HK1103639B - Dalbavancin compositions for treatment of bacterial infections - Google Patents

Description

Dalbavancin compositions for treatment of bacterial infections

Technical Field

The present application relates to dalbavancin (dalbavancin) compositions and methods of using the same in methods of treating bacterial infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on international application serial No. 892,280-225 filed on 26.4.2005, which is a continuation-in-part application serial No. 10/834,395 filed on 27.4.2004, which is a continuation-in-part application serial No. 10/714,261 filed on 14.11.2003, which claims rights to U.S. provisional patent application serial No. 60/427,654 filed on 18.11.2002, No. 60/485,694 filed on 8.7.2003, No. 60/495,048 filed on 13.8.13.2003, and No. 60/496,483 filed on 19.8.2003, all of which are incorporated herein by reference in their entirety.

Background

According to the united states center for disease control and Prevention (u.s.center for disease control and Prevention), nosocomial bloodstream infections are the leading cause of death in the united states. Approximately 5% of 7 million Central Venous Catheters (CVCs) inserted annually in the united states are associated with at least one bloodstream infection event (approximately 350,000 pieces per year). Catheter-related bloodstream infections occur when bacteria enter the bloodstream through an intravenous catheter and can be life threatening.

Skin and Soft Tissue Infections (SSTI), also known as complicated and/or uncomplicated (non-complicated) Skin and Skin Structure Infections (SSSI), are common medical conditions and often occur after trauma and surgical procedures. Staphylococcus aureus and streptococcus pyogenes are the most commonly isolated pathogens from deep soft tissue infected patients, although any pathogenic organism can cause an infection, including those found on healthy skin. Many SSTIs are mild or moderate in severity, allowing successful treatment with oral antiseptics and topical washes. In contrast, more severe or complex infections that often occur in patients with potential risk factors (e.g., vascular injury, diabetes) and/or infections caused by refractory or multidrug-resistant bacteria may require robust intravenous antimicrobial therapy and aggressive surgical debridement.

Staphylococci are clinical and therapeutic problems and have been increasingly associated with nosocomial infections since the early 60 s of the 20 th century. Methicillin-resistant staphylococcus aureus (MRSA) of the coagulase-positive species has long been a problem with community-acquired and nosocomial infections, and several coagulase-negative staphylococci have been identified as opportunistic human pathogens, especially in the treatment of critically ill patients in the intensive care unit. Another major cause of clinical concern is the increased isolation of penicillin-resistant Streptococcus pneumoniae strains in many parts of the world.

The glycopeptide antibiotics vancomycin and teicoplanin have been used to combat serious nosocomial infections caused by the following organisms: multidrug-resistant gram-positive pathogens, especially MRSA, coagulase-negative staphylococci (cos), and enterococci. Vancomycin and teicoplanin were available for MRSA-induced infections and until recently all isolates were sensitive to them. However, it has been reported that the frequency of isolation of staphylococcus aureus strains with moderate sensitivity or resistance to teicoplanin and vancomycin gradually increases. A variety of vancomycin resistant strains classified as "VanA", "VanB" or "VanC" based on the resistance mechanism have been reported. Therefore, alternative treatment options are needed.

Teicoplanin is at least as active as vancomycin for most gram-positive bacteria and appears to cause fewer adverse events. Both forms of treatment require at least once daily administration to achieve complete recovery. Currently, treatment options for severe infections caused by some of these pathogens are quite limited. The emerging resistance of gram-positive pathogens to vancomycin makes it highly desirable to have new antibiotics with increased potency potential.

Furthermore, less frequent dosing regimens compared to currently available treatments are needed to increase patient comfort, especially for parenteral (e.g., intravenous or intramuscular) administration of antibiotics. Hospitalization is sometimes necessary for parenteral administration of antibiotics for multiple days, and less frequent administration would be advantageous to allow the treatment to be administered to outpatients.

While less frequent dosing is a desirable feature of an antibiotic dosing regimen, the "therapeutic window" (i.e., toxicity profile) of the antibiotic administered must be sufficiently acceptable to allow the administration of larger individual doses without causing serious adverse effects in the treated patient that compromise treatment. Furthermore, even when the antibiotic exhibits a suitable therapeutic window, less frequent administration of the drug may be possible only when the antibiotic exhibits an appropriate serum half-life to maintain therapeutic efficacy within the desired dosing interval. The serum half-life of the antibiotic determines the life of the drug in vivo and the length of time after administration that the serum level will reach a minimum trough level that is still effectively bactericidal. The serum trough levels that develop over time after administration of a first dose of antibiotic determine when another dose must be administered to maintain a minimum bactericidal level of antibiotic in vivo.

Recently, successful glycopeptide antibiotics have been rationally synthesized from natural glycopeptides. For example, the semisynthetic glycopeptide dalbavancin was synthesized from the natural antibiotic a40926, originally isolated from a culture of actinomadura (Malabarba et al, 1998, U.S. patent No. 5,750,509). Dalbavancin has shown greater efficacy against various bacterial strains than vancomycin or the antibiotic linezolid and represents a promising new therapy for skin and soft tissue infections (see, e.g., Jabes et al, 2004, AntOrganics chemither.48: 1118-1123). According to U.S. Pat. No. 5,750,509, dalbavancin is at its N¹⁵Glycopeptide antibiotic having a methyl moiety on the amino group (see numbering in FIG. 1), N¹⁵The monomethylamino group may be free (i.e. -NHCH)₃) Or by an amino protecting group such as t-butoxycarbonyl, benzyloxycarbonyl, aralkyl or benzyl. The method of preparing certain dalbavancin compositions reported in the' 509 patent also produced small amounts of the N of dalbavancin¹⁵，N¹⁵Dialkyl analogues, but these molecules have not been characterized.

In view of the above pathogens, other antibiotics having activity against one or more microorganisms, including antibiotic-resistant bacteria, would be of commercial value and would satisfy a long-felt need in the art.

Summary of The Invention

The present invention provides compositions, methods and kits for treating or preventing bacterial infections with dalbavancin. Surprisingly, it has been found that stable dalbavancin dosage forms have a pharmaceutical window and extended serum half-life to allow for a treatment regimen of about once every 5-7 days or longer while maintaining antibacterial properties in vivo.

Accordingly, in one aspect, a pharmaceutical composition is provided comprising a unit dose of dalbavancin, a stabilizing agent, and a pharmaceutically acceptable carrier, wherein the amount of dalbavancin is sufficient to provide a therapeutically or prophylactically effective plasma level of dalbavancin in an individual for at least 5 days.

The pharmaceutical compositions of the invention are typically formulated for administration to a subject in a pharmaceutically acceptable form, e.g., a pharmaceutically acceptable aqueous formulation. The pharmaceutical composition is preferably administered by a parenteral route, e.g., intravenously or intramuscularly. Thus, in this preferred embodiment, the pharmaceutical composition is generally sterile.

In some embodiments, the unit dose of dalbavancin is provided in a dry powder (e.g., lyophilized) form and reconstituted in a pharmaceutically acceptable carrier, such as a sterile aqueous formulation, prior to administration to a subject. In one embodiment, the pharmaceutically acceptable carrier comprises a 5% dextrose in water solution. The pharmaceutical compositions of the present invention may be administered to a mammal, such as a human, in need of treatment or prevention of a bacterial infection. In some embodiments, the pharmaceutical composition may include at least one antibiotic other than dalbavancin, such as an antibiotic effective (e.g., bactericidal) against a gram-negative bacterium and/or an antibiotic effective against a gram-positive species, such as a VanA vancomycin-resistant strain, against which dalbavancin is not effective.

The present invention provides compositions, methods of making the same, and methods of treating or preventing bacterial infections with room temperature stable dalbavancin pharmaceutical compositions.

In some embodiments, one or more stabilizing substances are used to inhibit degradation of one or more dalbavancin components during storage as a dry powder (e.g., lyophilized) formulation and/or as an aqueous formulation prior to administration to an individual. Degradation can lead over time to the undesirable formation of less active and/or inactive components, which can potentially cause adverse effects in vivo. Preferred stabilizers include nonionic components, such as sugars or sugar alcohols, for example mono-, di-or polysaccharides or derivatives thereof, for example mannitol, lactose, sucrose, sorbitol, glycerol, cellulose, trehalose, maltose or dextrose or mixtures thereof.

In one embodiment, the invention includes a pharmaceutical composition comprising the stable dalbavancin.

In another embodiment, the invention encompasses a pharmaceutical composition comprising dalbavancin and a stabilizing agent.

In yet another embodiment, the invention encompasses pharmaceutical compositions comprising dalbavancin and a stabilizer at a pH of about 1 to 7, more preferably 2 to 6. In another embodiment, the pH of the composition is about 3 to 5. The stabilizer may comprise a saccharide or an amino acid. The saccharide can be mannitol, lactose, or a combination of mannitol and lactose. Mannitol and lactose may be added in equal or unequal amounts. In one embodiment, equal amounts of mannitol and lactose are added and the pH is adjusted to about pH 4.5.

In another embodiment, the invention also includes a pharmaceutical composition comprising dalbavancin and mannitol at a pH of about 3. In one embodiment, the pH of the composition is about 3.3. In another embodiment, the composition may further comprise lactose. Lactose and mannitol can be added in equal or unequal amounts.

In another embodiment, the invention also includes a pharmaceutical composition comprising dalbavancin and a stabilizer, wherein the stabilizer comprises mannitol and lactose. Mannitol and lactose may be added in equal amounts. The pH of the composition may optionally be from 1 to 7, more preferably from 2 to 6, more preferably from 3 to 5, more preferably from 4 to 5, more preferably about 4.5.

Due to glycosidic linkages, glycopeptides, in particular dalbavancin, are very unstable. There may be some degradation at room temperature, more at 40 ℃. Some of the above formulations may require special storage conditions. In particular, refrigeration may be required (e.g., -40-10 ℃, alternatively, -20-9 ℃, more preferably 2-8 ℃). The formulation may additionally be sterilized. When administered, they will form a stable, clear, particle-free solution. The solution should be stable and free of precipitates.

Preferably, the above pharmaceutical composition degrades by no more than about 4%, more preferably no more than about 3%, more preferably no more than about 2%, more preferably no more than about 1%, more preferably no more than about 0.5% after about 2 years at about 25 ℃. Alternatively, the pharmaceutical composition has no more than about 4% MAG, more preferably no more than about 3% MAG, more preferably no more than about 2% MAG, more preferably no more than about 1% MAG, more preferably no more than about 0.5% MAG after about 2 years at about 25 ℃.

In another embodiment, the above-described pharmaceutical composition preferably degrades by no more than about 6%, more preferably by no more than about 5%, preferably by no more than about 4%, preferably by no more than about 3%, preferably by no more than about 2%, preferably by no more than about 1% after about 3-6 months at about 40 ℃. Alternatively, the above pharmaceutical composition preferably has no more than about 6% MAG, more preferably no more than about 5% MAG, preferably no more than about 4% MAG, preferably no more than about 3% MAG, preferably no more than about 2% MAG, and even more preferably no more than about 1% MAG, after about 3-6 months at about 40 ℃. It is desirable to obtain compounds that are stable at 40 ℃, particularly where the compounds cannot be stored in a refrigerator or at room temperature (e.g., third world countries and indian residences).

In yet another embodiment, the pharmaceutical composition degrades by no more than 3%, more preferably by no more than about 2%, more preferably by no more than about 1%, more preferably by no more than about 0.5% after about 2 years at about 2-8 ℃. Alternatively, the pharmaceutical composition has no more than 3% MAG, more preferably no more than about 2% MAG, more preferably no more than about 1% MAG, more preferably no more than about 0.5% MAG after about 2 years at about 2-8 ℃.

The present invention includes dalbavancin compositions that may include any combination of dalbavancin factors. These factors include dalbavancin factor a₀、A₁、B₁、B₂、C₀、C₁Iso B₀And a MAG.

The invention also includes a drying process for reducing the level of solvent in a dalbavancin composition. The method comprises the following steps: providing wet dalbavancin comprising dalbavancin, water, and a solvent, and drying the wet dalbavancin at about 30 ℃ or less under a vacuum pressure of about 50 mbar or less until the moisture content of the wet dalbavancin is less than about 20% (w/w). Water is then added to the wet dalbavancin and the drying step is repeated. These steps of adding water and drying the wet dalbavancin are repeated until the solvent level is below about 3.0% (w/w). These steps may be repeated one, two, three, four, five, six times or as many times as necessary to achieve the desired solvent content. Similarly, the present invention also includes dalbavancin compositions with low solvent levels. In one embodiment, the solvent level may be less than 3.0% (w/w), alternatively less than 2.5% (w/w), alternatively less than 2.0% (w/w), alternatively less than 1.5% (w/w), alternatively less than 1.0% (w/w), alternatively less than 0.5% (w/w), alternatively less than 0.1% (w/w). The solvent may be acetone. Alternatively, the solvent may be any of the following: ethanol, methanol, propanol, butanol, diethyl ether, dichloromethane, tetrahydrofuran, chloroform, 1, 4-dioxane, trichloroethylene, benzene, carbon tetrachloride, 1, 2-dichloroethane, 1, 1, 1-trichloroethane, acetonitrile, chlorobenzene, cyclohexane, dichloromethane, 1, 2-dimethoxyethane, N-dimethylacetamide, N-dimethylformamide, 1, 4-dioxane, 2-ethoxyethanol, ethylene glycol, formamide, hexane, 2-methoxyethanol, methyl butanone, methylcyclohexane, N-methylpyrrolidone, nitromethane, pyridine, dioxythiophene, tetrahydronaphthalene, toluene, 1, 1, 2-trichloroethane, xylene, acetic acid, anisole, 1-butanol, 2-butanol, tetrahydrofuran, chloroform, methanol, ethanol, methanol, butyl acetate, t-butyl methyl ether, cumene, dimethyl sulfoxide, ethyl acetate, diethyl ether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, propyl acetate, 1-diethoxypropane, 1-dimethoxymethane, 2-dimethoxypropane, isooctane, isopropyl ether, methyl isopropyl ketone, methyl tetrahydrofuran, petroleum ether, trichloroacetic acid and trifluoroacetic acid. Additionally, the drying process may be performed at a temperature of about 28 ℃ or less, alternatively at a temperature of about 26 ℃ or less, alternatively at a temperature of about 24 ℃ or less, alternatively at a temperature of about 22 ℃ or less, alternatively at a temperature of about 20 ℃ or less, alternatively at a temperature of about 15 ℃ or less, alternatively at a temperature of about 10 ℃ or less. The vacuum pressure of the drying process may be about 50 mbar or less, alternatively about 45 mbar or less, alternatively about 40 mbar or less, alternatively about 35 mbar or less, alternatively about 30 mbar or less, alternatively about 25 mbar or less, alternatively about 20 mbar or less, alternatively about 15 mbar or less, alternatively about 10 mbar or less, alternatively about 5 mbar or less. Additionally, the moisture content may be less than about 25% (w/w), alternatively less than about 20% (w/w), alternatively less than about 15% (w/w).

