiment described in Example 1 are pooled and the 1,2-dichloroethane is evaporated off to give 139 g of a solid. This solid is taken up with a minimum amount of 1,2-dichloroethane and placed on a silica column. Elution is carried out by flowing a mixture of 1,2-dichloroethane / methanol at a flow rate of 18 liters / hour for 6 hours, and during this chromatography the percentage of methanol (5% water content) is changed from 0 to 5%. To rise. 48 2.4 liter fractions were collected. After evaporating fractions 38 to 43, the solid is taken up with 300 cm ³ of ethanol. After recrystallization, 22 g of PI containing 40% of PIC is obtained. By continuously flowing through a methylene chloride / methanol (98: 2 volume) eluent in a chromatography experiment using silica (20-45 μm), 5 g of a solid is available, which is combined with methyl isobutyl ketone. After vigorous stirring and recrystallization in ethanol, its assay value for PIC is 95%.

  1000 grams of PI obtained as described in Example 1 above is dissolved in a minimum amount of chloroform and then purified by continuous fractionation using a column containing silica (20-45 μm). After eluting with chloroform containing 2 to 5% methanol, one product is obtained, which is concentrated to dryness. The product is then purified by passing the acetonitrile / water (60:40 volume) mixture through the column containing Diaion ™ resin twice in succession. These fractions are monitored by chromatography. Pool the fractions containing PID and concentrate to dryness. This gives about 3 g of product with an assay value for PID of 60%. Further purification is carried out by countercurrent chromatography using a solvent mixture of methyl isobutyl ketone / acetone / formic acid (40: 2: 40 volume). By concentrating and drying the fraction containing PID, 1 g of a solid having an assay value for PID of 95% can be obtained.

400 g Staphylomycine ™ (tablet form—initial composition: Virginiamycin S ₁ (S ₁ ): 3.4%, Virginiamycin S ₄ (S ₄ ): 0.9%) is placed in 4 liters of water. .

These tablets are disintegrated by stirring for 15 minutes at 20 ° C. After 1 liter of methylene chloride is added, stirring is continued for 1 hour. Next, after settling, the methylene chloride phase is separated, filtered, and poured into stirred hexane having a volume of 5 liters over 30 minutes. After stirring for 1 hour, the suspension is filtered and the solid is collected and washed 3 times with 250 cm ³ of hexane. After drying, 52 g of solid are recovered and suspended in 370 cm ³ of ethyl acetate. The suspension is vigorously stirred twice at 20 ° C. for 18 hours. The filtrate corresponding to each stirring treatment is taken out and dried, and then dissolved in 850 cm ³ of methanol under reflux. After the temperature is gradually lowered to -20 ° C over 16 hours, the solid is collected by filtration and washed with a small amount of methanol. After drying this solid at 35 ° C. under reduced pressure, 9 g of Factor S (Virginiamycin S) is obtained.
Assay value: 96% (S ₁ : 75.4%, S ₄ : 20.6%)
Yield on factor S ₁ (Virginiamycin S ₁ ): 50%.

1 g of Factor S obtained as described in Example 5 above was dissolved in acetonitrile at a ratio of 125 mg / cm ³ and 2 cm ³ volume was dissolved in a Nucleosil 5C8 ™ column (height 25 cm, external Purification is performed by injecting into chromatography using 2.54 cm diameter) and eluting the water / acetonitrile (60:40 volume) mixture at a flow rate of 7.5 cm ³ / min four times. The ones with factor S ₁ are collected and their volume is 120 cm ³ in each case, which corresponds to a total of 480 cm ³ . By repeating this chromatography operation four times, the whole 1 g of factor S is processed. In this way, the _one containing factor S ₁ is collected and its volume is about 500 cm ³ . Acetonitrile is removed using a rotary evaporator. The aqueous phase is extracted 3 times with 50 cm ³ of dichloromethane. The methylene chloride phases are combined, washed with 50 cm ³ of distilled water, dried over sodium sulfate and then filtered. Dichloromethane is removed under reduced pressure (5 mm mercury) using a rotary evaporator. In this way, 0.67 g of a factor S ₁ with an assay value of 99.6% is obtained.
Production of crude group A ingredients

  In 50 liters of methyl isobutyl ketone, 500 g of crude pristinamycin [pristinamycin IA (PIA): 20.7%, pristinamycin IB (PIB): 3.9%, pristinamycin IC (PIC) : 0.6%, pristinamycin ID (PID): 0.3%, pristinamycin IIB (PIIB): 8%, pristinamycin IIA (PIIA): 45%]. This solution is extracted 5 times with an aqueous phase composed of 2.5 liters of water and 2.5 liters of 1N sulfuric acid and then washed 3 times with 10 liters of water. Next, this methyl isobutyl ketone is treated with 7.5 liters of an aqueous sodium hydrogen carbonate solution containing 35 g / liter and then washed with 5 liters of water. In each case, the aqueous phase is mixed with the organic phase and allowed to settle, and then the aqueous phase is separated.

