A method for the treatment of staphylococcal infections in a mammal (options)

 

Proposed: new ways of treatment of endocarditis, osteomyelitis or bacteremia, caused by a strain of staph that is resistant to vancomycin (or as an option - resistant to methicillin), including intravenous or subcutaneous administration of lysostaphin or its variants. The invention expands the Arsenal of methods of treatment of these diseases. 2 N. and 12 C.p. f-crystals, 2 ill., 8 table.

The technical field to which the invention relates

The invention relates to pharmaceutical preparations and their use in medicine and, in particular, relates to the use of lysostaphin in the treatment of staphylococcal infections in mammals, including man, as well as pharmaceutical preparations used in this way.

The level of technology

Lysostaphin is an enzyme identified in bacteria of the species Staphylococcus simulans (formerly known as S. staphylolyticus), which possesses antimicrobial activity due to its proteolytic activity against glycine-containing bridges in peptidoglycan cell wall of bacteria [Zygmunt et al., Progr. Drug. Res., 16:309-333 (1972)].

In vitro, lysostaphin is especially active against Staphylococcus aureus in consequence of relativity in relation to other staphylococci species (Ibid.).

The activity of lysostaphin was also investigated in models of infections in animals. Studies that intraperitoneal administration of the drug followed by intraperitoneal infection, similar to in vitro experiments and in this description are not considered. There were two reports of 50% survival of treated mice, according to which animals were infected intraperitoneally with subsequent single or multiple subcutaneous injections of a total dose of approximately 1 mg/kg of the preparation of lysostaphin [Schuhardt et al., J. Bacteriol., 88:815-816 (1964); Harrison et al., Can. J. Environ., 13:93-97 (1967)]. In one of these studies reported that the total dose of 6 mg/kg protects 100% of mice [Harrison et al., Ibid.]. Virulence to control bacterial contamination, used in both studies, it appears extremely low, since none of untreated infected mice did not die within a short period of time.

In some experiments used a model of subacute infection in mice to determine the bacterial load in the kidneys after infection by a strain of S. aureus Giorgio [Dixon et al., Luxurious Jale J. Biol. Med., 41:62-68 (1968); Schaffner et al., Yale J. Biol. Med., 39:230-244 (1967); Harrison et al., J. Bacteriol., 93:520-524 (1967)]. When intravenous drug lit mg/kg or higher. However, developed infections were more resistant, it was observed only a very slight decrease in the number of bacteria in cases when treatment was delayed for 24 hours or more, even at doses of 125 or 250 mg/kg of the preparation of lysostaphin. The effect of multiple doses has not been studied.

In the only study of Goldberg et al., Antimicrob. Ag. Chemother., 1967:45-53 (1967) used a limited number of dogs in the model uncharacteristic of endocarditis. The model on dogs in the future were developed. Experiment Goldberg et al. was not comparative, and therefore is of limited use in assessing the introduction of lysostaphin. However, high doses of lysostaphin (at least 50 mg/kg/introduction) were only moderately effective, which was estimated by the condition of the dogs and the reduction in the number of bacteria in heart valves and kidneys.

In accordance with this from the data obtained in studies of the prior art using animal models, it is not necessary that the use of lysostaphin would be an effective and practical approach to the purification of various organs from developing infections.

The purpose of the limited research conducted on human beings, was the elimination of nasal carriage of S. aureu, 1:791-797 (1968); Quickel et al., Appl. Environ, 22:446-450 (1971)]. Nasal carriage in itself is not a painful condition. It is a risk factor in terms of infection of patients colonized with bacteria employees health care, or simontacchi in the case of a colonized patient.

In the prior art described treatment of a patient with very severe disease with a single dose of parenteral entered lysostaphin with the subsequent introduction of the antibiotic gentamicin in three days. The patient died, but the manifestation reduce bacteremia [Stark et al., N. Engl. J. Med., 291:239-240 (1974)].

Immunogenic manifestations observed during cycle studies of animals and humans, were noted as requiring special attention. Cause at least some of these phenomena may be contaminated drugs lysostaphin extraneous substances.