In addition to low solvent content, the drying process may also reduce the amount of MAG present in the dalbavancin composition. MAG may be present in an amount less than about 5% HPLC distribution, alternatively less than about 4.5% HPLC distribution, alternatively less than about 4.0% HPLC distribution, alternatively less than about 3.5% HPLC distribution, alternatively less than about 3.0% HPLC distribution, alternatively less than about 2.5% HPLC distribution, alternatively less than about 2.0% HPLC distribution, alternatively less than about 1.5% HPLC distribution, alternatively less than about 1.0% HPLC distribution, alternatively less than about 0.8% HPLC distribution, alternatively less than about 0.6% HPLC distribution, alternatively less than about 0.5% HPLC distribution, alternatively less than about 0.4% HPLC distribution, alternatively less than about 0.3% HPLC distribution, alternatively less than about 0.2% HPLC distribution, alternatively less than about 0.1% HPLC distribution.

The invention also includes the component B containing dalbavancin factor₂And iso B₀The pharmaceutical composition of (1). B is₂And iso B₀The respective amounts may independently not exceed about 3.0% HPLC profile, alternatively may independently not exceed about 2.5% HPLC profile, alternatively may independently not exceed about 2.0% HPLC profile, alternatively may independently not exceed about 1.5% HPLC profile, alternatively may independently not exceed about 1.0% HPLC profile, alternatively may independently not exceed about 0.5% HPLC profile, or alternatively may independently not exceed about 0.1% HPLC profile.

In addition, the invention also comprises a dalbavancin factor B₂Iso B₀And MAG. B is₂Iso B₀And MAG may each independently comprise no more than about 3.0% HPLC profile, alternatively may independently comprise no more than about 2.5% HPLC profile, alternatively may independently comprise no more than about 2.0% HPLC profile, alternatively may independently comprise no more than about 1.5% HPLC profile, alternatively may independently comprise no more than about 1.0% HPLC profile, alternatively may independently comprise no more than about 0.5% HPLC profile, or alternatively may independently comprise no more than about 0.1% HPLC profile.

The present invention also includes a method of treating a bacterial infection comprising providing to a patient in need of treatment at least one of the above pharmaceutical compositions and administering to the patient a therapeutically effective amount of sterile, stable, particle-free, clear dalbavancin. The method may further comprise administering a single subsequent therapeutically effective dose. A single subsequent therapeutically effective dose can be administered about 5-10 days or about one week after the initial dose. A single subsequent therapeutically effective dose may also be administered about 5-10 days or about one week after the initial dose, without any intervening dalbavancin dose. In another embodiment, the method may comprise administering a plurality of subsequent doses. Multiple subsequent doses may be administered at approximately 5-10 day intervals or at weekly intervals. Multiple subsequent doses may also be administered at approximately 5-10 day intervals or weekly intervals, without any intervening dalbavancin doses. The method may further comprise the step of further monitoring the infection after administration of the first dose, and optionally adjusting subsequent doses accordingly.

The invention also includes a method of treating a bacterial infection in a kidney injury patient comprising administering to the patient a therapeutically effective amount of sterile, stable, particle-free, clear dalbavancin. The lesions may be mild to severe. In one embodiment, the therapeutically effective dose achieves a peak concentration (C) in the patient of at least 100mg/L_max). In another embodiment, a therapeutically effective dose achieves a patient exposure (area under the curve) of at least 13,000 mg.h/L. The method may comprise administering a single dose of about 300 and 1200mg of dalbavancin, alternatively about 400mg, alternatively about 500mg, alternatively about 600mg, alternatively about 700mg, alternatively about 800mg, alternatively about 900mg, alternatively about 1000mg, alternatively about 1100mg, alternatively about 1200 mg.

The method of treating a bacterial infection in a kidney injury patient may further comprise administering multiple doses of dalbavancin. In one embodiment, two doses may be administered with an interval of about 5 to about 10 days, such as about one week, or alternatively about 10 to about 18 days, such as about 2 weeks (or 14 days). Alternatively, the dosing frequency may be, for example, two doses a week, three doses a week, or multiple doses a week. Alternatively, the dosing interval may be, for example, any of: about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more days apart. The number of doses administered may be, for example, one, two, three, four, five, six or more doses, each dose following the initial dose being administered after a selected dose interval.

In one embodiment, the first dose may be about 750mg and the second (or subsequent) dose may be about 250 mg. In another embodiment, the first dose may be about 750mg and the second (or subsequent) dose may be about 150 mg. In another embodiment, the first dose may be about 750mg and the second (or subsequent) dose may be about 125 mg. In another embodiment, the first dose may be about 1000mg and the second (or subsequent) dose may be about 500 mg.

In another embodiment, the first dose may be from about 200mg to about 1500mg, alternatively from about 200mg to about 1300mg, alternatively from about 100mg to about 1500mg, alternatively from about 200mg to about 1400mg, alternatively from about 300mg to about 1300mg, alternatively from about 400mg to about 1200mg, alternatively from about 500mg to about 1100mg, alternatively from about 600mg to about 1000mg, alternatively about 950mg, alternatively about 900mg, alternatively about 850mg, alternatively about 800mg, alternatively about 750mg, alternatively about 700mg, alternatively about 650mg, alternatively about 600mg, alternatively about 550mg, or alternatively about 500 mg. The second or subsequent dose may be from about 200mg to about 1500mg, alternatively from about 200mg to about 1300mg, alternatively from about 100mg to about 1500mg, alternatively from about 200mg to about 1400mg, alternatively from about 300mg to about 1300mg, alternatively from about 400mg to about 1200mg, alternatively from about 500mg to about 1100mg, or alternatively from about 600mg to about 1000mg, alternatively about 600mg, alternatively about 550mg, alternatively about 500mg, alternatively about 450mg, alternatively about 400mg, alternatively about 350mg, alternatively about 300mg, alternatively about 250mg, alternatively about 200mg, alternatively about 150mg, or alternatively about 100 mg. It will be appreciated that any of the above first doses may be combined with any of the above second or subsequent doses in a dosing regimen.

In a multiple dosing regimen for a kidney injury patient or a normal patient, the ratio of the first dose to the second (or subsequent) dose can be defined in terms of fold. The amount of the first dose may be about 1-fold greater than the amount of the second dose. Alternatively, the first dose may be about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, or about 6 times greater than the amount of the second (or subsequent) dose.

Similarly, the amount of the second (or subsequent) dose may be about 6 times less than the amount of the first dose. Alternatively, the amount of the second (or subsequent) dose may be about 5.5 times less, alternatively about 5.0 times less, alternatively about 4.5 times less, alternatively about 4.0 times less, alternatively about 3.5 times less, alternatively about 3.0 times less, alternatively about 2.5 times less, alternatively about 2.0 times less, alternatively about 1.5 times less, alternatively about 1.0 times less than the amount of the first dose.

A method of treating a bacterial infection in a kidney injury patient may comprise administering a single dose of dalbavancin. The patient may have mild, moderate, severe or end-stage renal injury. The single dose may be in an amount of about 1200mg, alternatively about 1150mg, alternatively about 1100mg, alternatively about 1050mg, alternatively about 1000mg, alternatively about 950mg, alternatively about 900mg, alternatively about 850mg, alternatively about 800mg, alternatively about 750mg, alternatively about 700mg, alternatively about 650mg, alternatively about 600mg, alternatively about 550mg, alternatively about 500 mg. A single dose may also be in an amount of from about 250mg to about 1300mg, alternatively from about 300mg to about 1200mg, alternatively from about 350mg to about 1100mg, alternatively from about 400mg to about 1000mg, alternatively from about 450mg to about 1000mg, alternatively from about 500mg to about 1000mg, alternatively from about 550mg to about 950mg, alternatively from about 600mg to about 900mg, alternatively from about 650mg to about 850 mg.

The invention also includes a method of preparing the pharmaceutical composition described above, comprising providing dalbavancin and adding a stabilizer. In one embodiment, the stabilizing agent is a saccharide or sugar. In another embodiment, the stabilizing agent is mannitol, lactose, or a combination thereof.

In yet another embodiment, the method further comprises the step of adjusting the pH of the composition, if desired. In one embodiment, the pH is adjusted to about 1 to 7, more preferably about 2 to 6, more preferably about 3 to 5. The pH can be adjusted with a pH adjusting agent. The pH regulator comprises alkali goldInorganic bases of the genus and alkaline earth metals, e.g. NaOH, Ca (OH)₂KOH and Mg (OH)₂. Inorganic oxides, carbonates, bicarbonates of alkali metals and alkaline earth metals may also be used as pH regulators. Weak inorganic acids and amphoteric acids such as alkali metal and base salts of phosphoric acid, boric acid, sulfuric acid and all other sulfur-containing acids can also be used as pH adjusters. Amino acids such as lysine, meglumine, arginine, n-methylglucamine may also be used to adjust the pH. Organic bases, including all amines, phenols, weak carboxylic acids, dicarboxylic acids, hydroxycarboxylic acids, and salts thereof, may also be used to adjust the pH of the composition.

In another aspect, there is provided a method of treating a bacterial infection in an individual in need thereof, comprising administering at least one unit dose of dalbavancin in an amount sufficient to provide a therapeutically effective plasma level of dalbavancin in the individual for at least 5 days, and a pharmaceutically acceptable carrier. Therapeutically effective plasma levels of dalbavancin are typically at least about 4mg dalbavancin per liter of plasma. In one embodiment, the dose of dalbavancin administered is a clinically effective amount and it also has reduced adverse side effects relative to standard care with drugs such as teicoplanin and vancomycin.

Dalbavancin may be administered in a single dose or in multiple doses. In some embodiments, a single dose of about 100mg to about 4000mg, e.g., 3000mg, of dalbavancin is administered. In various embodiments, a single dalbavancin dose may include at least about any one of 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5, or 3 grams.

In other embodiments, two doses are administered at about 5 to about 10 day intervals, such as about one week intervals. The first dose may be about 500 to about 5000mg of dalbavancin and the second dose may be about 250mg to about 2500mg of dalbavancin. Typically, the first dose comprises about 1.5 to about 3 times, usually at least about 2 times the amount of dalbavancin contained in the second dose. For example, the first dose may be about 1000mg, and the second dose may be about 500mg dalbavancin. In another embodiment, the first dose may be about 1200mg and the second dose may be about 600 mg. In the method of administering two doses, the plasma trough level of dalbavancin in an individual prior to administration of the second dose is typically about 4mg, often at least about 10mg, often at least about 20mg, more often at least about 30mg, more often at least about 40mg dalbavancin per liter of plasma.

Generally, the methods of the invention include parenteral administration, such as intravenous administration. In some embodiments, the administration is intravenous and the rate of administration is controlled such that administration takes at least about 30 minutes or more.

The methods of the invention are useful for treating gram positive bacterial infections, such as staphylococcus aureus or streptococcus pyogenes skin and soft tissue infections. In some embodiments, the infection is penicillin-resistant and/or multi-resistant.

In another aspect, a method of preventing a bacterial infection is provided, the method comprising administering at least one unit dose of dalbavancin in an amount sufficient to provide a prophylactically effective plasma level of dalbavancin in an individual for at least about 1 day, 3 days, 5 days, a week, 10 days, or longer, and a pharmaceutically acceptable carrier. The dose of dalbavancin may be, for example, about 100mg to about 1000 mg. In some embodiments, the dalbavancin is administered before, during, or after a medical treatment or hospitalization.

The therapeutic or prophylactic method of the invention may comprise the administration of at least one antibiotic other than dalbavancin, preferably an antibiotic effective against gram-negative bacteria and/or an antibiotic effective against gram-positive bacteria against which dalbavancin is not effective, such as the VanA strain.

In another aspect, a kit is provided that includes at least one unit dose of dalbavancin in an amount sufficient to provide a therapeutically effective plasma level of dalbavancin for at least about 5 days or a prophylactically effective plasma level of dalbavancin for at least about 1 day in an individual and instructions for a method of treatment or prevention of a bacterial infection. The kit may comprise two unit doses, wherein the first dose comprises 1.5-3 times, often at least about 2 times the amount of dalbavancin comprised in the second dose. The kit may also include a non-dalbavancin antibiotic, preferably an antibiotic that is effective against gram-negative bacteria.

In one embodiment, a kit is provided that includes a first container containing a dry powder (e.g., lyophilized) dalbavancin composition and a second container containing a predetermined amount of a physiologically acceptable aqueous solution for mixing with the dalbavancin composition. The solution is preferably a sterile aqueous solution. In one embodiment, the kit includes a delivery device, such as a syringe or intravenous administration device, for administering the dalbavancin composition to the subject.

The invention also provides N useful for infectious microorganisms¹⁵，N¹⁵-a dialkyl antibiotic compound. The compounds of the present invention are useful for the treatment and/or prevention of microbial infections, such as SSTI and other bacterial infections.

The present invention is based, in part, on the discovery of N¹⁵，N¹⁵A dialkyl antibiotic compound having at least N which is comparable to a previously known compound¹⁵-a monoalkyl antibiotic compound such as a dalbavancin compound, or even greater antimicrobial activity. In addition, as described in the following examples, when compared to previously known N¹⁵Monoalkyl antibiotic compounds such as N¹⁵Certain N of the present invention as compared to monomethyldalbavancin compounds¹⁵，N¹⁵Dialkyl antibiotic compounds show a broader spectrum of antimicrobial activity.

