EA013864B1 - Method of enhancement antimicrobal activity of cephalosporin antibiotics - Google Patents

Abstract

The invention relates to chemico-pharmaceutical industry, the claimed method of antibacterial activity of cephalosporin antibiotic enhancement by their mechanical treatment provides to enhance antibacterial activity by at least two times due to enrichment of commercially produced antibiotics by biologically-active enantiomers. For that, powder-like substance of cephalosporin antibiotics is treated by intensive shock-abrasive action in ball mills controlling the change of specific angle of optical rotation measured by standard technique with wave length 589 nm, which must be at least 2°.

Description

The invention relates to the pharmaceutical industry, in particular to a method for increasing the basic antibacterial activity of cephalosporin antibiotics and creating highly effective drugs.

Cephalosporin antibiotics are widely used in modern medical practice for the treatment of infectious and inflammatory diseases.

Cephalosporins are beta-lactam antibiotics, their chemical structure is based on 7-aminocephalosporanic acid (7-ACA) [1] (Fig. 1). In medicine, semi-synthetic cephalosporin antibiotics are used, which differ in the nature of substituents B and B '.

SOOYa

FIG. one

From a structural chemical point of view, cephalosporins are rather complex large molecules that can have isomeric forms that differ in biological activity.

In FIG. 2, the structure of the Δ ³ isomer of the cefemic nucleus of cephalosporin antibiotics is shown. However, it is possible to change the position of the double bond (see Fig. 3). Such structures are called Δ ² isomers.

FIG. 2

SOOIa

FIG. 3

It is the Δ ³ isomers that exhibit antimicrobial activity, while the Δ ² isomers are inactive [2].

Another type of isomerism of cephalosporin antibiotics that affect their biological activity is the geometric syn- and anti-isomerism of the position of the Ν-oxime group = Ν-Ο-Β in the side chain attached at position 7 of the cefemic nucleus. This is shown by the example of 3rd generation antibiotic structures. The syn configuration is shown in FIG. 4, and the anti-configuration in FIG. 5.

FIG. 4 to.

N

SOOYa

FIG. 5

For the most popular cephalosporin antibiotics, this type of isomerism is possible for cefotaxime, cefuroxime, ceftazidime, cefepime, and ceftriaxone. Using cefotaxime as an example, the antibacterial activity of syn- and anti-isomers was compared [3-6]. It is shown that the activity of the syn-isomer against beta-lactamase-producing gram-positive bacteria is 80 times

- 1 013864 higher than the anti-isomer.

A known method of isomerization of cefotaxime [7] and cefuroxime [8] by UV irradiation of their aqueous solutions. As a result of irradiation, a definite photostationary relationship is formed between the blue anti-isomers. Thus, if the initial state of the antibiotic is a low activity isomer (anti-), as a result of irradiation, a mixture of syn and anti-measures is formed, which has increased antimicrobial activity. These studies of the mutual transitions between biologically active and inactive forms under the influence of a physical factor - UV irradiation - are analogues of the present invention.

The disadvantage of this method is the need to transfer antibiotics to the liquid phase. Considering that their gradual decomposition occurs in solution [9], for storage of irradiated antibiotics, their isolation in dry form is necessary. This greatly complicates the industrial implementation of the described isomerization process.

A known method of enrichment with the biologically active geometric isomer cis-7-amino-3 [(E) -2- (4-methyl-5-thiazoline) vinyl] -3-cephem-4-carboxylic acid, described in [10], which also is an analogue of our invention. It consists in obtaining its ammonium salt in an aqueous solution and its subsequent precipitation. In this case, the cis and trans isomers precipitate to varying degrees and the precipitate is enriched mainly in the cis isomer, which has increased activity against gram-negative bacteria.

The disadvantage of this method is associated with the translation of the original substance of the antibiotic in an aqueous solution, followed by isolation of the dry product. In addition, it is applicable only for a specific antibiotic - 7-amino-3 - [(E) -2- (4-methyl-5-thiazoline) vinyl] -3-cefem-4-carboxylic acid and is not known for other cephalosporins.