No further elaboration of the enzyme as a therapeutic agent was not performed due to lack of desired efficiency in the studies described. In addition, it may be due to the difficulty of obtaining and purification of lysostaphin.

Staphylococcal gene of lysostaphin by now sequeiros by fermentation of non-pathogenic recombinant strain of Bacillus sphaericus, from which it was easily cleaned up.

The invention

Task for specialists remains the creation of a therapeutic agent that can be injected parenterally and which can be used in the treatment of staphylococcal infections in General, and also infections of specific tissues, as in endocarditis.

The problem is solved by applying lysostaphin to treat staphylococcal infections in mammals, including man, as well as pharmaceutical drugs used in this treatment. This invention relates to methods for the treatment of certain medical conditions, including staphylococcal endocarditis, Staphylococcus and staphylococcal bacteremia infection of the kidneys, lungs, skin, bones, burns, wounds, and prosthetic devices. The invention encompasses the use of lysostaphin in a broad sense, including not only lysostaphin wild-type and recombinant lysostaphin, options lysostaphin with the amino acid sequence that differs from the published "natural sequence of the Mature peptide (U.S. patent No 4931390), due to genetic mutations such as substitutions, insertions, and deletions), posttranslational processing, genetic engineering chem is Athena (up to 50 mg/kg) parenterally is an extremely effective therapeutic method for the treatment of staphylococcal infections especially infections that are resistant to treatment and/or is usually associated with high morbidity and mortality. In addition, lysostaphin, as shown, is effective against staphylococcal bacteria, which are at least partially resistant to available antimicrobial agents, such as-lactam antibiotics, including the penicillinase-resistant penicillins, vancomycin, etc.,

The invention further includes the combination therapy containing alternating or simultaneous introduction of lysostaphin and one or more other antimicrobial agents. Especially preferred antibiotics for injection in combination with lysostaphin, according to this invention, are rifamycins (isolated from microorganisms or derived synthetic or semi-synthetic means, such as rifampin) and glycopeptides (group of molecules, in which natural molecules usually contain heptapeptide and one or more structures of sugar), as obtained and isolated from natural sources (such as vancomycin, teicoplanin and others) and semi-synthetic drugs.

The availability of cloned, recombinant lysostaphin and their variants further broadens the scope of this the image is instead.

Cloning and sequencing of the gene of lysostaphin allows you to select variants of the enzyme, which may have properties similar or different from the properties of lysostaphin wild type. Was characterized by one of these modified enzymes, supporting the change of one amino acid and is the result of this work, and shows that he has a strong antistaphylococcal activity in vitro and in models of infection in animals.

Other analogues of lysostaphin, including existing in naturally occurring enzymes with sequence homologous to lysostaphin and activity endopeptidase or even chimeric enzymes obtained at the confluence of the binding domain of the enzyme with a catalytic domain of the other, will be a powerful agents that can treat intractable bacterial disease caused by staphylococci or other pathogenic bacteria.

List of drawings and other materials

In Fig.1 graphically presents the bactericidal activity of lysostaphin against methicillin-resistant strain of S. aureus compared with vancomycin.

In Fig.2 presents a bar graph showing the bactericidal activity of lysostaphin against several strains of S. aureus with different the foster

Definition

Used in this application, the terms used, where possible, in their normal and ordinary use. Some of the terms used to describe a class action or connections to obtain a General description of certain moments or scientific phenomena, which are logically grouped together.

The analogue of lysostaphin - any enzyme, including lysostaphin (wild type), any mutant or variant of lysostaphin, any recombinant or near the enzyme that retains proteolytic ability in vitro and in vivo proteolytic effect on the glycine-containing bridges in peptidoglycan cell wall of staphylococci. Variants can be created by post-translational processing of proteins (or enzymes present in the strain-producer, either by enzymes or reagents introduced at any stage of the process) or by mutation of the structural gene. Mutations can include targeted deletions, insertions, deletion of the domain and mutations associated with replacement. Analogues of lysostaphin described in this invention can be expressed by recombinant or otherwise.

Parenteral introduction by injection, including vnutriorganne, or delivery to the organs or tissues by injection (e.g., intramedullary).