In one aspect, the invention provides N of formula (I)¹⁵，N¹⁵-a dialkyl antibiotic compound:

n of the invention¹⁵，N¹⁵The dialkylantibiotic compounds (atom numbering according to figure 26) contain two alkyl substituents on the amino terminal nitrogen. In other words, in formula (I), R¹And R¹' is an alkyl group. In the most preferred embodiment of the invention, R¹And R¹' is methyl. Other substituents of formula (I) are described in detail below. Preferred substituents include those found on antibiotic A40926 compounds and/or dalbavancin compounds known to those skilled in the art. For example, in a preferred embodiment, G and M are glycosyl moieties, such as aminoglucuronyl and mannopyranosyl moieties described in the following sections. In certain embodiments, the glucosamine uronic acid moiety is acylated, for example with a fatty acid, and in certain embodiments, the mannopyranosyl moiety is acetylated.

In certain embodiments, X is OH, and in further embodiments, X is aminoalkylamino. The aminoalkylamino group can be any aminoalkylamino group known to those skilled in the art, including those described in U.S. patent No. 5,750,509. In a preferred embodiment, X is N, N-dimethylaminopropylamino. As described in detail in the following examples, compounds of formula (I) wherein X is aminoalkylamino exhibit antibacterial activity comparable to or greater than that of the corresponding dalbavancin compounds such as those described in U.S. patent No. 5,750,509. Although compounds in which X is aminoalkylamino are preferred, compounds in which X is OH are also useful, for example, for preparing compounds of the invention in which X is aminoalkylamino, and may themselves be used in the treatment and/or prophylaxis of microbial infections.

In another aspect, the invention provides a composition comprising N of the invention¹⁵，N¹⁵-a combination of a dialkyl antibiotic compound and a second compound. The second compound may be any compound known to those skilled in the art. In a particular embodiment, the second compound is an antibiotic compound, such as an antibiotic a 40926 compound or a dalbavancin compound. In a further embodiment, the second compound is also N of the invention ¹⁵，N¹⁵-a dialkyl antibiotic compound. Exemplary compositions of the invention include a mixture of the antibiotic A40926 compound and/or the dalbavancin compoundThe compound further comprises a compound of formula (I). In particular embodiments, the compositions are enriched with N of the invention¹⁵，N¹⁵-a dialkyl antibiotic compound. Enrichment may be relative to the antibiotic a40926 compound and/or dalbavancin compound of the composition, or relative to other compounds of the composition, or both. In certain embodiments, the invention provides compositions comprising purified N of the invention¹⁵，N¹⁵A dialkyl antibiotic compound, isolated N of the invention¹⁵，N¹⁵A dialkyl antibiotic compound, or purified and isolated N of the invention¹⁵，N¹⁵-a combination of dialkyl antibiotic compounds.

In a further aspect, the invention provides a pharmaceutical composition comprising N of the invention¹⁵，N¹⁵-pharmaceutical compositions of dialkyl antibiotic compounds. Preferred compositions comprise compounds of formula (I) wherein X is aminoalkylamino. The compositions may further comprise other active ingredients, including the antibiotic a40926 compound and/or dalbavancin compounds known to those skilled in the art. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent. In particular embodiments, the present invention provides pharmaceutical unit doses of the compounds of the invention for use, e.g., in the treatment and/or prevention of microbial infections.

In a further aspect, the present invention provides a pharmaceutical composition comprising a composition comprising dalbavancin-containing factor B suitable for reconstitution with a pharmaceutically acceptable excipient₀And B₂Is sterile, stable, and free of particulate dalbavancin powder dosage forms. Name B₂、C₂And N¹⁵，N¹⁵-dimethyl dalbavancin B₀May be used interchangeably. In one embodiment, the factor B₀Is not less than about 75% HPLC profile, alternatively not less than about 80% HPLC profile, alternatively not less than about 85% HPLC profile, alternatively not less than about 90% HPLC profile, alternatively not less than about 95% HPLC profile. B is₂Is present in an amount of not less than about 4.0% HPLC distribution, alternatively not less than about 5.0% HPLC distribution, alternatively not less than about 6.0% HPLC distribution, alternatively not lessAt about 7.0% HPLC distribution, alternatively not less than about 8.0% HPLC distribution, alternatively not less than about 9.0% HPLC distribution, alternatively not less than about 10.0% HPLC distribution, alternatively not less than about 15.0% HPLC distribution, alternatively not less than about 20.0% HPLC distribution, alternatively not less than about 25.0% HPLC distribution, alternatively not less than about 30.0% HPLC distribution, alternatively not less than about 40.0% HPLC distribution. The percentage HPLC distribution of a particular component can be calculated by comparing the area of each individual component to the total chromatographic area. The dosage form may further comprise at least one additional factor, such as dalbavancin factor a ₀、A₁、B₁、C₀Or C₁。

In a further aspect, the invention provides a pharmaceutical composition comprising dalbavancin factor B₀And B₂The pharmaceutical composition of (1). In one embodiment, the factor B₀Is not less than about 75% HPLC profile, alternatively not less than about 80% HPLC profile, alternatively not less than about 85% HPLC profile, alternatively not less than about 90% HPLC profile, alternatively not less than about 95% HPLC profile. B is₂The content is not less than about 3.0% HPLC distribution, alternatively not less than about 4.0% HPLC distribution, alternatively not less than about 5.0% HPLC distribution, alternatively not less than about 6.0% HPLC distribution, alternatively not less than about 7.0% HPLC distribution, alternatively not less than about 8.0% HPLC distribution, alternatively not less than about 9.0% HPLC distribution, alternatively not less than about 10.0% HPLC distribution, alternatively not less than about 15.0% HPLC distribution, alternatively not less than about 20.0% HPLC distribution, alternatively not less than about 25.0% HPLC distribution, alternatively not less than about 30.0% HPLC distribution, alternatively not less than about 40.0% HPLC distribution. As defined above, the percentage HPLC distribution of a particular component can be calculated by comparing the area of each individual component to the total chromatographic area. The pharmaceutical composition may further comprise at least one additional factor, such as dalbavancin factor a ₀、A₁、B₁、C₀Or C₁。

In another aspect, the invention also provides B₂De-concentration of fractions. In one embodiment, the factor B₂In a small amountAt about 5.0% HPLC distribution, alternatively no more than about 4.5% HPLC distribution, alternatively no more than about 4.0% HPLC distribution, alternatively no more than about 3.5% HPLC distribution, alternatively no more than about 3.0% HPLC distribution, alternatively no more than about 2.5% HPLC distribution, alternatively no more than about 2.0% HPLC distribution, alternatively no more than about 1.5% HPLC distribution, alternatively no more than about 1.0% HPLC distribution, alternatively no more than about 0.5% HPLC distribution, alternatively no more than about 0.1% HPLC distribution.

In another aspect, the present invention provides a method of treating and/or preventing a microbial infection in a subject in need thereof. The method may comprise administering to the subject an effective amount of N of the invention¹⁵，N¹⁵-a dialkyl antibiotic compound or composition. The invention includes the prevention or treatment of gram-positive or antibiotic-resistant bacterial infections, such as bacillus, corynebacterium, listeria, enterococcus, staphylococcus, streptococcus, neisseria or clostridium infections, particularly staphylococcus aureus, staphylococcus epidermidis, staphylococcus haemolyticus, streptococcus pyogenes, streptococcus pneumoniae, group a and group C streptococci, enterococcus faecalis, bacillus subtilis, neisseria gonorrhoeae, or clostridium difficile. Can use the present invention N ¹⁵，N¹⁵Other infections that are prevented or treated by the dialkyl antibiotic compounds, compositions and methods include gram-negative bacterial infections, such as infections by bartonella, brucella, campylobacter, enterobacter, escherichia (and other Proteobacteria), francisella, helicobacter, haemophilus, klebsiella, legionella, leptospira, morganella, moraxella, proteus, providencia, pseudomonas, salmonella, serratia, shigella, stenotrophomonas, vibrio and yersinia, especially infections by escherichia coli, proteus vulgaris, pseudomonas aeruginosa, and yeasts such as candida albicans.

Brief description of the drawings

FIG. 1A depicts component B of dalbavancin versus time in different pharmaceutical compositions with and without mannitol at 25 ℃₀The amount of (c).

FIG. 1B depicts the amount of MAG over time in different pharmaceutical compositions with or without mannitol at 25 ℃.

FIG. 2A depicts component B of dalbavancin versus time in different pharmaceutical compositions with and without mannitol at 40 deg.C ₀The amount of (c).

FIG. 2B depicts the amount of MAG over time in different pharmaceutical compositions with or without mannitol at 40 ℃.

FIG. 3A depicts component B of dalbavancin versus time in different pharmaceutical compositions containing mannitol and/or lactose at 25 deg.C₀The amount of (c).

Figure 3B depicts the amount of MAG over time in different pharmaceutical compositions containing mannitol and/or lactose at 25 ℃.

FIG. 4A depicts component B of dalbavancin versus time in different pharmaceutical compositions containing mannitol and/or lactose at 40 deg.C₀The amount of (c).

Figure 4B depicts the amount of MAG over time in different pharmaceutical compositions containing mannitol and/or lactose at 40 ℃.

FIG. 5 depicts the plasma concentration of dalbavancin versus time after a single intravenous infusion of 1000mg dalbavancin.

Figure 6 depicts isothermal titration calorimetry data for dalbavancin binding to human serum albumin (upper panel), and results determined from dalbavancin: the protein is 2: 1 (lower panel) plot of data fitted to curves of the binding model.

FIG. 7 depicts an electrospray ionization mass spectrum of dalbavancin.

FIG. 8 is a graph of dalbavancin concentration versus the overall ratio of dalbavancin multimers to monomers, and depicts that the overall ratio of dalbavancin multimers to monomers increases with increasing dalbavancin concentration.

FIG. 9 is a graph of pH versus overall ratio of dalbavancin multimers to monomers and depicts that the overall ratio of dalbavancin multimers to monomers increases with increasing pH.

FIG. 10 depicts an electrospray ionization mass spectrum of dalbavancin 5mM ammonium formate pH 5 solution.

FIG. 11 depicts an electrospray ionization mass spectrum of dalbavancin 50mM ammonium formate pH 5 solution.

FIG. 12 depicts an electrospray ionization mass spectrum of dalbavancin ammonium formate in 100mM pH 5 solution.

FIG. 13 depicts electrospray ionization mass spectra of teicoplanin (50 μ g/mL) in water.

FIG. 14 depicts electrospray ionization mass spectra of teicoplanin (100 μ g/mL) in water.

FIG. 15 depicts the effect of HSA on the apparent dissociation constant for dalbavancin/tripeptide binding at 26 deg.C (pH 7.4).

FIG. 16 depicts a comparison of isothermal calorimetry (ITC) data for tripeptides in combination with vancomycin and dalbavancin under the same conditions using the same tripeptide solution.

FIGS. 17A and 17B depict possible interactions of dalbavancin monomers and multimers (including dimers) with tripeptide ligands and HSA. Figure 17A depicts monomer-dimer equilibrium dalbavancin as a monomer in solution binding to two independent sites on HSA. Figure 17B depicts ligand binding to dalbavancin dimer in solution and weaker binding to dalbavancin monomer linked to HSA.

Figure 18 provides mean dalbavancin plasma concentration versus time curves.

Figure 19 provides mean dalbavancin plasma concentration versus time curves.

Figure 20 provides additional mean dalbavancin plasma concentration-time curves.

Figure 21 provides a graph of dalbavancin exposure versus creatinine clearance over the relative treatment periods.

Fig. 22 provides a comparison of exposure during relative treatment periods versus overall exposure.

Figure 23 provides dalbavancin concentration-time curves for normal renal function subjects and subjects with severe renal injury.

Figure 24 provides a time plot of dalbavancin plasma concentration.

Figure 25 provides a comparison of dalbavancin exposure throughout the treatment period.

FIG. 26 provides the structures of antibiotic A40926 compound and dalbavancin compound, including the numbering of selected atoms;

FIG. 27 provides dalbavancin B₀The structure of (1);

FIG. 28 provides an exemplary N of the present invention¹⁵，N¹⁵-the structure of a dimethyl antibiotic compound;

FIGS. 29-31 provide illustrations of embodiments of the present invention demonstrating the present invention N¹⁵，N¹⁵-the structure of a dimethyl antibiotic compound;

FIGS. 32-33 provide confirmation N¹⁵，N¹⁵-ESI and HPLC chromatograms of the structure of the dimethyl antibiotic compound;

FIG. 34 provides dalbavancin B ₂The structure of (1);

FIG. 35 provides dalbavancin B₂(N¹⁵，N¹⁵-dimethyl dalbavancin B₀) (ii) infrared spectroscopy;

FIG. 36 provides dalbavancin B₂(N¹⁵，N¹⁵-dimethyl dalbavancin B₀) ESI-MS spectrum of (a); and

FIG. 37 illustrates HPLC tracing of compositions of the invention.

FIG. 38 provides iso B₀Mass spectrum of (2).

FIG. 39 illustrates iso B₀HPLC trace of (3).

FIGS. 40A-C provide iso B₀Mass spectrum of (2).

FIG. 41 provides iso B₀Is/are as follows¹H-NMR spectrum.

FIG. 42 provides the iso B₀Is/are as follows¹³C-NMR spectrum.

Figure 43 provides identification of the proton localization of dalbavancin.

Figure 44 provides plasma pharmacokinetics of dalbavancin in femoral infected mice.

Figure 45 provides the effect of a single dose of dalbavancin on killing streptococcus pneumoniae in vivo over time.

Figure 46 provides the effect of a single dose of dalbavancin on killing staphylococcus aureus over time in vivo.

FIG. 47 provides the relationship between dalbavancin dosing interval and efficacy against Streptococcus pneumoniae.

FIG. 48 provides a relationship between dalbavancin dosing intervals and efficacy against Staphylococcus aureus.

FIG. 49 provides a relationship between the PK/PD parameters for dalbavancin and efficacy against Streptococcus pneumoniae.

FIG. 50 provides the relationship between the PK/PD parameters for dalbavancin and efficacy against Staphylococcus aureus.

Figure 51 provides dose response curves of dalbavancin against various strains.

FIG. 52 provides dose response curves for dalbavancin against Streptococcus pneumoniae and Staphylococcus aureus.

FIG. 53 provides dose response curves of dalbavancin in normal and neutropenic mice infected with Streptococcus pneumoniae.

Figure 54 provides dose response curves for dalbavancin in a lung and thigh infection model.

Detailed description of the invention

Definition of

When describing the compounds of the present invention, pharmaceutical compositions containing the compounds, and methods of using the compounds and compositions, the following terms have the following meanings, unless otherwise specified.