Another type of isomerism is optical isomerism due to the disseminative structure of cephalosporin molecules [11]. Thus, regardless of the nature of the substituents, in the cefemal core of cephalosporins there are two optically active centers — asymmetric quaternary carbon atoms at positions 6 and 7, see FIG. 1. As a result, all cephalosporin antibiotics have optical activity - chirality. The introduction of various substituents B and B 'complicates the spatial structure of the molecules and makes possible the existence of additional optically active configurations. Most drugs have a close relationship between the spatial structure and pharmacological activity, that is, the stereospecificity of the action. Many synthetic drugs exist as a mixture of two, and often more, spatial isomers that differ in their biological effects. Previously, theoretical calculations of optical activity for cephalosporins have not been carried out, and for most cephalosporins, the absolute configuration of molecules has not been established. This makes it impossible to construct plausible molecular models that would be useful even for a qualitative representation of possible stereochemically / optically active configurations. In this situation, the existence of many optically isomeric states of cephalosporin molecules — their enantiomeric forms — is likely. There are no data in the literature on the biological / antimicrobial activity of individual enantiomers of cephalosporins, as well as their mixtures.

Conventional methods for controlling the quality of cephalosporin substances are based on HPLC [12] using achiral phases and are not sensitive to enantiomeric composition. The only characteristic that is sensitive to the ratio of enantiomers is the specific optical rotation index [α], measured in solutions of the studied substances on special instruments - polarimeters. However, this indicator allows variation over a fairly wide range [12] (for example, for cefazolin from -10 to -24 °) or is not used at all in pharmacopoeial quality standards. At the same time, for enantiomerically pure compounds (for example, ascorbic and tartaric acids, aspartame), the accuracy of the task [α] is no worse than ± 1 °. Based on this, it can be assumed that the enantiomeric composition of industrial cephalosporin antibiotics is a variable value and depends on the conditions of the particular technology used for their synthesis. This assumption explains the experimentally observed differences in the antimicrobial activity of cephalosporin substances from different manufacturers [13]. There is no evidence of the effect of optical isomerization on the antimicrobial activity of cephalosporin antibiotics.

However, optical isomerization processes occurring in solutions under the influence of various chemical and physical factors are widely known for organic substances [11]. A disadvantage that impedes their industrial use is the need to transfer substances into the liquid phase (dissolution) and subsequent isolation from the solution in the form of a dry, storage-stable substance.

The closest technical solution chosen for the prototype of the present invention is the mechanochemical process of optical isomerization (racemization) of phenylalanine described in [14]. For this, a mixture of the Ό or L optical isomers of phenylalanine and an inorganic material powder (talc, kaolin, silicon oxide, aluminum or magnesium) taken in a weight ratio of 1:10 in dry form is subjected to mechanical processing in a ball mill. Analysis of the treated mixture indicates

- 2 013864 on racemization (isomerization) of the starting phenylalanine. In the absence of inorganic powders, the racemization process fails.

The disadvantage of this technical solution is the need to use the above inorganic additives, since for further practical use it is necessary to isolate pure organic matter - in relation to the purpose of the present invention, the antibiotic. Such purification is possible only with the participation of liquid phase stages, for example, dissolution, filtration / precipitation from inorganic materials and drying. These unavoidable technological operations significantly complicate the industrial implementation of the described isomerization process.

The objective of the present invention is to increase the antimicrobial activity of cephalosporin antibiotics by changing the enantiomeric composition of industrially produced substances.

The problem is solved due to the fact that in the inventive method of increasing the antibacterial activity of cephalosporin antibiotics by optical isomerization, the dry drug substance is subjected to shock-abrasion, while the processing is carried out until the specific angle of the optical rotation of the antibiotic is measured according to the standard method at a wavelength of 589 nm not less than 2 ° from the initial value and the shock-abrasive effect of the antibiotic is carried out in ball mills at room temperature re.