Staphylococcus aureus is a highly virulent human pathogen. He is the cause of several human diseases, ranging from localized skin infections to life-threatening bacteremia and infection of vital organs. Without the adoption of quick response S. aureus infection can quickly spread from the original area of infection to other organs. Although the centers of infection may not be obvious, the authorities are particularly susceptible to infection include heart valves, kidneys, lungs, bones, meninges and skin in patients with burns. Surgical or traumatic wounds and any area in which there is a foreign body, is also often infected. These infections, which can occur in the community or during the hospital stay, are the cause of high morbidity and mortality, which may reach 60% in the case of severe infections in some populations even when using the very best treatments available. Other species of staphylococci (coagulase-negative staphylococci such as S. epidermidis) are less virulent, but can colonize catheters or prosthetic devices is Dechen valve.

Resistance to available antimicrobial agents, there appears to be especially easy to Staphylococcus, since resistance to penicillin in S. aureus; this resistance appeared at the dawn of the era of antibiotics. In fact, all staphylococcal infections that appear both in society and in hospitals, are no longer sensitive to penicillins first-generation products due penitsillinazy; strains that are also resistant to the penicillinase-resistant penicillins such as methicillin), are also currently an important issue, especially in the case of nosocomial infections. [Centers for disease control and prevention (Centers for Disease Control and Prevention), 1997. Reduced sensitivity of Staphylococcus aureus to vancomycin (Reduced susceptibility of Staphylococcus aureus to vancomycin are) - Japan, 1996. Weekly report the morbidity and mortality (Morbidity and Mortality Weekly Report), 46:624-626 (1997)].

Vancomycin has been the drug first line of defense against staphylococcal infections, especially in hospitals. However, as it is evident from the high levels of morbidity, none available at this time, treatment is not ideal for a number of diseases, such as caused by S. aureus endocavitary heart and other organs, the infection often is filled with blood. One reason for the failure of currently available drugs is that they are relatively slow, especially in vivo, when rapid sterilization of infected areas may be necessary for a full and speedy recovery of the patient. In such life-threatening situations and for a variety of other infections (for example, when the treatment is very long, such as osteomyelitis) new therapeutic drugs or new combinations of therapeutic drugs can significantly improve the clinical outcome of the patient.

It was shown that lysostaphin is highly active in moderate doses. This is illustrated below on the model is very heavy well characterized infection in animal endocarditis in rabbits caused by methicillin-resistant S. aureus (MRSA). In particular, we demonstrated a complete sterilization of the vegetation in the heart valve almost all the animals were treated according to one of the schemes of the drug, which is an unprecedented result is not observed when using the currently available antimicrobial agents. Further we have shown in this context that the combination even more the efficiency of the components in this model system.

Dose of lysostaphin that we used were much lower than the dose that would have previously been shown only a limited effect purification of the bacteria in animal models [Zygmunt et al., Progr. Drug. Res., 16:309-333 (1972); Goldberg et al., Antimicrob. Ag.Chemother., 1967:45-53(1967)].

Below us was also demonstrated activity against staphylococci in vitro and in a model of acute infection in mice modified form of lysostaphin obtained by mutagenesis of recombinant Bacillus sphaericus strain carrying a gene of lysostaphin. As a consequence, other implemented aspect of the invention to provide pharmaceutical analogues of lysostaphin or lysostaphin or other enzymes with activity endopeptidase peptidoglycan, including genetically modified enzymes containing from one to five substitutions of amino acids, enzymes with deletions or insertions of up to 10 amino acids, including deletions or insertions at the N end, or chimeric enzymes, which are the result of the merger of the catalytic and binding domains of various enzymes as therapeutic agents for the treatment of infections in humans and animals.

For example, it was described the other glycylglycine-endopeptidase (ALE-1 from Staphylococcus capi J. Bacteriol., 179:1193-1202 (1997)]. Another hydrolases peptidoglycan with a lesser degree of homology with lysostaphin, but also possessing activity of endopeptidase is zeocin And produced by Streptococcus zooepidemicus 4881 [Simmonds et al., Applied and Environmental Microbiology, 62:4536-4541 (1996); Simmonds et al., Gene, 189:255-261 (1997)]. Chimeric proteins can be obtained by merging the data domain, or loved ones of enzymes with domain analogue of lysostaphin.