The resulting organic phase is contacted with 750 g of alumina, filtered and concentrated to a volume of about 4 liters and then taken up with 5 volumes of hexane. The resulting precipitate is filtered off and dried. 300 g of product are obtained, which is suspended in 1 liter of isopropanol. After stirring for 45 minutes at 55 ° C., the suspension is filtered at 4 ° C. These filtered mother liquors are concentrated to dryness and the residue is taken up with 500 cm ³ of methyl isobutyl ketone, into which 5 volumes of hexane are poured. The precipitate is filtered off, washed with hexane and dried at 40 ° C. under reduced pressure. 69 g of crude PIIB containing 36% PIIB and 6% PIIA but no longer containing PIA is obtained.

60 g of the crude PIIB obtained as described in Example 7 above is chromatographed on a Nucleosil 5C8 ™ column (column diameter 5 cm, height 30 cm), water / acetonitrile (60:40 volume) eluent. Purify by performing several flow-throughs using In this way, 250 mg of pristinamycin IIF (PIIF) is obtained.
In the following examples preparation of the products obtained by co-crystallization, X-rays diffraction spectrum indicated the product was the co-crystallization, crystals of this component alone in the same solvent group B component contains It was shown to be different from the spectrum shown by

The crude PIIB obtained in Example 7 above was dissolved in 190 cm ³ of acetone. Add 33 g of purified PI (PIA: 81.1%, PIB: 12%, PIC: 2.6%, PID: 1.1%). After stirring for 17 hours at 20 ° C., a suspension is obtained, which is filtered at 4 ° C. The product is washed and then dried. After recrystallization at a concentration of 100 g in 1 liter of acetone, 10 g of white crystals with an assay value for PIIB + PIIF + PIIG of 55% and an assay value for PIA + PIB + PIC + PID of 43% are obtained.

250 mg of purified PIIB obtained as described in Example 18 below is dissolved in 17 cm ³ of ethyl acetate. Add 300 mg of purified PI (PIA: 81.1%, PIB: 12%, PIC: 2.6%, PID: 1.1%). After stirring for 20 hours at 20 ° C., filtered, washed and dried, 125 mg of white crystals are obtained.
Assay value for PIIB + PIIF + PIIG: 56% [this PIIB is 54%]
Assay value for PIA + PIB + PIC + PID: 43%.

560 mg of pure PIIB obtained as described in Example 18 below is dissolved in 5 cm ³ of acetone. Add 480 mg of PIA (assay value 99.8%). After stirring for 20 hours at 20 ° C., the suspension is filtered. After washing with 1 cm ³ of acetone and drying at 40 ° C. under reduced pressure (<1 kPa) for 30 hours, 590 mg of white crystals are obtained.
Assay value for PIIB + PIIF + PIIG: 56% [this PIIB is 54%]
Assay value for PIA: 43%.

  The mother liquor obtained from the crystallization is collected and PIIB is added in an amount of 560 mg. The PIIB / PIA mass ratio at this point is about 4. After stirring for 20 hours, filtering, washing and drying, 195 mg of crystals having the same purity and composition as the crystals obtained in the first harvest are obtained.

  Using the procedure described in Example 11 above except that PIA is replaced with 480 mg PIB (97% assay value), the assay value for PIIB + PIIF + PIIG is 56% (this PIIB is 54%) and the assay value for PIB is 820 mg of 43% white crystals are obtained.

Using the procedure described in Example 11 above, except that 680 mg PIIB and 580 mg PIC (assay value 95%) in 4 cm ³ of acetone was used, the assay value for PIIB + PIIF + PIIG was 57% PIIB is 55%) and 315 mg of white crystals are obtained with an assay value for PI of 42% (37% of which is PIC).

  Using the procedure described in Example 11 above, except that PIA is replaced with 480 mg PID (assay value 95%), the assay value for PIIB + PIIF + PIIG is 55% (its PIIB is 53%) and the assay value for PID is 475 mg of 39% white crystals are obtained.

The procedure described in Example 11 above was used except that 450 mg PIIB and 380 mg Factor S (S ₁ : 75.4%, S ₄ : 20.6%) in 4 cm ³ acetone were used. This gives 550 mg of white crystals with an assay value for PIIB + PIIF + PIIG of 58% (its PIIB is 56%) and an assay value for factor S of 41% (37% of this is S ₁ ).

Using the procedure described in Example 11 above, except that PIA is replaced with 480 mg Factor S ₁ , the assay value for PIIB + PIIF + PIIG is 58% (its PIIB is 54%) and the assay value for Factor S ₁ is 41% 750 mg of white crystals are obtained.