Stereoisomerization (the transition from one enantiomeric form to another) is carried out in the present invention by mechanical processing of dry substances of cephalosporin antibiotics (without any additives) by abrasive effects realized in various kinds of mills. The degree of change in the enantiomeric composition is quantitatively controlled by measuring the specific angle of optical rotation at a wavelength of 589 nm.

The basis of the invention is the phenomenon we have discovered of increasing the antimicrobial activity of cephalosporin antibiotics after processing their dry substances with shock-abrasive effects carried out in ball mills. In this case, a change in antimicrobial activity occurs only when the angle of specific optical rotation of the antibiotic is changed by at least 2 ° compared with the initial value. The remaining quality indicators of the pharmacopeia substance remain within the limits admissible by the quality standards.

The antimicrobial activity of the initial as well as mechanically processed cephalosporin antibiotics was determined in special microbiological tests according to the recommendations [15]. The minimal inhibitory concentration of antibiotics was studied by the method of serial dilutions in agar (Mie11eg Ηίηΐοη 2 Adag, Wushepeich).

Museum reference strains of microorganisms were used: E. soy ATCC 25922, 8. igeeik ATCC 29213, 8. rhizome ATCC 49619 and R. aetidshock ATTCC 27853, as well as microorganisms that do not have acquired resistance, isolated from patients of the Novosibirsk Regional Clinical Hospital: 8a1 K1ebk1e11a rpeishoshaye and 8. aigeik krr. (SY).

The substances of the following cephalosporin antibiotics were studied: cefuroxime, cefotaxime, ceftriaxone, ceftazidime, cefepime (produced by several companies - WatFaGatsha, Italy; Nepsh 1 Yeshe Sesh., South Korea; Otsy Sesh., India; Natsh Ryatsh. Otoir, China).

The mechanical treatment of antibiotics was carried out in ball mills at room temperature for several hours. The change in the content of the basic substance was determined using HPLC by the methods of [12] and did not exceed 5%. With a longer and more intensive treatment in mills, a significant chemical decomposition of antibiotics occurs, which excludes the possibility of their further use. As apparatuses for machining, mainly ball, planetary, vibration, centrifugal, etc. mills with shock-abrasive action can be used. It is possible to use grinding media other than balls, for example rods. The process scales from ball laboratory mills with a load of several grams to flow vibrocentrifugal mills with a capacity of up to 100 kg / h and more [16]. There are no special requirements for controlling the humidity of the premises, since the substances are processed in the sealed internal volume of the mills. In all cases, the process is carried out in virtually one stage, does not require the participation of liquid phases, expensive equipment and is easy to implement. The specific parameters of technological processes depend on the physicochemical and physico-mechanical properties of the components used.

The specific optical rotation angle is measured in aqueous solutions of cephalosporin antibiotics at a concentration of 2-4 wt.% On standard polarimeters (in our case, Po1AAg 3005, manufactured in Great Britain) at a wavelength of λ = 589.44 nm. During mechanical processing, a change in optical activity [α] can occur within significant limits - in absolute value up to 15 ° (in the case of cefepime) and relative changes - up to 66% (in the case of ceftazidime). The minimum change [α], at which an increase in antibacterial activity is observed, is 2.0 ° (for cefotaxime). In the case of changes [α] of less than 2 °, an increase or decrease in the antimicrobial activity of cephalosporins is not observed. In addition, such small changes [α] are within the error of its determination. It is important that cephalosporin antibiotics of different manufacturers exhibit

- 3 013864 different ability to optical isomerization and, accordingly, to the possibility of modifying their antimicrobial properties. Obviously, this is due to differences in the enantiomeric composition of industrial substances, which is determined by the production technology used.

With regard to the mechanism of changes in optical activity, it must be borne in mind that there are several optically active centers in the molecules of cephalosporins. Through racemic transformations of even one of them, transitions between various enantiomers are carried out, accompanied by a change in the total specific optical rotation of the studied substances. It is this phenomenon that we observe during the mechanochemical treatment of a number of cephalosporin antibiotics. This assumption is also consistent with a change in biological (antimicrobial) activity. Thus, our experiments show that the mechanochemical effect enriches the industrially produced substances of cephalosporins with biologically active enantiomers.