"antibiotic A40926 compound" refers to glycopeptide antibiotics known to those skilled in the art. Typical antibiotic A40926 compounds are described in U.S. Pat. Nos. 4,935,238, 4,868,171 and 4,782,042, the contents of which are hereby incorporated by reference in their entirety.

"dalbavancin compounds" refers to glycopeptide antibiotics described in U.S. patent No. 5,750,509 and U.S. patent application publication No. 2004/0142883, the contents of which are hereby incorporated by reference in their entirety. A typical systematic name for the dalbavancin compounds known to those skilled in the art is ristocetin A aglycone, 5, 31-dichloro-38-de (methoxycarbonyl) -7-demethyl-19-deoxy-56-O- (2-deoxy-2- ((10-methyl-1-oxoundecyl) amino) -beta-D-glucopyranosonosis 1) -38- (((3- (dimethylamino) propyl) amino) -carbonyl) -42-O-alpha-D-mannopyranosyl-N ¹⁵-methyl-. Examples include those described in fig. 26 and 27. Dalbavancin compounds include those compounds having an optional sugar moiety at positions 56 and 42 and an optional acyl group, such as a fatty acid, on the sugar. The dalbavancin compounds may be derived from the natural antibiotic a-40926 known to those skilled in the art. Typical dalbavancin compounds include those described in U.S. patent application publication No. 2004/0142883, such as dalbavancin a₀、A₁、B₀、B₁、C₀And C₁。

"N" as described in detail in the following section¹⁵，N¹⁵By a "dialkyl antibiotic compound" is meant a compound of the invention, i.e. at its N¹⁵Antibiotic compounds having two alkyl groups on the nitrogen. N of the invention¹⁵，N¹⁵The dialkyl antibiotic compound may be derived from the antibiotic A40926 compound or from a dalbavancin compound such as dalbavancin A₀、A₁、B₀、B₁、C₀And C₁。

"N" as described in detail in the following section¹⁵，N¹⁵By a "dimethyl antibiotic compound" is meant a compound of the invention, i.e. in its N¹⁵Antibiotic compounds having two methyl groups on the nitrogen.

"acyl" refers to the group-C (O) R, where R is alkyl.

"alkyl" refers to a monovalent saturated aliphatic hydrocarbon group, preferably having from 1 to about 11 carbon atoms, more preferably from 1 to 8 carbon atoms, and even more preferably from 1 to 6 carbon atoms. The hydrocarbon chain may be straight or branched. Examples of this term are groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, n-octyl and tert-octyl. The term "lower alkyl" refers to an alkyl group having 1 to 6 carbon atoms.

"alkylene" refers to a divalent saturated aliphatic hydrocarbon group, preferably having 1 to 11 carbon atoms, more preferably 1 to 6 carbon atoms, which may be straight or branched. Examples of such terms are, for example, methylene (-CH)₂-) ethylene (-CH₂CH₂-), propylene isomers (e.g. -CH₂CH₂CH₂-and-CH (CH)₃)CH₂-) and the like.

"amino" refers to the group-NH₂。

"alkylamino" refers to the group-NH-alkyl or-N (alkyl)₂。

"aminoalkylamino" refers to a group of the form-NR- (alkyl) -NR 'R ", wherein R, R' and R" each independently represent hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl (cycloheteryakyl), substituted heterocycloalkyl, heteroaryl, or substituted heteroaryl. Preferred R, R 'and R' groups include hydrogen and alkyl groups. Typical aminoalkylamino groups are described in U.S. patent No. 5,750,509, the contents of which are hereby incorporated by reference in their entirety.

"carboxyl" refers to the group-C (O) OH.

"dialkylamino" refers to the group-NRR ', where R and R' each independently represent alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, heteroaryl, or substituted heteroaryl as defined herein.

"halo" or "halogen" refers to fluorine, chlorine, bromine and iodine. Preferred halogen groups are fluorine or chlorine.

The "percent HPLC distribution" of a particular component is calculated by comparing the peak area corresponding to the particular component to the total chromatographic area.

"pharmaceutically acceptable" means approved by the federal or state government (through a regulatory agency for research or commercial use) or listed in the U.S. pharmacopeia or other pharmacopeia generally recognized for use in animals, and more particularly in humans.

"pharmaceutically acceptable salt" refers to a salt of a compound of the present invention which is pharmaceutically acceptable and which possesses the desired pharmacological activity of the parent compound. These salts include: (1) acid addition salts with organic or inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, caproic acid, cyclopentanepropionic acid, glycolic acid, glutaric acid, pyruvic acid, lactic acid, malonic acid, succinic acid, sorbic acid, ascorbic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, picric acid, cinnamic acid, mandelic acid, phthalic acid, lauric acid, methanesulfonic acid, ethanesulfonic acid, 1, 2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphoric acid, camphorsulfonic acid, 4-methylbicyclo [2.2.2] -oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, pivalic acid, t-butylacetic acid, lauryl sulfuric acid, gluconic acid, benzoic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) a salt formed when an acidic proton present in the parent compound encounters: (a) substituted with metal ions such as alkali metal ions, alkaline earth metal ions or aluminum ions, or substituted with alkali metal or alkaline earth metal hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide and barium hydroxide, amines, or (b) coordinated with organic bases such as aliphatic, alicyclic or aromatic organic amines such as methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, etc. (see, for example, U.S. patent No. 5,606,036).

Salts further include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, and when the compound contains a basic functional group, salts of non-toxic organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, and the like. The term "pharmaceutically acceptable cation" refers to a non-toxic pharmaceutically acceptable cationic counterion of the acidic functionality. Examples of such cations are sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium cations and the like.

"pharmaceutically acceptable excipient" refers to a diluent, adjuvant, excipient, or carrier with which a compound of the invention is administered.

"solvate" refers to a compound of the invention or a salt thereof, which further comprises a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. When the solvent is water, the solvate is a hydrate.

"Preventing" or "prevention" refers to reducing the risk of acquiring a disease or disorder (i.e., causing at least one clinical symptom of a disease not to occur in a subject that may be exposed to or predisposed to the disease but has not yet experienced or exhibited symptoms of the disease). Preferably, prevention refers to the use of a compound or composition in a subject that is not affected by a disease or disorder or that does not yet exhibit symptoms of a disease or disorder, e.g., a subject that is not yet infected or that does not yet exhibit symptoms of an infection.

A "subject" includes a human. The terms "human", "patient" and "subject" are used interchangeably herein.

By "therapeutically effective amount" is meant an amount of a compound or composition sufficient to effect treatment of a disease when administered to a patient to treat the disease. The "therapeutically effective amount" may vary depending on, inter alia, the compound, the disease and its severity, the age, weight, etc. of the subject to be treated.

"treating" or "treatment" of any disease or disorder refers in one embodiment to ameliorating the disease or disorder present in a patient (i.e., inhibiting or reducing the development of the disease or at least one clinical symptom thereof). In another embodiment, "treating" or "treatment" refers to improving at least one physical parameter, which may be indistinguishable by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically (e.g., stabilizing a discernible symptom), physiologically (e.g., stabilizing a physical parameter), or both. In yet another embodiment, "treating" or "treatment" refers to delaying the onset of the disease or disorder.

It is understood that compounds having the same molecular formula but differing in the nature or order of bonding of their atoms or the arrangement of their atoms in space are referred to as "isomers". Isomers whose atoms differ in their arrangement in space are referred to as "stereoisomers".

Stereoisomers that are not mirror images of each other are referred to as "diastereomers", and stereoisomers that are non-superimposable mirror images of each other are referred to as "enantiomers". When a compound has an asymmetric center, for example when it is bonded to four different groups, a pair of enantiomers may be present. Enantiomers can be characterized by the absolute configuration of their asymmetric centers and designated as (R) or (S) according to Cahn and Prelog rules, or can be characterized in a manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., the (+) -or (-) -isomers, respectively). The chiral compounds may exist as individual enantiomers or as mixtures thereof. Mixtures containing equal proportions of enantiomers are referred to as "racemic mixtures".

In certain embodiments, the compounds of the present invention may have one or more asymmetric centers; the compounds can thus be produced as the (R) -or (S) -enantiomers alone or as a mixture thereof. Unless otherwise indicated, for example by specifying the stereochemistry at any position of the general formula, the specification or naming of a particular compound in the specification and claims is intended to include both the individual enantiomers and mixtures thereof (racemic or not). Methods for determining stereochemistry and separating stereoisomers are well known in the art. In certain embodiments, the present invention provides base-treated stereoisomers of the compounds described herein.

The present invention provides novel dalbavancin pharmaceutical compositions, methods of preparing pharmaceutical compositions, and methods of treating bacterial infections using the novel compositions. In particular, the present invention provides stable dalbavancin compositions with bactericidal activity that can be refrigerated or stored at room temperature for extended periods of time, more preferably at least one year at room temperature, more preferably at least two years at room temperature, without significant degradation of the dalbavancin active ingredient.

The invention also provides improved dosage regimens and novel dalbavancin compositions, and improved methods of treating antibiotic-resistant bacterial infections. In particular, the present invention provides dalbavancin compositions having activity against one or more antibiotic-resistant strains, such as MRSA, that can be administered in a dosing regimen of once every 5 to 7 days or longer.

Dalbavancin, also known as BI 397 or VER001 in the scientific literature, is a semi-synthetic glycopeptide mixture whose properties have been reported in U.S. patent nos. 5,606,036, 5,750,509, 5,843,679 and 5,935,238.

As used herein, the term "dalbavancin" refers to a composition comprising: one or more, preferably two or more, in some cases three or more, in some cases four or more, in some cases five or more, are referred to as "A" in close association as described below ₀″、″A₁″、″B₀″、″B₁″、″C₀″、″C₁″、″B₂"or" C₂″、″D₀"and" D₁"or a monomer, multimer (i.e., dimer or higher order multimer), tautomer, ester, solvate, or pharmaceutically acceptable salt thereof. As used herein, "dimer" or "multimer" refers to a homodimer or a homomultimer, i.e., a dimer or multimer composed of the same monomers of dalbavancin homologs; or a heterodimer or heteromultimer, i.e., a dimer or multimer composed of at least two different dalbavancin homolog monomers. The factor is distinguished from the fatty acid side chain structure of the N-acylglucosamine uronic acid moiety, except for C₂(also called B)₂) And (c) other than. C₂Mass spectra of the fractions indicated the presence of additional methylene groups on the terminal amino groups. Dalbavancin often includes "MAG," which is a non-homolog variant lacking an acylglucuronamine (acylglucuronamine) moiety, as described below. Individually, the dalbavancin homologs and MAGs are sometimes referred to herein as "dalbavancin components".

Such as Malabarba and Donadio (1999) Drugs of the Future24 (8): 839-846, dalbavancin was prepared by chemically modifying native glycopeptide complex A-40, 926. The main components of dalbavancin are due to Seed B₀It represents > 75% of the total complex.

The amounts of the components present in the dalbavancin composition are determined by a variety of factors, including, for example, the fermentation conditions used in the preparation of the native glycopeptide complex a-40926 (which is a precursor of dalbavancin, see, e.g., U.S. patent No. 5,843,679), the conditions used to recover a-40926 from the fermentation broth, the chemical reaction used to selectively esterify the carboxyl group of the sugar moiety of a-40926, the conditions used to amidate the peptidyl carboxyl group, the conditions used to saponify the carboxyl ester of the N-amidoglucuronate functional group, the conditions used to recover dalbavancin from the synthesis mixture, and the like.

In a preferred embodiment, the dalbavancin composition comprises at least about 80 to about 98% by weight of B₀And (4) components. In a particularly preferred embodiment, dalbavancin comprises B in the following amount₀：

TABLE 1 Dalbavancin composition B₀Preferred amounts of Components

¹Each range represents B relative to the total dalbavancin components present in the dalbavancin composition including MAG₀Mole% of (c).

In one embodiment, the dalbavancin composition or formulation comprises a minimum amount of component C₀、C₁And C₂(B₂) If any. In another embodiment, the dalbavancin composition or formulation does not comprise any component C ₀、C₁And C₂(B2)。

Each dalbavancin factor had been previously purified by HPLC and characterized by NMR. In U.S. Pat. No. 5,750,509, Malabarba et al describe derivatives of antibiotic A40926 characterized by an N-acyl aminoglucuronyl moietyHaving a carboxyl group, (C)₁-C₄) Alkoxycarbonyl, aminocarbonyl, (C)₁-C₄) An alkylaminocarbonyl or hydroxymethyl substituent, and having a hydroxyl or polyamine substituent at position 63 of the molecule. The compounds of the invention were found to have high activity against glycopeptide-resistant enterococci and staphylococci in vitro. However, Malabarba et al neither recognized a combination of pharmaceutically beneficial factors nor identified or characterized degradation products lacking the acyl glucuronamide moiety. Malabarba et al never monitored MAG, or produced a sterile form. Malabarba et al purified only a small amount by HPLC and did not perform quantitative material analysis.

The chemical structures of several dalbavancin components are described below in II:

all of the above dalbavancin components have bactericidal activity against a variety of gram-positive bacteria. However, a non-syngeneic dalbavancin component, which lacks the acylglucuronamine moiety present in the other components, referred to as "MAG" has lower bactericidal efficacy both in vivo and in vitro than the other dalbavancin components (see tables 2 and 3). MAG is believed to be a decomposition product of one or more other dalbavancin components. Thus, in preferred embodiments, the amount of MAG in the dalbavancin is less than about 4, 3.5, 3, 2.5, 2, 1.5, 1, or 0.5 mole percent of all dalbavancin components present, including MAG.

TABLE 2 ED of MAG against murine septicemia of Staphylococcus aureus compared to dalbavancin and vancomycin₅₀

Treatment: treatment once in 10 minutes of infection by the subcutaneous route

TABLE 3 microbiological Activity

Other modifications in the final drug substance result from the fermentation process, subsequent chemical steps, or drying steps. Hetero B₀(RRT 0.76) is dalbavancin B₀The diastereomer of (a), which is caused by epimerization of the labile proton within the dalbavancin core structure. It is produced during the deacetylation of the precursor A-40926 with a base, forming the diastereoisomer of A-40,926. The diastereomer is subsequently converted into iso-B in a subsequent chemical modification step₀. Hetero B₀It was well resolved in the HPLC release method and was the only stereoisomer detected in the API. Laboratory data have demonstrated that heteroB₀Is not formed during the alkaline hydrolysis process that converts the ester form (MA-A-1) to dalbavancin. The iso-B has been characterized by NMR and MS₀And it has been demonstrated to be in vivo compatible with dalbavancin B₀The biological activity of the phase.