Because certain immunological side effects, noted in a much earlier studies, may be important in some but not in other situations (such as critical or emergency situation), it is expected that appropriately purified preparations analogues of lysostaphin, obtained by fermentation harmless recombinant strains of bacteria will be less capable of inducing immunogenic or other side effects.

Effective pharmaceuticals data of antimicrobial enzymes include aqueous solutions or dry preparations (for example, freeze-dried, crystalline or amorphous, whether or not containing more soluble substances for osmotic balance) for recovery of liquids suitable for parenteral delivery of outdoor activities is rebrushing (i.p.) ways or podvoloshino, or by inhalation or direct instillation into the infected area to provide a blood or tissue levels above the minimum inhibitory concentration (MIC) of the active agent, and thus reduce the titers of bacteria with the purpose of cure or relief of the current infection.

Moreover, an active analog of lysostaphin can be put together, simultaneously or sequentially with other antimicrobial agents for more effective treatment of infectious diseases. Drugs can be or to be recovered in small volumes of liquid, suitable for bolus intravenous or peripheral injection or adding more volume for intravenous drip, or may be, or to be restored to a greater extent for administration by slow intravenous infusion. Agents for co-injection with lysostaphin or other antibacterial enzymes can be in the same drug with the enzyme in the form of fixed combinations or can be used for preparation before applying any available and used in practice drugs and any route of administration known as providing adequate levels Yes is on the severity of the infection and the sensitivity of the infecting organism and, in the case of combination therapy may depend on the particular antimicrobial agent(s) used in combination. The dose can be in the range from 0.5 to 200 mg/kg/day, preferably from 3 to 25 to 50 mg/kg/day administered as single or divided doses, preferably by continuous infusion or divided into two to four doses per day.

Examples

All experiments are conducted using analogs of lysostaphin, obtained by fermentation of recombinant strains of C. sphaericus, designed in such a way that they contain the gene of lysostaphin described Recsei (U.S. patent N 4931390) or its mutant. In particular, the analogues of lysostaphin, obtained by fermentation of C. sphaericus, differ from the published sequences that have 2 amino acids or less up to two additional amino acids at N-end.

In particular, the data largely derived from studies using analogues of drugs lysostaphin obtained recombinant manner in which the main component is a structure that lacks the two N-terminal amino acids relative to the published sequence. However, the results are not limited to data prep the th.

Example 1

The activity of lysostaphin in vitro

As shown in table 1, the experiments show that the drug of lysostaphin is active and bactericidal in vitro against clinical isolates of S. aureus; the minimum inhibiting concentration (MIC) and minimum bactericidal concentration (MBC) is defined as <1.0 microgram/ml using standard methods microdesmidae in broth [national Committee for clinical laboratory standards (National Committee for Clinical Laboratory Standard), 1993. Approved standard M7-A3. Methods of analysis of antimicrobial sensitivity of bacteria to aerobic growth using dilutions (Approved Standard M7-A3. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically) - Third edition. National Committee for clinical laboratory standards, Villanova, PA; national Committee for clinical laboratory standards (National Committee for Clinical Laboratory Standards), 1992.

Experimental manual M26-T (Tentative Guideline M26-T). Methods for determining bactericidal activity of antimicrobial agents (Methods for determining bactericidal activity of antimicribial agents). National Committee for clinical laboratory standards (National Committee for Clinical Laboratory Standards, Villanova, PA].

Moreover, show that lysostaphin is active against sterowany isolates. The MIC is defined as the lowest concentration studied, which completely inhibits visible growth of bacteria, and MBC is the lowest concentration that kills 99.9% of the original inoculum within 24 hours of processing. As shown in table 1, the sensitivity to lysostaphin not affected by resistance or reduced sensitivity to methicillin and/or vancomycin. Strains of S. aureus that are methicillin-resistant, and have only average sensitivity to vancomycin, the last time arise in the United States. [Centers for disease control and prevention, 1997 (Centers for Disease Control and Prevention, 1997), Ezhenedelny report on morbidity and mortality (Morbidity and Mortality Weekly Report), 46:813-815 (1997)]

.