This fact is an unexpected technical result, not following from the prior art. The effectiveness of the method is demonstrated by an increase in the antimicrobial activity of substances of cephalosporin antibiotics treated with impact abrasion in a ball mill.

The prototype of the present invention [14] describes the optical isomerization of phenylalanine under conditions of mechanochemical effects. This process took place in the presence of inorganic fillers, whose active surface catalyzes stereochemical transitions.

The phenomenon of changes in the specific angle of optical rotation of cephalosporins — their optical isomerization — when we treat substances with shock-abrasive mechanical influences without special additives, which we discovered, meets the novelty criterion for this particular series of antibiotics.

Thus, the developed method of optical isomerization in industrially obtained substances of cephalosporins by mechanical treatment of antibiotic substances with shock-abrasive effects provides the following technical results:

increase in specific antimicrobial activity of cephalosporin antibiotics not less than 2 times;

creation of a low-cost and non-waste method for the production of substances of increased antimicrobial activity.

The present invention is illustrated by the following examples.

Example 1. Cefuroxime.

When machining in a ball mill VM-1 (drum volume 0.5 l, loading of the processed substance 20 g, grinding media loading - 15 steel balls with a diameter of 12 mm) for 2 hours, the angle of specific polarization rotation [α] changes from 61, 2 to 82.2 °. The content of the basic substance determined by HPLC analysis after machining is 98.6%. Antimicrobial activity of mechanically treated cefuroxime against E. coP. 8a1tope11a ep1epbb1k and K1ebk1e11a pseudomonas increases by 2 times.

Example 2. Cefotaxime.

When machining in a ball mill VM-1 for 3 hours, the angle of specific polarization rotation [α] varies from 63.2 to 76.2 °. The content of the basic substance determined by HPLC analysis after machining is 96.4%. Antimicrobial activity of mechanically treated cefotaxime against 8. aigeik, 8. aigeik krr. (ΟΝ), E. coP and K1eBk1e11a pituitus increases by 2 times.

Example 3. Ceftriaxone.

When machined in a VM-1 ball mill for 2 hours, the angle of specific polarization rotation [α] changes from -118.1 to -124.2 °. The content of the basic substance determined by HPLC analysis after machining is 98.8%. Antimicrobial activity of the mechanically treated ceftriaxone against 8. aigeik, 8. aigeik krr. (ΟΝ), 8. ppeitosiae, E. co11 and K1ebk1e11a ppeitoshae increases by 2 times.

Example 4. Ceftazidime.

When machined in a VM-1 ball mill for 2 hours, the angle of specific polarization rotation [α] changes from -17.8 ° to -6.0 °. The content of the basic substance determined by HPLC analysis after machining is 95.4%. The antimicrobial activity of the mechanically treated ceftazidime against 8. aigeik, 8. pheitosome, R. aegidshock, E. coP, and K1eBk1e11a pseudomonas increases by 2 times.

Example 5. Cepepime.

When machined in a VM-1 ball mill for 2 hours, the angle of specific polarization rotation [α] changes from -35.3 to -50.3 °. The content of the basic substance determined by HPLC analysis after machining is 95.6%. Antimicrobial activity of mechanically treated cefepime against 8. aigeik, 8. aigeik krr. (ΟΝ), R. aegidshock, E. co11 and 8a1topope11a ep (er111P1k increases by 2 times.

- 4 013864

Thus, the above examples confirm the claims.

Literature

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Claims (2)

CLAIM 1. A method of increasing the antibacterial activity of cephalosporin antibiotics by optical isomerization, characterized in that the dry drug substance is subjected to impact-abrasion, the treatment process being carried out until the specific angle of the optical rotation of the antibiotic, measured by the standard method at a wavelength of 589 nm, is not less than 2 ° from the original value. 2. The method according to claim 1, characterized in that the shock-abrasive effect of the antibiotic is carried out in ball mills at room temperature. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2