Iso B in dalbavancin compositions₀The level is less than about 3.5%, alternatively less than about 3.0%, alternatively less than about 2.5%, alternatively less than about 2.0%, alternatively less than about 1.5%, alternatively less than about 1.0%, alternatively less than about 0.5%, alternatively less than about 0.4%, alternatively less than about 0.3%, alternatively less than about 0.2%, or alternatively less than about 0.1%.

Iso B in dalbavancin compositions₀The level may optionally be from about 0.5% to about 3.0%, optionally from about 0.5% to about 2.8%, optionally from about 0.5% to about 2.5%, optionally from about 0.5% to about 2.3%, optionally from about 0.5% to about 2.0%, optionally about 0.5%-about 1.8%, alternatively from about 0.5% to about 1.5%, alternatively from about 0.5% to about 1.3%, alternatively from about 0.5% to about 1.0%, alternatively from about 0.1% to about 0.5%.

Iso B in dalbavancin compositions₀The level is optionally no less than about 1.0%, optionally no less than about 1.5%, optionally no less than about 2.0%, optionally no less than about 2.5%, optionally no less than about 3.0%, optionally no less than about 3.5%, optionally no less than about 4.0%, optionally no less than about 4.5%, or optionally no less than about 5.0%.

Dalbavancin is thought to inhibit bacterial cell wall biosynthesis by binding to the D-alanyl-D-alanine-terminal precursor of peptidoglycan. Dimers or higher order multimers of dalbavancin may have further antibacterial properties through the interaction of lipophilic side chains with the bacterial cytoplasmic membrane. See, e.g., Malabarba and Ciabatti et al (2001) CurrentMedicina1 Chemistry 8: 1759-1773. Further studies on dalbavancin multimers can be found in U.S. series 10/714,166 entitled "dalbavancin compositions for treating bacterial infections" attorney docket No. 34231-20052.00 filed on 4.11.2003, the disclosure of which is hereby incorporated by reference in its entirety.

In vitro, non-clinical and clinical data indicate that dalbavancin is beneficial in the treatment of severe gram-positive infections caused by MRSA and cos and all streptococci and non VanA enterococci (including VanB and VanC phenotypes that are less sensitive or resistant to vancomycin).

Dalbavancin is more active in vitro against staphylococci (including some teicoplanin-resistant strains) than teicoplanin and vancomycin. Dalbavancin has better activity against streptococci (including penicillin-resistant strains) than teicoplanin or vancomycin. Dalbavancin is active against a variety of gram-positive bacteria, including most resistant strains, both in vitro and in vivo.

Dalbavancin is typically administered to an individual as a dalbavancin composition. As used herein, the term "dalbavancin composition" or "dalbavancin formulation" refers to a composition, typically a pharmaceutical composition, comprising the dalbavancin described above and one or more other components of the dalbavancin, such as a pharmaceutically acceptable carrier, stabilizer, buffer, or other similar component.

As shown in example 1, dalbavancin was effective at dosing intervals of 1 week. Thus, the advantage of dalbavancin over other treatment options is the ability to administer the antibiotic on a weekly basis, thereby maximizing patient compliance and potentially minimizing the need for, or shortening the hospital stay for, parenteral antibiotic administration. Less frequent administration generally allows for outpatient treatment, thus reducing treatment costs. As further shown in example 1, a second dose of dalbavancin about 1 week after administration of the first dose unexpectedly provides a significant improvement in therapeutic efficacy, wherein the second dose is about half of the first dose.

Application method

Various dosing regimens of dalbavancin have been previously reported, including single dose and multiple dosing regimens. Leighton et al reported multiple dose administration with an optimal dose ratio (loading dose (LD)/Maintenance Dose (MD)) of 10: 1. In this study, the dose was scaled up to 1120mg Single Dose (SD) and multiple dose regimen until day 1 of 500mg BID, followed by 100mg daily for 6 consecutive days. Leighton et al, "Dalbavancin: phase I Single and multiple-Dose plant Controlled Intra Safety, pharmaceutical Study in health volumes, "41^st ICAACAbstracts，Chicago，IL，September 22-252001，Abstract No.951，p.25。

Other single and multiple dose administrations are also described by Leighton et al. In a single dose study, the reported dose was scaled up through a series of 140mg, 220mg, 350mg, 500mg, 630mg, 840mg and 1120 mg. In the multiple dose phase, the administration consisted of a loading dose, administered as two equal doses at 12 hour intervals, followed by a maintenance dose. The initial dosing regimen was 150mg BID minusThe loading dose, followed by a 30mg daily maintenance dose, was 6 days. The dose escalation was performed as follows: 200mg BID/40mg, 300mg BID/60mg, 400mg BID/80mg and 500mg BID/100 mg. Leighton et al, "Dalbavancin: phase I Single and Multiple-dose PlaceboControled Intra venous Safe in health volumes "41 ^st ICAAC，Chicago，IL，December 2001，Poster No.951。

White et al reported a single 0.5 hour intravenous infusion of a 70mg, 140mg, 220mg or 360mg dosing regimen. The multiple dosing regimen included administration of 70mg daily for 7 days. White et al "V-Glycopeptide: phase 1 Single and multiple-Dose plant Controlled Intra venous Safety, Pharmacokinetic, and Pharmacokinetic Study in Healthy Subjects, "40^thICAAC, Toronto, Canada, September 17-20, 2000, Poster No.2196 and Abstract No. 2196. All of the above references are hereby specifically incorporated by reference in their entirety.

Novel methods of administering dalbavancin to an individual in need of treatment for a bacterial infection are provided. Treatment may include prophylaxis, treatment or cure. The method comprises administering one or more unit doses of dalbavancin in a therapeutically or prophylactically effective amount.

As used herein, "therapeutically effective amount" refers to the amount of dalbavancin that will provide the desired therapeutic result (e.g., reduce or eliminate bacterial infection). A therapeutically effective amount may be administered in one or more doses. A "prophylactically effective amount" refers to an amount of dalbavancin that is sufficient to prevent or reduce the severity of a future bacterial infection when given to an individual who is susceptible to and/or likely to be infected with the bacterial infection, e.g., due to a medical procedure or hospitalization or exposure to the individual with the bacterial infection. Dalbavancin is typically administered in a pharmaceutically acceptable carrier.

Dalbavancin is typically provided as a hydrochloride salt that is readily soluble in water.

Typically, dalbavancin is administered as a "unit dose" in a dalbavancin formulation that includes an amount of dalbavancin sufficient to provide a therapeutically or prophylactically effective plasma level of dalbavancin for several days (usually at least about 5 days, 1 week, or 10 days) when administered to an individual.

As used herein, "individual" refers to a vertebrate, typically a mammal, often a human.

All of the homologs of dalbavancin described above exhibit an extended half-life in plasma, usually 9 days or more, although MAG is believed to have a shorter half-life than the other homologs. The long half-life allows for longer dosage intervals than either vancomycin or teicoplanin. As described in example 1, once weekly administration of dalbavancin is effective in controlling bacterial infections as compared to a twice daily dosing schedule often used for vancomycin or a once daily dosing schedule often used for teicoplanin. Administration of dalbavancin at a lower frequency may provide significant therapeutic advantages over vancomycin and teicoplanin, particularly in terms of improved convenience and patient compliance with the treatment regimen. Abnormally high doses (i.e., resulting in abnormally high and sustained serum levels) may be administered at a lower frequency of administration than other available treatment options. Because dalbavancin exhibits minimal adverse effects in vivo at concentrations required to achieve lower frequency dosing (demonstrating a large drug window), and further because dalbavancin blood levels are maintained above the minimum bactericidal level throughout the treatment regimen (demonstrating a prolonged dalbavancin plasma half-life), new dosing regimens useful for dalbavancin can improve efficacy. The combination of extended serum half-life and large drug window allows for less frequent administration of dalbavancin.

Additionally, it is preferred that the dalbavancin be formulated with a stabilizer that inhibits degradation of one or more of the dalbavancin components. In a preferred embodiment, the dalbavancin is formulated in a 1: 2 ratio by weight of mannitol to dalbavancin. In another preferred embodiment, the dalbavancin is formulated in a 1: 4 weight ratio of mannitol, lactose, dalbavancin.

In some embodiments, the dalbavancin formulation is administered at a dose that results in therapeutically effective (i.e., bactericidal) plasma levels of the drug for several days (often at least about 5 to about 10 days, often at least about 1 week). Generally, the minimum bactericidal concentration of dalbavancin in plasma is maintained at or above about 4mg/l for at least 5 days. Dalbavancin is often maintained at a plasma level of at least about 5mg/l, often at least about 10mg/l, often at least about 20mg/l, often at least about 30mg/l, often at least about 40mg/l for at least 5 days, often for at least about 1 week or more. Dalbavancin plasma levels can be determined by methods well known in the art, such as liquid chromatography, mass spectrometry, or microbiological biological assays. An example of a method for quantifying dalbavancin in plasma is provided in example 5.

The upper limit for the plasma concentration level of dalbavancin is generally dictated by the dose that inhibits an unacceptable adverse effect in the patient population being treated.

The dalbavancin composition may be administered in a single dose or in multiple doses. When administered as a single dose, the dalbavancin composition is preferably formulated to contain sufficient dalbavancin to achieve bactericidal activity in vivo for at least 5 days, preferably at least 7 days, and more preferably at least 10 days.

When multiple doses are employed, dalbavancin may be administered once a week for 2 weeks or more. In one embodiment, dalbavancin is administered in at least 2 doses, often 2 doses spaced about 5 to about 10 days apart, more often once per week for 2 weeks. As shown in example 1, the dosing regimen provides significant advantages over conventional antibiotic treatment regimens.

The dalbavancin composition may also be administered in multiple doses, or in one or more once-a-week doses, two or more days apart or at least one week apart. In some embodiments, the dalbavancin composition is administered once a week, followed by once every two weeks or once a month. In some embodiments, dalbavancin is administered once every other week for 2, 3, 4, 5, 6 weeks or more.

Most advantageously, daily dosing is not required because higher, less frequent doses are used. For example, a single or multiple dose may be about 0.1 to about 5 grams. A single dose of about 0.1 to about 4 grams, e.g., about 3 grams, may be administered for the treatment of various infections. When multiple doses are administered, e.g., once per week, each dose can range, e.g., from about 0.25 to about 5.0 grams.

For embodiments in which a single dose is administered to treat an infection, the dose may be, for example, from about 0.1 to about 5 grams, or from about 0.5 to about 4 grams, or from about 1 to about 3.5 grams, or from about 2 to about 3 grams, e.g., about 3 grams. In some embodiments, a single dose of about 1, 1.5, 2, 2.5, or 3 grams is administered to treat a bacterial infection. For embodiments in which a single dose is administered for prophylaxis, the dose may be, for example, from about 0.1 to about 3 grams, or from about 0.1 to about 1 gram, e.g., about 0.5 or about 0.25 grams.

In a dosage regimen comprising multiple doses, the individual doses may be the same or different. In some embodiments, a first dose that is higher than one or more subsequent doses is administered, i.e., e.g., about 1.5-10 times higher, in some cases 9 times higher, in some cases 8 times higher, in some cases 7 times higher, in some cases 6 times higher, in some cases 5 times higher, in some cases 4 times higher, in some cases 3 times higher, in some cases 2 times higher. For example, the first dose may be about 0.5 grams to about 5 grams and the second dose about 0.25 grams to about 2.5 grams, the first dose may be about 0.8 to about 2 grams and the second dose about 0.4 to about 1 gram, or the first dose may be about 0.4 to about 3 grams and the second dose about 0.2 to 1.5 grams.

In some embodiments, at least 2 doses are administered, wherein the first dose comprises about twice the subsequent dose of dalbavancin. In one embodiment, the first dose comprises about 1 gram of dalbavancin and the subsequent dose comprises about 0.5 gram. In another embodiment, the first dose comprises about 0.5 grams of dalbavancin and the subsequent dose comprises about 0.25 grams.

In some embodiments, the dalbavancin composition is administered in 2 doses of the same or different amounts two or more days apart or at least about one week apart. Two doses of about 0.2 to about 1.5 grams of dalbavancin are often administered about 5 to about 10 days apart, more often about 1 week apart. In one embodiment, a first dose of about 1 gram of dalbavancin and a second dose of about 0.5 gram of dalbavancin are administered about 1 week apart.

In a multiple dose regimen, the time between doses may be, for example, in the range of about 5 to about 10 days, often about one week, alternatively about 2 weeks. The frequency of administration may be, for example, two doses per week or more doses per week. The dosing interval or time between doses can be, for example, any of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more days. The number of doses given can be, for example, one, two, three, four, five, six or more doses, with each dose after the initial dose being administered after a selected dose interval.

In a multi-dose dosing regimen, the "trough level" or plasma level of dalbavancin after a first dose of dalbavancin and just prior to administration of a second dose is at least about 4 mg/l. Preferably, the trough level at the end of a dosing interval, such as about one week, is preferably at least about 20mg/l, more preferably at least about 30mg/l, even more preferably at least about 40 mg/l.

Dalbavancin may be administered parenterally, for example intramuscularly (i.m.), intravenously (i.v.) (bolus injection or slow infusion), subcutaneously (s.c), intraperitoneally (i.p.), or intrathecally (i.t). The dosing schedule and actual dose administered may vary depending on factors such as the nature and severity of the infection, the age, weight, general health of the patient, and the tolerance of the particular patient to dalbavancin, but this is determinable by a health professional. In one embodiment, a 0.5 gram intravenous dose is administered one week after 1 gram intravenous administration of dalbavancin.

The drug may be administered and delivered to the patient at a controlled rate, e.g., intravenously, so that the concentration in the blood does not increase too quickly or precipitate. In some embodiments, dalbavancin is administered at an appropriate rate so that the drug forms a complex with an endogenous protein in the bloodstream. Without being bound by a particular theory, it is believed that endogenous proteins such as human serum albumin can form complexes with one or two dalbavancin homolog monomeric molecules in vivo. When a sufficient amount of dalbavancin is present, it is believed that at most two molecules of dalbavancin homolog will bind to the endogenous protein, and it is further believed that this complex is formed by binding separate molecules of dalbavancin homolog at two different binding sites. Alternatively, it is also possible that the dimeric dalbavancin binds to a single binding site on the endogenous protein. Further detailed studies of the DALBAVANCIN-endogenous protein complex discussed above can be found in U.S. series No. 10/713,924, attorney docket No. 34231-20053.00 entitled "COMPOSITIONS and methods FOR supporting BACTERIAL inhibitors with DALBAVANCIN COMPLEXES," filed on 11/4/2003, the disclosure of which is hereby incorporated by reference in its entirety.