Lysostaphin sticks to plastic materials and can be deduced from the solution, which can affect its determined activity. As a result, some definitions MICK carried out with addition of 0.1% bovine serum albumin (BSA) to the diluent. In other respects the method is identical to the one described above. As shown in table 2, the activity of lysostaphin in vitro against tested strains increases in 8-64 times when assayed in the presence of BSA. On the whole, the Staphylococcus strains are more sensitive to lysostaphin, than previously described.

These results demonstrate the very high activity of lysostaphin against contemporary clinical isolates of Staphylococcus aureus with multiple resistance to antibiotics.

The bactericidal activity of lysostaphin against S. aureus is also studied in experiments with relation to the death of time. In one experiment of this type strain of S. aureus AG461, methicillin-resistant clinical isolate from Genoa, Italy, bring the broth Mueller-Hinton (Difco) and grown at 37With careful shaking to obtain approximately 108viable cells/ml (CFU/ml (CFU - colony forming units)) that is determined by absorption of the culture at 600 nm. Then the culture is diluted in fresh broth to about 106CFU/ml and aliquots (5 ml) placed in a number of different retorts for processing various concentrations of antibacterial agents. Incubation continue with gentle shaking at 37With and draw samples through the time intervals for determination of viable cells. Serial 10-fold dilution of the samples do in sterile saline (0.9% NaCl in distilleria the soy agar (Remel), using the method of introduction into the agar. (In this way placed on the Cup make an aliquot 2.5 ml of agar for the upper layer, which is stirred and poured into the Cup. Agar for the top layer consists of molten tripticase soy agar (Difco) dissolved in 2 times trypticase soy broth Difco to obtain a final concentration of agar is 0.75%, wt./vol.). Cup incubated for 24-48 h at 36To manually count the colonies. All breeding lysostaphin do in the presence of 0.1-0.2% BSA to prevent adsorption of lysostaphin on the plastic materials. Vancomycin (Sigma Chemical Co.) diluted in sterile distilled water.

As shown in Fig.1, lysostaphin at concentrations of 0.004 and to 0.032 µg/ml has a rapid bactericidal action, destroying at least 99.9% of bacteria within one hour of contact. Compared to the bactericidal action of vancomycin is low and significantly slower at a very low level of destruction of bacteria, celebrated for three hours contact, even when using vancomycin in significantly higher concentrations (2 and 16 ág/ml). Different concentrations of lysostaphin and vancomycin, which use the x methicillin-resistant clinical isolate of S. aureus and fourth strain 27619VR obtained in the laboratory strain "VISA" (i.e., the strain with average resistance to vancomycin), isolated from methicillin-resistant clinical isolate, bring the broth Mueller-Hinton with the addition of cations (Becton Dickinson) and grown at 37°C over night. They were then diluted in fresh broth and incubated at 37With careful shaking up until you determine that they have reached the logarithmic phase of growth. As shown in Fig.2, the titers of bacteria at a given time are in the range of 2106to 9107CFU/ml. Lysostaphin added to each culture at a concentration of 1 μg/ml intervals selected samples, make a serial dilution in 0.9% NaCl and plated on agar Mueller-Hinton (Becton Dickinson). Cups with agar incubated for 48 h at 37To manually count the colonies. As shown in Fig.2, all of these strains are quickly killed by the action of lysostaphin.

These results demonstrate that lysostaphin has a strong and rapid bactericidal activity against contemporary clinical isolates of S. aureus, including strains resistant to methicillin, and strains, cramps which the shaft

Example 2

Comparative efficacy of lysostaphin on the model of S. aureus infection in mice

The effectiveness of lysostaphin compare with the effectiveness of vancomycin in a model of acute infection in mice. S. aureus Smith cultivated during the night under moderate stirring in veal broth (Difco) and diluted broth containing 5% hog gastric mucin (Difco). Male mice of Swiss-Webster (Taconic Farms, Germantown, NY) weighing approximately 20 g infect intraperitoneally 105-106viable cells, which is approximately ten times higher than the AMF inoculum, which reproducibly kills all untreated animals within 48 hours Each treatment group contained six mice. Lysostaphin injected (0.1 ml of 5% dextrose for injection) or subcutaneously (0.2 ml) within 10 min after infection. Vancomycin is administered subcutaneously.