Using the procedure described in Example 11 above, except that 224 mg PIIF and 192 mg Factor S in 2 cm ³ acetone was used, the assay value for PIIF was 55% and the assay value for Factor S was 220 mg of white crystals of 39% (factor S ₁ : 31%, factor S ₄ : 5%) are obtained.
X-ray diffractograms of the products of Examples 9 to 17 Table IV below shows the relative intensities of the main lines. X-ray diffractograms are obtained using a Phillips PW1700 diffractometer equipped with a cobalt counter-cathode. Assign a standard value of 100 to the 15.8cm line. Relative values are estimated by measuring the line height after subtracting the continuous background.

These X-ray diffractograms are similar regardless of the co-crystallized product (there is no significant difference between the planes of the main lines).
Purification of Group A ingredients

9.3 g of the product obtained in Example 9 is dissolved in 490 cm ³ of methyl isobutyl ketone. This solution is extracted twice with 370 cm ³ of 0.5 N aqueous sulfuric acid solution and then washed twice with 150 cm ³ of water. The organic phase is concentrated to a volume of about 80 cm ³ and poured into 5 volumes of hexane. The resulting precipitate is washed, filtered off and dried. Next, for the purpose of removing this methyl isobutyl ketone, the precipitate is taken up to a concentration of 100 g per liter of acetone, poured into 10 volumes of hexane, washed and dried. 3.5 g of product containing purified PIIB but no longer containing PI is obtained.
Assay value for PIIB + PIIF + PIIG: about 95% (92% of this is PIIB).

  The present invention also provides a combination with a group A component of general formula (II) in the pure form or in the presence of one or more compatible pharmaceutically acceptable diluents or adjuvants. There is also provided a pharmaceutical composition that can be used as a human or veterinary drug, comprising as an active product a novel purified streptogramin combination comprising at least one group B component of streptogramins. These compositions can be used orally or topically. These may comprise combinations according to the invention in the form of a physical mixture of powders, coprecipitates or cocrystals.

  As a composition for oral administration, a tablet, a hard gelatin capsule, a pill, a powder, a lyophilizing agent or a granule can be used. In these compositions, the active product according to the invention may be mixed with one or more inert diluents or adjuvants such as sucrose, lactose or starch. These compositions may also contain materials other than diluents, such as lubricants such as magnesium stearate.

  The composition for topical administration may be, for example, a cream, ointment or lotion.

  In human or veterinary treatment, the compositions according to the invention are particularly infections caused by bacteria, in particular severe infections caused by gram-positive cocci: Staphylococcus infections (especially infections caused by methicillin-resistant Staphylococcus) Have shown efficacy in the treatment of Streptococcus infections (especially against penicillin-resistant and macrolide-resistant pneumococci), and these also include Haemophilus, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria gonorrhoeae (Chlamydia trachomatis), Mycoplasma hominis, Mycoplasma pneu moniae) and ureaplasma urealyticum are particularly effective in the treatment of infections caused by Ureaplasma urealyticum.

  The composition according to the invention treats upper and lower respiratory infections (eg treatment of pulmonary infections), treatment of skin infections, long-term treatment of bone or joint infections, treatment or prevention of endocarditis in dental and urological surgery It can be used in the treatment of sexually transmitted diseases, and also in the treatment of opportunistic bacterial and parasitic infections that occur in AIDS, and in the prevention of the risk of staphylococcus in immunosuppressed patients.

  Generally speaking, the physician will determine the dosage deemed most appropriate according to age, weight, degree of infection, and other factors unique to the subject to be treated. In general, the dose for oral administration to adults is 0.4 to 3.5 g of active product taken 2 to 3 times a day.

The following examples illustrate compositions according to the present invention.
<Example A>
According to conventional techniques, an opaque hard gelatin capsule containing a 250 mg dose of the co-crystallized PIB / PIIB combination is prepared.
<Example B>
According to conventional techniques, an opaque hard gelatin capsule containing a 250 mg dose of the present co-crystallization factor S / PIIB combination is prepared.
<Example C>
According to conventional techniques, a tablet is prepared having the following composition containing a 384 mg dose of the active product.
-PIB / PIIB (45% / 55%) 384mg
-Hydroxypropyl methylcellulose 25mg
-Magnesium stearate 35 mg
-Colloidal silica 14mg
-Starch qs 700mg
<Example D>
According to conventional techniques, a tablet is prepared having the following composition containing a 384 mg dose of the active product.
-Factor S / PIIB (45% / 55%) 384mg
-Hydroxypropyl methylcellulose 25mg
-Magnesium stearate 35 mg
-Colloidal silica 14mg
-Starch qs 700mg

Claims (1)

Purification form formula:

[Where:
R ″ is a hydrogen atom or an ethyl group]
Streptogramins of group A component represented by