The infusion duration may be, for example, from about 1 minute to about 2 hours, or alternatively, from about 15 minutes to about 2 hours. For example, infusion durations of about 30 minutes may be used, with dosages of about 0.5 to about 1 gram. Intravenous administration under controlled rate conditions can produce a far greater excess of dalbavancin concentration in vivo than can be achieved in solution phase at physiological pH in vitro. While not wishing to be bound by theory, this may be attributed to the complex formation of dalbavancin with endogenous proteins such as serum albumin, which may increase the solubility of dalbavancin in plasma compared to in vitro studies.

Formation of the dalbavancin complex in vitro or ex vivo may allow for faster administration, e.g., at least about 1 minute, at least about 10 minutes, or at least about 20 minutes. The complex may be obtained by mixing human serum albumin and/or another endogenous protein with dalbavancin to form a complex in vitro or ex vivo, and then administering this complex to the patient to be treated. Alternatively, human serum albumin or other endogenous proteins may be obtained from autologous sources, or by expression from microorganisms modified to contain the genes for the proteins.

The amount of dalbavancin administered may be any dosage disclosed herein. The dose of dalbavancin is generally selected so that the drug is present at therapeutically or prophylactically effective (i.e., bactericidal) plasma levels for an extended period of time, often at least 5 days, more often about a week or more. It is preferred to administer a dose of dalbavancin that will produce and maintain a bactericidal concentration for at least about one week (or about 5 to about 10 days). The bactericidal concentration is defined as the concentration of dalbavancin required to kill at least 99% of the bacteria initially present in the in vitro experiment over a 24 hour period. The minimum bactericidal concentration of plasma dalbavancin is typically about 4 mg/l.

Examples of treatable indications include recurrent and non-recurrent Skin and Soft Tissue Infections (SSTI) (also known as complex and uncomplicated Skin and Skin Structure Infections (SSSI)), bloodstream infections (BSI), catheter-associated bloodstream infections (CRBSI), osteomyelitis, prosthetic joint infections, surgical prophylaxis, endocarditis, nosocomial or community-acquired pneumonia, pneumococcal pneumonia, empirical therapy for febrile neutropenia, joint cavity infections, and device infections (e.g., pacemakers and internal cardiac defibrillators). Gram-positive or antibiotic-resistant bacterial infections can be treated, for example staphylococcal, streptococcal, neisserial or clostridial infections, in particular staphylococcus aureus, staphylococcus epidermidis, staphylococcus haemolyticus, streptococcus pyogenes, group a and group C streptococci, neisseria gonorrhoeae or clostridium difficile.

The present invention provides methods of treating Skin and Soft Tissue Infections (SSTIs). Patients who may benefit from this treatment may have deep or superficial infections. SSTI can involve deeper soft tissue and/or require significant surgical intervention, such as severe abscesses, infected ulcers, severe burns, or deep and extensive cellulitis. Also can be used for treating infected surgical wound. The efficacy of dalbavancin in treating skin is an unexpected and surprising result, as dalbavancin complexes with proteins in vivo (see example 5).

The clinical manifestations of skin and skin structure infections can vary from mild folliculitis to severe necrotizing fasciitis. Acquisition patterns may also vary with community-acquired skin and skin structure infections that are often preceded by injuries caused by occupational exposure or recreational activities and are often associated with a large pathogen diversity. Hospital acquired skin and skin structure infections are commonly associated with surgery, decubitus ulcers and catheterization. Post-operative infection is the third most common nosocomial infection and accounts for 17% of all nosocomial infections reported to the national nosocomial infection monitoring system (NNIS). The most common source of infection is the endogenous flora of the patient. Staphylococcus aureus, coagulase-negative staphylococcus, and enterococcus are the most frequently isolated pathogens from SSTI.

Symptoms of SSTI infection can include erythema, tenderness or pain, heat or localized warmth, fluid drainage or drooling, swelling or hardening, redness or wave movement. Patients that may benefit from treatment with the methods of the invention include patients with deep or complex infections or infections requiring surgical intervention, or patients with underlying diabetes or peripheral vascular disease. The infection is often caused by gram-positive bacteria, such as staphylococci or streptococci, such as staphylococcus aureus or streptococcus pyogenes. A method of treating a bacterial infection of the skin or soft tissue comprising administering to a subject in need thereof a therapeutically effective amount of dalbavancin in an amount and according to the dosing regimen described above. In some embodiments, the dalbavancin composition is administered intravenously in two doses, often about 5 to about 10 days apart, more often about 1 week apart. In some embodiments, the first dose comprises at least twice as much dalbavancin as the second dose. In one embodiment, the first dose is about 1000mg and the second dose is about 500 mg. In another embodiment, the first dose may be about 1200mg and the second dose may be about 600 mg.

The invention also provides methods for prophylactically preventing the occurrence of a bacterial infection, such as an infection caused by Staphylococcus aureus, or by a bacterium of the Neisseria or Clostridium genera. In the prophylactic methods of the invention, a prophylactically effective amount of dalbavancin is administered to an individual who may be susceptible to a bacterial infection, for example, by a medical procedure. Dalbavancin is often administered in an amount sufficient to provide a prophylactically effective plasma level for at least about 1 day, at least about 3 days, at least about 5 days, or at least about one week or more. The dalbavancin compositions may be administered, e.g., parenterally, such as intramuscular (i.m.), intravenous (i.v.), intraperitoneal (i.p.), subcutaneous (s.c), or intrathecal (i.t.) injection, either before or after surgery as a prophylactic step against infection. The dalbavancin composition may be administered to prevent infection immediately before or after an invasive medical procedure such as surgery or hospitalization in a medical care facility such as a hospital for one or more days or about one week, or during. The prophylactic method may be used in any situation in which an individual is likely or likely to be infected with a bacterial infection, including situations in which an individual has been exposed or is likely to be exposed to a bacterial infection. For prophylactic methods, the dalbavancin composition may be administered as a single dose, or as two or more doses of the same or different amounts spaced several days to about one week apart. In one embodiment, the dalbavancin composition may be administered prior to or concurrently with insertion into an intravenous catheter to prevent blood flow related infections.

For prophylactic methods, the dalbavancin composition may be administered in a single dose or multiple doses according to any of the dosing regimens described above. The dalbavancin compositions are often administered as a single dose comprising about 0.1 to about 3 grams, or about 0.1 to about 1 gram, for example about 0.25 grams or about 0.5 grams. In one embodiment, a single dose of about 0.25 grams is administered intravenously over a time period of about 2 minutes to about 1 hour (e.g., about 30 minutes). In another embodiment, the dalbavancin composition is administered intravenously concurrently with administration of another drug (e.g., antibiotic) therapy.

In any of the foregoing methods of treatment or prevention, the dalbavancin composition may be administered simultaneously or sequentially with at least one other antibiotic. In some embodiments, at least one other antibiotic is administered in addition to dalbavancin, which is effective (e.g., bactericidal) against one or more gram-negative bacterial species and/or gram-positive strains against which dalbavancin is not effective. In some embodiments, the dalbavancin and at least one antibiotic effective against (e.g., bactericidal) at least one gram-negative bacterial species are administered as a mixture in a dalbavancin composition.

Pharmacokinetics

Animal studies of dalbavancin were performed using mice, rats, rabbits, dogs, and mini-pigs.

Various validation methods were used to quantify dalbavancin. The DABAVAXIN API is composed of 5 homologs (A)₀、A₁、B₀、B₁And B₂) And (4) forming. All microbiologically active dalbavancin fractions were measured using an antimicrobial activity-based method. Measuring main component B by chromatography₀Combination of component B₁(90% -95% of dalbavancin total), the results are reported as total dalbavancin. Liquid Scintillation Counting (LSC) and liquid chromatography coupled with radiochemical detection (LC/RC) can detect dalbavancin and metabolites in plasma, urine and feces of animals administered a radiolabeled drug. The concentration-time curves for the drug and radioactivity can be superimposed each time the dalbavancin and drug-derived radioactivity are quantitated in the plasma of an animal administered a clinically relevant dose. This indicates that virtually all drug-derived radioactivity in plasma is the complete drug.

Following IV dosing, the drug was widely distributed throughout the body, to all organs and tissues examined. Dalbavancin permeated into the skin of rats and mini pigs. Dalbavancin kinetic performance patterns are predictable across species. Plasma Clearance (CL) was found to be proportional to the body weight of the species. In addition, dalbavancin crosses the rat placenta and appears in the fetal rat plasma.

Dalbavancin is not a substrate, inhibitor, or inducer of the hepatocyte cytochrome P450 isozyme. The medicine is not applied to rat, dog or human liver microsomes in vitro; rat, dog or human hepatocytes; or human kidney microsomal metabolism. Dalbavancin does not affect the metabolism of the labeled substrate by the isolated human liver microsomes. Administration of dalbavancin to rats did not induce any P450 activity.

Similar dalbavancin metabolite profiles were observed in multiple species. Two (2) metabolites (OH-dalbavancin and MAG) have been observed in rat, dog and human urine. Both metabolites are undetectable or near detection limits (< 0.4mg/L) in human plasma, are less antibacterial than dalbavancin, and contribute only slightly, if at all, to in vivo activity. Similar results were commonly observed in animal plasma with clinically relevant doses.

The drug has dual excretion pathways (kidney and feces) and is excreted as a complete drug and metabolite. Dalbavancin is excreted in urine and feces of rats, dogs and humans. Most of the dose is excreted as intact drug in the urine. OH-dalbavancin and MAG are also found in urine. Some intact drug was found in animal feces along with trace metabolites. Human feces contain a microbiologically active dalbavancin component. Dalbavancin is also excreted in the rat milk. Thus, dalbavancin is eliminated from the body via metabolism, urinary excretion, and fecal excretion. This would expect that only minimal, if any, dose adjustments would be required, depending on the patient's demographics. Surprisingly, patients with severe renal failure (creatinine clearance CL) _CRLess than 30mL/min) compared to normal renal function patients (creatinine clearance CL)_CRGreater than 80mL/min) to higher levels. The levels of severe patients receiving a 1g dose on day 1 appear to be similar to normal patients receiving 100mg on day 1 and 500mg on day 8.

Pharmaceutical composition

The present invention provides a pharmaceutical composition formulated for administration of dalbavancin according to the methods described above. The pharmaceutical compositions of the present invention may be in unit dosage form of dalbavancin, which includes an amount of dalbavancin sufficient to provide a therapeutically or prophylactically effective plasma level of dalbavancin for several days (often at least about 3 days, at least about 5 days, or at least about one week or more) when the composition is administered to an individual, and a pharmaceutically acceptable carrier. Generally, therapeutically or prophylactically effective plasma levels of dalbavancin are at least about 4mg per liter of plasma. Dalbavancin plasma levels can be measured by methods well known in the art, such as those described above.

Dalbavancin may optionally be in a pharmaceutically acceptable form for administration to an individual, optionally as a pharmaceutically acceptable non-toxic salt.

Examples of suitable salts of dalbavancin include salts formed by standard reactions with organic and inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, succinic acid, citric acid, ascorbic acid, lactic acid, maleic acid, glutamic acid, camphoric acid, glutaric acid, glycolic acid, phthalic acid, tartaric acid, lauric acid, stearic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, sorbic acid, picric acid, benzoic acid, cinnamic acid, and the like. Representative examples of bases which can form salts with dalbavancin include alkali metal or alkaline earth metal hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and barium hydroxide; ammonia and aliphatic, alicyclic, or aromatic organic amines such as methylamine, dimethylamine, diethylamine, ethanolamine, and picoline (see, for example, U.S. patent No. 5,606,036).

In some embodiments, pharmaceutically acceptable aqueous formulations of dalbavancin are provided that are suitable for parenteral administration, e.g., intravenous injection. For the preparation of the aqueous formulation, methods well known in the art may be used, and any pharmaceutically acceptable carrier, diluent, excipient or other additive commonly used in the art may be used. In one embodiment, a pharmaceutically acceptable aqueous formulation for intravenous injection comprises 5% dextrose.

Dalbavancin may be administered parenterally, i.e., the route of administration is injection under or through one or more layers of skin or mucosa. Since this route encompasses these highly effective protective barriers in humans, a particularly pure parenteral dosage form free of microorganisms and insoluble particles must be achieved. The process for preparing the dosage form must embody pharmaceutical manufacturing quality control specifications that will produce and maintain the desired quality of the product in terms of sterility and therapeutic efficacy. In addition, the dosage form should be stable when practical dosage forms and convenient dosage forms are stored at room temperature.

There are several conventional methods that are commonly available for converting bulk pharmaceutical materials into dosage forms suitable for parenteral administration. These methods are generally listed in Remington's pharmaceutical Sciences, eighteenth edition, 1990 ("Remington").

Steam sterilization

USP defines steam sterilization as the use of saturated high pressure steam at a minimum of 121 ℃ for at least 15 minutes in a high pressure vessel. The solid form of the drug may be steam sterilized in an autoclave. The medicament in liquid form may be placed directly in an autoclave or contained in a sealed container and placed in an autoclave for the same type of steam sterilization.

Dry heat sterilization

In dry heat sterilization, bulk pharmaceutical materials are subjected to high temperatures at relatively low humidity. Since dry heat is less effective for sterilization than moist heat, longer exposure times and higher temperatures than used in steam sterilization are required. The goal is to kill the microorganisms through an oxidative process. Although it is not conventional to establish an exact and correct time-temperature cycle, typical temperatures used are 140 ℃ and 170 ℃ for 1 to 3 hours.

Radiation sterilization

Radiation sterilization may utilize electromagnetic radiation or particle radiation. Electromagnetic radiation containing photon energy includes ultraviolet, gamma, x-ray and cosmic radiation. Gamma radiation emitted from radioactive materials such as cobalt-60 or cesium-137 is the most commonly used source of electromagnetic sterilization. The most widely used particle radiation for sterilization is beta particles or electron radiation.