As shown in the table.3, lysostaphin protects 100% of infected mice at the dose of 0.16 mg/kg or intravenous dose of 2.5 mg/kg after subcutaneous administration. Vancomycin, which in mice is completely bioavailable following subcutaneous injection and has similar activity as in subcutaneous and intravenous has 100% efficacy at a dose of 2.5 mg/kg All untreated mice die less Chechelnyk against infection S. aureus in a model of acute infection in mice using highly virulent loading dose of bacteria. Intravenous effective are extremely low doses of purified recombinant human lysostaphin. When assessing the weight of lysostaphin 16 times more effective than vancomycin, polyarnosti - lysostaphin approximately 200 times more effective than vancomycin.

Example 3

The activity of the variant enzyme lysostaphin in vitro and in vivo

The Bacillus sphaericus strain containing the cloned gene of lysostaphin, which is described in U.S. patent N 4931390, mutagenicity processing N,N’-nitrosoguanidine. Conduct screening of surviving colonies in the presence of lytic activity, placing them on the lawn killed by heating cells of strain S. aureus RN4880 and inquira during the night when 32C. Colony forming large areas of lysis retain.

One of these clones is characterized next. The gene of lysostaphin is sequenced and find that it contains a single mutation G-A (instead of G) in the codon corresponding to position 218 of the Mature protein of lysostaphin that changes the codon from GGT (glycine) to GAT (aspartic acid). During the fermentation of this mutant strain get enough S. aureus in vitro, although the drug of lysostaphin wild-type is somewhat more active. In this experiment, MICK determined by makroyavleny in broth in a final volume of 1 ml in a glass tube. Other aspects of the method describe above.

As shown in the table.5, a variant of the enzyme lysostaphin is also highly active against S. aureus in a model of acute infection in mice. In this case again the option is somewhat less active than lysostaphin wild-type, but he is more active than vancomycin.

Example 4

Antimicrobial activity of rabbit serum, treated with lysostaphin

New Zealand white rabbit weighing about 5 kg carry out intravenous infusion of 125 mg of lysostaphin. Blood samples taken at intervals up to 4 h and prepare serum; make a twofold serial dilution and determine the bactericidal titer of the serum against methicillin-resistant strain of S. aureus (MRSA 27619). Bactericidal titer serum represents the maximum dilution that kills 99.9% of inoculum for 24 hours In this test the survival of bacteria determined mainly in the method of determining the min is possible serum instead of different concentrations of the solution of purified antimicrobial agent.

As shown in the table.6, the serum contains highly bactericidal concentration of lysostaphin during the whole period of time. In particular, at the points of time from 30 min to 120 min titer greater than 1:256 (the maximum of the used dilution), indicating that the cultivation of at least 256 times retain the ability to kill 99.9% of bacteria. In the most recent point in time, 240 minutes, the titer is 1:64.

This example demonstrates that lysostaphin supports bactericidal activity in serum of rabbits, and that he is present and remains active in the bloodstream for at least 4 h after injection.

Example 5

The effectiveness of lysostaphin against experimental endocarditis in rabbits

Endocarditis of the aortic valve is obtained from the new Zealand rabbits weighing approximately 3 kg to Rabbits give anesthesia and the right carotid artery is surgically isolated and kanyoro polyethylene catheter, which is pushed into the left ventricle. After at least 24 h rabbits intravenously infect 106-107cells methicillin-resistant strain of S. aureus (MRSA 27619). Through on the Troll (9 rabbits); the positive control, vancomycin, 30 mg/kg, twice a day (15); lysostaphin, 5 mg/kg, three times a day (11); lysostaphin 5 mg/kg, once a day (10); lysostaphin 5 mg/kg, once a day + vancomycin, 30 mg/kg, twice a day (11). All rabbits that do not confirm infection by preliminary analysis of the culture of blood, eliminate. In addition, all rabbits included in the analysis at autopsy confirms developed infection endocarditis, which is determined by the presence of vegetation in the aorta, indicating existing or pre-existing disease state.