Sterile filtration

A method for removing, without destroying microorganisms, from a liquid stream by sterile filtration. This filtration is the method of choice for solutions that are unstable to other types of sterilization methods.

Aseptic Freeze drying (Freeze-drying)

The process employs sterile filtration, followed by the step of separating the sterilized drug substance from the solution by sublimation of the frozen solution, leaving the drug substance behind. The method generally comprises the steps of:

1) dissolving a large amount of drug in an aqueous solution

2) Sterilization of the solution by membrane filtration

3) Filling the sterilized solution in an open, pre-sterilized vial, and placing in a lyophilization chamber

4) Freezing the solution in the vial

5) Discharging indoor air to sublimate ice at low temperature

6) The temperature is raised to room temperature or higher to remove residual moisture.

Sterile Freeze drying (lyophilization) with addition of sterile Water (steam)

The process employs sterile filtration, followed by the steps of isolating the sterile drug from solution by freeze-drying (lyophilization), and adding sterile water in vapor form.

Sterile precipitation

The process employs sterile filtration followed by the step of precipitating the sterile drug from solution. In more detail, a quantity of the drug is first dissolved in water at an elevated temperature (above room temperature), the heated solution is then filtered under sterile conditions to remove any microorganisms, and the filtered solution is then cooled to precipitate the drug from the solution. The precipitated drug is then separated from the solution by filtration or centrifugation and filled into a container by powder filling in a sterile state. In order for powder filling to be achieved, the drug must have good flow properties-the powder should generally be granular, amorphous, and of uniform particle size.

Pharmaceutical compositions for parenteral administration include dalbavancin and a physiologically acceptable diluent, such as sterile or deionized water for injection, physiological saline, 5% dextrose, water miscible solvents (e.g., ethanol, polyethylene glycol, propylene glycol, and the like), non-aqueous vehicles (e.g., oils such as corn, cottonseed, peanut, and sesame oils), or other common diluents. The formulation may additionally include solubilizers such as polyethylene glycol, polypropylene glycol, or other known solubilizers, buffers for stabilizing the solution (e.g., citrate, acetate, and phosphate), and/or antioxidants (e.g., ascorbic acid or sodium bisulfite) (see, e.g., U.S. patent No. 6,143,739). Other suitable pharmaceutical carriers and formulations thereof are described in "Remington's pharmaceutical Sciences" of e.w. martin. The pharmaceutical formulations of the present invention may also be prepared to contain acceptable levels of particulates (e.g., particle-free) and prepared to be pyrogen-free (e.g., to meet the requirements for injectables in the U.S. pharmacopoeia), as is known in the art.

In one embodiment, the pharmaceutical composition, which often contains a stabilizer or mixture of stabilizers, is provided by dissolving a dried (e.g., lyophilized) dose of dalbavancin in an amount of water and preferably deionized water in a volume sufficient to dissolve the lyophilizate. The amount of water sufficient for dissolution is typically about 10mL and the resulting pH of the dalbavancin solution is above 3.0 and about 3.5 to 4.5. The solution is further diluted in a second diluent, typically comprising 5% dextrose, such as the amount contained in a drip bag for intravenous administration, raising the pH of the dalbavancin solution to about 5-5.5. In another embodiment, the pH of the dalbavancin solution in the drip bag is about 4.5. The second diluent may be deionized and sterile water for injection. In one embodiment, the aqueous diluent is 5% dextrose.

Pharmaceutical compositions for parenteral use can be constituted in sterile vials containing a therapeutically or prophylactically effective amount of one or more unit doses of dalbavancin as described above, optionally including excipients, under conditions in which the bactericidal effectiveness of dalbavancin is maintained. The compositions may be in the form of a dry (e.g., lyophilized) powder. Prior to use, the powder may be reconstituted with a physiologically acceptable diluent and the solution withdrawn via syringe for administration to a patient. The above pharmaceutical formulations may be sterilized by any acceptable means including, for example, electron beam or gamma sterilization or by sterile filtration.

Typical formulations for parenteral administration may include dalbavancin at a concentration, for example, of about 0.1 to about 100mg, about 0.5 to about 50m g, about 1 to about 10mg, or about 2 to about 4mg dalbavancin per ml of the final formulation.

In some embodiments, the pharmaceutical compositions of the present invention comprise a mixture of dalbavancin and one or more additional antibiotics. Preferably, at least one non-dalbavancin antibiotic in the mixture is effective against (e.g., bactericidal) one or more gram-negative bacteria (such as thaumamide) and/or effective against (e.g., bactericidal) one or more gram-positive strains against which dalbavancin is not effective (such as linezolid or daptomycin). The mixture may also include a pharmaceutically acceptable carrier as described above.

In some embodiments, the pharmaceutical compositions of the present invention include one or more stabilizing substances that inhibit the degradation of one or more dalbavancin components to a less active or inactive material, homolog, or related substance (e.g., MAG). As used herein, "stabilizing substance" or "substance" refers to the stabilization of one or more of the constituent dalbavancin components of a composition (e.g., B)₀) A stabilizer at the level of (a). By "stabilizing effective amount" is meant an amount of a stabilizing agent sufficient to increase the long term stability of one or more components of the dalbavancin composition. In some embodiments, a stabilizing effective amount may be provided by a mixture of two or more stabilizing materials, where each material alone is not present in an amount sufficient to provide a stabilizing effect.

Examples of stabilizers include, for example, nonionic substances, such as sugars, e.g. mono-, di-or polysaccharides or derivatives thereof; a sugar alcohol; or a polyol. The stabilizing substances include, for example, mannitol, lactose, sucrose, sorbitol, glycerol, cellulose, trehalose, maltose, raffinose, dextrose, low and high molecular weight dextrans or mixtures thereof.

Besides sugars, stabilizers may also be amino acids. Amino acid stabilizers include natural and synthetic amino acids and amino acid derivatives. In a preferred embodiment, the amino acids are glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan and asparagine. The stabilizers may also be cyclodextrins, cyclic amides such as niacinamide and benzamide, salicylic acid and ortho-, meta-, para-substituted hydroxy acids and esters thereof.

In addition to the addition of stabilizers, it has also been found that adjusting the pH of the compound can improve the stability of the composition. The particular pH of the composition that enhances or maximizes stability depends on the type and amount of stabilizer added. The pH may preferably be about 1 to 7, more preferably 2 to 6, more preferably 3 to 5, more preferably 4 to 5, more preferably about 4.5.

In one embodiment, the pharmaceutical composition comprises mannitol dalbavancin in a weight ratio of 1: 2. In another embodiment, the pharmaceutical composition comprises mannitol, lactose, dalbavancin a weight ratio of 1: 4. Surprisingly, it has been found that the combination of mannitol and lactose provides a better stabilizing effect than either of the substances alone. The pH of the pharmaceutical compositions of the invention, for example, is often from about 3 to about 5, e.g., about 3.5 or about 4.5.

In some embodiments, MAG formation may be reduced using one or more methods. For example, dalbavancin may be freeze-dried in the presence of a stabilizing substance such as mannitol and/or lactose for reducing the amount of MAG formed.

The dalbavancin compositions are often stored at a temperature below ambient temperature (e.g., at about 5 ℃) to improve stability.

Drying

Removal of acetone from high molecular weight natural products can be very difficult because it can form adducts with high molecular weight natural products. The removal of acetone can be accomplished in the presence of water, which replaces the acetone in the solid product. Moreover, the process of drying dalbavancin is more elaborate as MAG may form, depending on pH, temperature, vacuum and time. To minimize MAG formation and speed up the drying process, low endotoxin water is added to or sprayed on the product to replace acetone during the drying process. Subsequent drying for several more hours reduces excess water.

Drying was carried out under vacuum (internal temperature < 25 ℃). When most of the acetone had been removed from the product, about 20% (w/w) of low endotoxin water was sprayed on the solid product and drying was continued under the same conditions. To follow the drying process, acetone and karl fischer (K.F.) analyses were performed and additional water was sprayed on the solid product until the residual acetone was below 1.5%.

Study 25

Drying at 20-30 deg.C under 50 mbar

The process was first simulated in the laboratory using a rotary evaporator. Batch 027 of dalbavancin was dried without water at 30 ℃ to give a product containing 6.4% MAG and 3.2% acetone. 126g of this dry dalbavancin batch (batch 027, HCl/dalbavancin ratio 1.7mols/mol) were charged into a 1L round bottom flask, and the solid was sprayed every 2 hours with 25mL of water, maintaining a water bath temperature of 20-30 ℃ and a vacuum of 50 mbar. Samples of dalbavancin were taken at specific times and analyzed for acetone, k.f. and MAG. The resulting data are reported in table 4A. All MAG percentages reported in the table are the difference between the actual amount and the starting amount.

TABLE 4A

The experiment was completed by drying under the same conditions for an additional few hours to reduce the water content to below 15%.

Drying at 30 ℃ under 10 mbar

125g of the 027 th batch of dalbavancin, MAG 6.4% (HPLC area%) were dried at 30 ℃ and 10 mbar for 24 hours using the same apparatus as in the previous experiment.

The results obtained are reported in table 4B.

TABLE 4B

From these laboratory experiments it can be concluded that the drying process can be simplified by adding water to the solid product. These results are unexpected because the degradation of dalbavancin to MAG is a hydrolysis step, one would expect that an increase in water would increase the degradation process. In this way, acetone is quickly removed from the product, reducing drying time and avoiding the formation of additional MAG. It can also be observed (table 4B) that the lower residual pressure significantly reduced the drying process. It is also surprising that the acetone level is reduced in the presence of large amounts of water.

Drying dalbavancin in a tumble dryer

The wet dalbavancin was placed in a tumble dryer DR 216 and dried at 29-32 ℃ (external temperature) at max vac (< 50 mbar). Initially, the internal temperature was reduced from 30 ℃ to 17-18 ℃, and then, as the temperature started to increase, a sample of the product was taken for analysis (water 17.2%, acetone 9.5%). At this point, low endotoxin water (about 20%, w/w) was sprayed into the dryer, onto the stirred product, and sampled again on time for analysis. After 2 hours, low endotoxin water was sprayed again until the amount of acetone was below 0.5% (table 4C).

TABLE 4C

External temperature 29-30 ℃ and pressure 10-15 mbar

Final weight 2592g, assay # 200201027: 13.93% of water (K.F.), 0.12% of acetone and 0.38% of MAG. (ii) spraying water into the drum dryer after sampling

Comparison of drying in the Presence of Water and absence of Water

The study was a comparison between the drying process for the 027 th batch of dalbavancin and the 027 th/R batch of dalbavancin in the absence of water (027 th batch) and in the presence of water (027 th/R batch). The 027 th batch of dalbavancin (14.6Kg) and the 027 th/R batch of dalbavancin (8.3Kg) were vacuum dried using a tumble dryer (DR 216) at 25-35 ℃ (internal temperature). Batch 027 was dried without water. Instead, four 400mL portions of low endotoxin water were sprayed onto the 027/R batch. Samples of dalbavancin were taken at specific times during each drying run and analyzed. The results are reported in table 5. For lot 027 MAG was determined only at the end of the drying process.

TABLE 5

(^*) Assay #200101392(°) assay #200101913

Full scale production data

Table 6 below is a summary of the methods and MAG levels in full-scale production of 2002 and 2004.

TABLE 6

In the 2002 process, the dalbavancin product is vacuum dried at a temperature below 30 ℃. Low endotoxin water was sprayed on the product at various times. Drying was continued until the acetone content was below 0.5%.

In the 2004 process, the dalbavancin API was recovered by centrifugation, washed with cold acetone (0-10 ℃) and dried under vacuum (internal temperature < 25 ℃). MAG is the major degradation product of dalbavancin, the formation of which is temperature dependent. Water was added during the drying process to displace the acetone and finally achieve an acetone content of less than 1.5% (GC). It is evident from table 5 that cryodrying in the presence of water results in an unexpectedly low content of acetone (compare 0.27% in Lot027/R and 3.14% in Lot 027) and an unexpectedly low content of MAG degradation products (compare 1.81% in Lot027/R and 6.46% in Lot 027).

Thus, the method of drying dalbavancin comprises the step of adding water to the dalbavancin product to displace the acetone. The preferred amount of water added is about 20% of the assumed final dry product. Alternatively, the amount of water may be about 15%, alternatively about 25%, alternatively about 30%, alternatively about 40%, alternatively about 45%, alternatively about 50% of the final dry product as assumed.

The method of drying dalbavancin may include the step of drying the dalbavancin product at about 100 torr at a temperature of less than about 25 ℃ for about 2 more hours without adding any water. The low endotoxin water was then sprayed onto the product, and the product was then dried for about 2 hours. Samples were then taken and checked for acetone levels. If the level is above about 1.5%, additional water is sprayed on the product and the product is dried again until the acetone level is below about 1.5%.

Manufacture of

All bulk solution manufacturing operations were performed in the 100,000 class (class D) zone. Aseptic filling was performed in a class 100 (class a) laminar flow region.

A suitably manufactured vial is filled with about 80% of the theoretical batch volume of water for injection. The solution was mixed and the temperature was maintained at 15-30 ℃. Add dalbavancin and mix until dalbavancin is dissolved. At least one stabilizer is added to the solution and mixed until dissolved. Water for injection was added to bring the solution to final volume and the pH was adjusted to the appropriate pH with 0.1N HCl or 1.0N NaOH if necessary. The bulk solution was sterilized by filtration through two successive layers of 0.2 μm sterilizing filters and filled into sterilized receiving vials. (a prefilter may be used if desired to aid in filter clarity, or to reduce particulate loading on the sterilized filter). The solution was aseptically bottled in sterile/depyrogenated type I glass vials. The sterile siliconized lyopilization plug portion is inserted into the lyophilization station and the vial is transferred to the lyophilization chamber.

The lyophilization process was monitored on a representative vial using a thermocouple probe. The vials were frozen at-45 ℃ and held for 3 hours, after which vacuum was applied. The shelf temperature was adjusted to-25 ℃. When all thermocouples were at-29 ℃ or higher, the rack temperature was adjusted to 0 ℃. When all thermocouples were at-5 ℃ or higher, the rack temperature was adjusted to +30 ℃. When all thermocouples were at +27 ℃ or higher, the temperature of the vial was maintained for 14. + -. 2 hours. The chamber pressure was returned to atmospheric pressure by introducing sterile nitrogen, which had been filtered through a 0.2 μm filter, and then collapsing the freeze-dried shelf to seal the vial. The vial was then removed from the chamber and aluminum sealed.