All intravenous administration make and spend for three days.

The health status of rabbits appreciate intervals. Rabbits killed 18 h after the last injection. Vegetation of the aorta is removed, weighed and processed to determine the number of viable bacteria, which is expressed as IgKOE/, the Limit of detection is 102CFU/g (IgKOE/g=2,0). Average titers of bacteria/g compared using one-way analysis of variance. The criterion of student-Newman-Keuls use to bring for multiple comparisons. Comparison of the velocities of the sterilization performed using Fisher's exact test. Statistike 5 mg/kg of lysostaphin three times a day is the most effective treatment. Impressive statistics are that this treatment is completely sterilizes the vegetation on the heart valve at all rabbits except one. It is much superior to the standard regimen used as a positive control in this model of infection 30 mg/kg of vancomycin twice a day. The scheme of injection of 5 mg/kg of lysostaphin once a day is less efficient than the scheme of injection three times a day, but almost as good as vancomycin, in reducing the number of bacteria in the vegetation; in fact, the effect is not statistically different from vancomicina group. When the scheme is the introduction of lysostaphin once a day also achieve complete sterilization of vegetation in some animals. The introduction of lysostaphin once daily in addition to standard treatment with vancomycin causes a sharp decrease in the average number of bacteria, almost to the level observed with the introduction of lysostaphin 3 times a day. However, in terms of the number of fully sterilized vegetation scheme is the introduction of lysostaphin three times a day is uniquely superior to all others.

Abscesses in the kidneys also assess for the presence of staphylococci. Scheme Noah band to less than 102CFU/g of tissue in the group of lysostaphin compared with more than 108CFU/g in the controls.

Observing animals shows that all rabbits treated according to the scheme of introduction of lysostaphin three times a day, good feel in the early stages of the treatment cycle.

These results could not be expected, based on previous research. In particular, sterilization virtually all vegetation has never been observed or has not been described previously with any antimicrobial agent in this model of infection. The fact that sterilization occurs within a relatively short period of treatment (3 days), indicates that lysostaphin acts in vivo very quickly and assumes that the analogues of lysostaphin with antimicrobial activity could significantly improve the results of treatment of patients with serious staphylococcal infections when you need a rapid reduction in bacterial load.

The above data demonstrate the effectiveness of analogues of lysostaphin against S. aureus, including MRSA (methicillin-resistant S. aureus). The strains resistant to methicillin and vancomycin, are recently the problem. Variant strain of this type of selected item is as described for the existing natural conditions of the VISA strains, the selected patients in the United States and Japan. (Centers for disease control and prevention (Centers for Disease Control and Prevention), weekly report the morbidity and mortality) Morbidity and Mortality Weekly Report, 46:813-815). Believe that the isolates of staphylococci are sensitive to vancomycin, if MICK is less than or equal to 4 µg/ml, and are completely resistant if the MIC is greater than or equal to 32 μg/ml (national Committee for clinical laboratory standards (National Committee for Clinical Laboratory Standards), 1993. Approved standard M2-A5. Existing standards for sensitivity tests using discs with antimicrobial agents (Approved Standard M2-A5. Performance standards for antimicrobial disk susceptibility tests - Fifth edition. National Committee for clinical laboratory standards (National Committee for Clinical Laboratory Standards, Villanova, PA).

As shown in the table.8, lysostaphin is effective in the treatment of rabbits with infective endocarditis, caused by methicillin-resistant strain VISA.

Against the strain VISA, lysostaphin at the dose of 5 mg/kg three times a day has the same efficiency as vancomycin, concerning reduction of the bacterial load in the vegetation of the aorta. Lysostaphin at the dose of 15 mg/kg three times a day I have chimo more effective than lysostaphin at the dose of 5 mg/kg three times a day. Moreover, vancomycin, even when the dose of 30 mg/kg twice a day, could not ensure complete sterilization of the heart valve vegetation none of the investigated animals. On the other hand, complete sterilization is achieved in some animals when the scheme is the introduction of lysostaphin three times a day.

The model of endocarditis in rabbits at the present time very well standardized and adopted as a tough test on the ability of antimicrobial agents to treat serious infections in humans. Previous work with lysostaphin on developed infections shows a limited reduction in bacterial load in the kidneys on the model in mice and in heart valves, and other bodies on the model of endocarditis in dogs at doses in the range from 50 to 250 μg/kg/treatment. Despite the high doses used in these previous studies, the effectiveness of the magnitude of the doses required for the treatment of severe staphylococcal infections, do not watch. Previously obtained results lead to the prediction of rapid total sterilisation really all vegetation cardiac valves, as now shown when used in the context, do not only demonstrate the surprising effectiveness of lysostaphin against endocarditis caused by S. aureus, but also show that this efficiency greatly exceeds the expected standard methods of treatment.

Currently available treatments are often not effective in life-threatening infections, which can lead to irreversible tissue damage and in which, consequently, rapid decline in the number of bacteria to prevent such damage, and the spread of infection to other vital organs. The above results indicate that the analogues of lysostaphin as monotherapy or in combination with other agents have the potential efficacy for the treatment of such infections. Moreover, on the basis of these results and activity in vitro of lysostaphin against staphylococci should be expected that the analogues of lysostaphin as monotherapy or in combination with other agents will be applied against the Staphylococcus species other than S. aureus. Additional agents suitable for use in conjunction with lysostaphin are vancomycin and other glycopeptides, rifampin, and other rifamycins and other anti-infective is designed not only for the treatment of staphylococcal endocarditis, but other potentially lethal staphylococcal diseases, such as bacteremia and infection of other vital organs, such as kidneys, lungs, skin and bones. These methods are also applicable in the treatment of infections of burns, wounds and prosthetic devices. These same techniques can be applied, in particular, in the treatment of diseases such as osteomyelitis, which is the result of infection type and severity, which requires long-term treatment currently used antimicrobial agents. The invention further covers the use of analogues of lysostaphin in the treatment of such infections and diseases caused by staphylococci resistant to traditionally used antibiotics.

Claims

1. A method of treatment of endocarditis, osteomyelitis or bacteremia in a mammal caused by a strain of Staphylococcus, with at least partial resistance to vancomycin, in which the mammal is administered by inhalation, or parenterally, or by direct instillation in the affected area an effective amount of lysostaphin or variant retains the ability of lysostaphin to the proteolytic action of the tion with at least one other antimicrobial agent.

2. The method according to p. 1, characterized in that an effective amount of lysostaphin choose 3-50 mg/kg/day.

3. The method according to p. 2, wherein the use amount of 3-25 mg/kg/day.

4. The method according to one of paragraphs.1-3, characterized in that use a combination of lysostaphin or its variant with another antimicrobial agent.

5. The method according to p. 4, characterized in that as another antimicrobial agent chosen rifamycin or glikopeptid.

6. The method according to one of paragraphs.1-5, characterized in that lysostaphin introduce the man.

7. The method according to one of paragraphs.1-6, characterized in that use lysostaphin or its variant, obtained by recombinant.

8. A method of treatment of endocarditis, osteomyelitis or bacteremia in a mammal caused metitsillinrezistentnye strain of Staphylococcus aureus that has average sensitivity to vancomycin, in which the mammal is administered by inhalation, or parenterally, or by direct instillation in the affected area an effective amount of lysostaphin or variant retains the ability of lysostaphin to proteolytic action on glycinamide bridges of peptidoglycan cell wall of staphylococci, as monotherapy or in combination the effective number of lysostaphin choose 3-50 mg/kg/day.

10. The method according to p. 9, wherein using the number of 3-25 mg/kg/day.

11. The method according to one of paragraphs.8-10, characterized in that use a combination of lysostaphin or its variant with another antimicrobial agent.

12. The method according to p. 11, characterized in that as another antimicrobial agent chosen rifamycin or glikopeptid.

13. The method according to one of paragraphs.8-12, characterized in that lysostaphin introduce the man.

14. The method according to one of paragraphs.8-13, characterized in that use lysostaphin or its variant, obtained by recombinant means.

 

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