All components and devices are sterilized by appropriate methods. The vials were washed and sterilized in a dry heat sterilizer at a temperature of not less than 255 ℃ for not less than 3 hours. The stopper was steam autoclaved at a temperature of 123 ℃ and 125 ℃ with a chamber pressure of about 33 psi. Residence times in the sterile range are generally from 50 to 60 minutes.

The sterilization process was validated using the "over kill" method for steam and dry heat sterilization cycles. AU sterilization cycles provide sufficient lethality to provide at least 10 no matter what naturally occurring microorganism(s)^-6The microbial survival rate of (1). To provide no less than 12 log reduction of mortalityThe rate designs all the periods. The dry heat cycle will provide a minimum of 3log endotoxin reduction.

Stability study

Dalbavancin was found to decompose during the freeze-drying process. During the stability studies, it was found that the addition of stabilizers reduces the active component B₀The amount of decomposition.

The stability of the various lyophilized formulations of dalbavancin over time at 25 ℃ and 40 ℃ is shown in figures 1-4. According to FDA guidelines, it is assumed that a product stable at 40 ℃ for 3 to 6 months will be stable at room temperature for 2 years. FIGS. 1A, 2A, 3A and 4A show the dalbavancin component B ₀The amount of the compound is reduced, and the compound is one of the bactericidal active components of dalbavancin. As discussed above, component B₀And is also one of the major components of most dalbavancin compositions. FIGS. 1B, 2B, 3B, and 4B show an increase in the amount of MAG, a less active component that is believed to be a decomposition product of one or more of the other dalbavancin components. Table 7 lists the composition of each formulation used in the stability study, the results of which are shown in figures 1-4. Any of these compositions can be used to produce stable, sterile, non-particulate dosage forms.

TABLE 7 composition of the respective dalbavancin formulations

As shown in fig. 1B and 2B, at T ═ 0, a significant amount (greater than 4%) of MAG was already present in composition D, which contained dalbavancin, no other non-dalbavancin components, and was not pH adjusted (pH about 3.01), at 25 ℃ and 40 ℃, respectively. At higher temperatures, MAG formation increases at a much greater rate. After 3 months and 6 months at 40 ℃, composition D had 21.0% MAG and 23.7% MAG, respectively (see fig. 2B). This suggests that pure dalbavancin is highly unstable and that merely lyophilizing dalbavancin will result in significant degradation. In addition, conventional drying also leads to the formation of MAG degradation products. Some dalbavancin formulations require storage at-20 ℃.

Stability will also increase as the pH increases without adding any other non-dalbavancin component. Composition D, without pH adjustment, had a pH of about 3.01. Composition B was adjusted to pH 3.69. Composition F was adjusted to pH 4.5. As shown in FIGS. 1A and 2A, B increased with time as the pH increased₀Less initial degradation and less overall degradation. Similarly, in FIGS. 1B and 2B, at both temperatures, there was less MAG formation in the compositions adjusted to the higher pH, both initially and over time.

The addition of mannitol also showed a significant improvement in the stability of dalbavancin. Factor B over time, compared to composition D (which did not adjust pH, nor contained mannitol)₀Degradation and MAG increase are also significantly reduced. As shown in composition E (62.5mg mannitol, about pH 3.01), there was a significant improvement in stability during initial lyophilization and over time, even without any pH adjustment. At T-0, approximately 2% MAG was present at T-25 ℃ (see fig. 1A) and T-40 ℃ (see fig. 2A), which was less than half the amount of MAG present in composition D at T-0.

The stability of dalbavancin was also improved when the pH was increased and the amount of mannitol was kept constant. Comparison of compositions E, C and G shows that the amount of dalbavancin degradation is also reduced when the pH is increased from about 3.01 to 3.8 to 4.5. As shown in FIGS. 1A and 2A, as the pH increased, there was less B over time ₀Initial degradation and less total degradation. Similarly, in FIGS. 1B and 2B, less MAG was formed in the compositions adjusted to higher pH, both initially and over time.

Increasing the amount of mannitol also leads to an increase in stability when the pH is kept constant. For example, although compounds L and K, which contain 62.5mg and 125mg of mannitol, respectively, at pH 4.5, have similar B at T ═ 0₀Content, but after 12 months, B₀Percentage ofThe ratio change was significantly smaller for compound K. A similar situation can be seen in compounds J and I, which contain 62.5mg and 125mg mannitol, respectively, at pH 5.0.

Although a change in the pH of a composition comprising mannitol alone does result in B₀The amount of degradation changes, however there is no predictable trend. Compounds L, J and H contained 62.5mg mannitol at pH 4.5, 5.0 and 5.3, respectively. After 12 months at 25 ℃, B₀The percent change of (a) was 1.0, 1.2 and 0.7, respectively. Compounds O, K and I contained 125mg of mannitol at pH 3.3, 4.5 and 5.0, respectively. After 12 months at 25 ℃ B of these compositions₀The percent change of (a) was 0.5, 0.1 and 0.3, respectively. It is noteworthy that, in compound O, component B takes place over a period of 2 months at 40 deg.C ₀The amount of (c) was reduced by only 1.9%, and by 1.0% in compound M (see fig. 4A).

Despite B of Compound O (125mg mannitol, pH 3.3)₀The percentage change was similar to the other differences found in the compositions containing 125mg mannitol (see compositions I and K), as shown in figure 3A, B at T ═ 0₀The amount of initial degradation was significantly less for compound O (88.3% B)₀). When combined with Compound K (pH 4.5) and Compound I (pH 5.0) (which each contained 85.3% B₀And 85.5% B₀) This is particularly evident when compared. B present in the compound O₀The difference between the amount of MAG and the amount of MAG (see FIGS. 3A and B) is most likely explained by the fact that, as explained before, B₀Is not the only dalbavancin component that degrades to form MAG.

Lactose also appears to be a suitable stabilizer for dalbavancin. For compound N containing 125mg lactose at pH 4.5, over 12 months at 25 ℃, B₀The percentage change of (c) is only 0.6. After 2 months at 40 ℃ B₀The percentage change of (c) is only 1.4. Lactose alone, however, does not appear to stabilize dalbavancin, as does mannitol. B of Compound K (125mg mannitol) at pH 4.5 at 40 ℃ over a period of 2 months ₀Component is reduced by only 1.0。

The combination of mannitol and lactose also appears to stabilize dalbavancin, which is particularly preferred. Mannitol and lactose have similar stabilizing properties. Mannitol, however, is a diuretic. Thus, in a preferred embodiment, the amount of mannitol is minimized. Compound M contained 62.5mg each of mannitol and lactose. As seen in fig. 3A, the percentage change in MAG was only 0.6 over 12 months at 25 ℃. In addition, as seen in FIG. 4A, the amount of MAG increased only 2.1% over the course of 3 months at 40 ℃. This is less than the degradation seen in both compounds N (125mg lactose) and K (125mg mannitol), both of which show a 2.9% increase in the amount of MAG over 3 months at 40 ℃. Surprisingly, the combination of half of each amount of mannitol and lactose used in the other formulations resulted in a greater increase in stability of dalbavancin.

Other stability data for dalbavancin stabilized with mannitol and lactose at various temperatures at about pH 4.5 are reported in tables 8A-D below.

TABLE 8A stability of dalbavancin Lot 554399 for injection at 40 ℃/75% TH (500mg vial, mannitol and lactose, pH 4.5)

n.d. not detected

- -No test at this time point, according to the stability protocol

TABLE 8B stability of dalbavancin Lot 554399 for injection at 30 ℃/65% RH (500mg vial, mannitol and lactose, pH 4.5)

n.d. not detected

- -No test at this time point, according to the stability protocol

TABLE 8C stability of DABAVAXIN Lot 554399 for injection at 25 deg.C/60% RH (500mg vial, mannitol and lactose, pH 4.5)

n.d. not detected

- -No test at this time point, according to the stability protocol

TABLE 8D stability of dalbavancin Lot 554399 for injection at 5 ℃ (500mg vial, mannitol and lactose, pH 4.5)

n.d. not detected

- -No test at this time point, according to the stability protocol

Further long term stability study data are presented in tables 9-11. The stability of the dalbavancin formulation was determined by reverse phase HPLC using a binary mobile phase gradient and UV detection system. External standards were used to determine dalbavancin content. The percentage distribution of the dalbavancin component was calculated by comparing the area of each individual component to the total area of all major drug components. The percentage distribution of impurities was calculated by comparing the area of each impurity to the total chromatographic area.

HPLC determination, bioassay, water content, microbial limit, pH, HPLC profile, or total impurities of the test sample. The results are shown in tables 9 to 11. The stability data for the 025 th batch is shown in tables 9A, 10A and 11A, which contains 1.5% MAG at T ═ 0. The stability data for the 020005/R batch is shown in tables 9B, 10B and 11B, which contains 0.8% MAG at T ═ 0, as a result of the different drying procedures described below.

Dalbavancin showed good stability during storage at-20 ℃ (see tables 9A-B) with no significant changes in bioassays, HPLC assays, HPLC profiles, or total impurities. It was observed that the pH remained unchanged and the water content increased slightly.

Degradation was observed at 5 ℃ over 36 months (see Table 10A), e.g. an increase in MAG content of about 2.9% and factor B₀A corresponding decrease of about 3.6%. No overall impurity trend was observed, the bioassay and pH remained essentially unchanged. Similar results were observed in the 020005 batch (see table 10B).

A more extensive degradation was observed during storage at 25 ℃ over 12 months (see Table 11A), with an increase of MAG of about 10.2%, factor B₀The reduction is about 8.5%. Other factors, related substances and pH levels remained unchanged. The water content increased by about 1.6%.

TABLE 9A. stability data (pH3.5) of unformulated dalbavancin batch 025 at-20 deg.C

- -No test at this time point, according to the stability protocol

^*Total aerobic count, total mold and yeast count

TABLE 9B stability data of unformulated dalbavancin Lot 020005/R at-20 deg.C

- -No test at this time point, according to the stability protocol

^*Total aerobic count, total mold and yeast count

TABLE 10A. stability data for the unformulated dalbavancin batch 025 at 5 deg.C

- -No test at this time point, according to the stability protocol

^*Total aerobic count, total mold and yeast count

TABLE 10B stability data of unformulated dalbavancin Lot 020005/R at 5 ℃

- -No test at this time point, according to the stability protocol

^*Total aerobic count, total mold and yeast count

TABLE 11A. 025 batch unformulated dalbavancin stability data at 25 ℃/60% RH

- -No test at this time point, according to the stability protocol

^*Total aerobic countTotal mold and yeast counts

TABLE 11B stability data of unformulated dalbavancin at 25 ℃/60% RH for the 020005/R batch

- -No test at this time point, according to the stability protocol

^*Total aerobic count, total mold and yeast count

Stability tests of the sterile product were also performed. Bulk and solution samples were tested for appearance, reconstitution time, pH, HPLC assay, bioassay, moisture content, sterility, HPLC profile, and related substances. The results are shown in tables 12 to 14.

The lyophilized product showed good stability with only minor changes in HPLC determination or HPLC profile when stored at 5 ℃ (see tables 12A and B). Factor B as the temperature increased (see tables 13 and 14)₀There is a decrease and a corresponding increase in MAG. Other factors appear to be unaffected by temperature. The product appearance and pH did not appear to be affected by temperature. It is also not possible to determine the clear trend of the reconstitution time, since it may take several seconds to take into account the visual inspection of the dissolved product.

TABLE 12A stability of dalbavancin Lot 149570 for injection at 5 ℃ (lyophilized, mannitol, pH 3.5)

- -No test at this time point, according to the stability protocol

^*The analysis was performed by developed HPLC. Dalbavancin B₁And B₂Cannot be distinguished in this way.

TABLE 12B stability of dalbavancin for injection Lot 442378 at 5 ℃ (lyophilized, mannitol, pH 3.5)

n.d. not detected

- -No test at this time point, according to the stability protocol

TABLE 13A stability of dalbavancin Lot 149570 for injection at 25 ℃/60% RH (lyophilized, mannitol, pH 3.5)

- -No test at this time point, according to the stability protocol

^*The analysis was performed by developed HPLC. Dalbavancin B₁And B₂Cannot be distinguished in this way.

TABLE 13B stability of DABAVAXIN Lot 442378 for injection at 25 ℃/60% RH (Freeze-drying, mannitol, pH 3.5)

n.d. not detected

- -No test at this time point, according to the stability protocol

TABLE 13C stability of DABAVAXIN Lot 442378 for injection at 30 ℃/65% RH (Freeze-drying, mannitol, pH 3.5)

- -No test at this time point, according to the stability protocol

TABLE 14A stability of dalbavancin Lot 149570 for injection at 40 ℃/75% RH (lyophilized, mannitol, pH 3.5)

- -No test at this time point, according to the stability protocol

^*The analysis was performed by developed HPLC. Dalbavancin B₁And B₂Cannot be distinguished in this way.

TABLE 14B stability of dalbavancin Lot 442378 for injection at 40 ℃/75% RH (lyophilized, mannitol, pH 3.5)

n.d. not detected

- -No test at this time point, according to the stability protocol

Light resistance

The lightfastness of dalbavancin for injection (500mg vial) in the labeled vial was also evaluated. The 10 labeled vials were placed in a light-tight chamber, along with 10 labeled vials wrapped in aluminum foil as dark controls. The sample is exposed for no less than 1.2 Mhr and the total near ultraviolet radiant energy is no less than 200 Watt-hours/meter². Samples were evaluated for appearance, solution appearance, pH, moisture and HPLC measurements. The evaluation was madeThe results are shown in table 15.

TABLE 15.500 immediate packaging lightfastness results for mg/vial drug product

n.d. not detected

Comparison of the light exposed samples and the dark control samples showed that the light exposed samples remained comparable, with slight variations in potency, API component levels, and total impurities.

The light exposed samples demonstrated very low levels of RRT 0.85 without determining the presence of impurities that were not detectable in the dark control samples. Evaluation of this fraction by HPLC/MS indicated that it was a mono-chlorinated derivative of the B homolog of dalbavancin.

Based on these results, dalbavancin for injection was considered photostable in its immediate packaging.

Improved efficacy and reduced side effects

As demonstrated in the examples herein, administration of dalbavancin at high dose levels (i.e., resulting in abnormally high and sustained serum levels) once a week shows an exceptionally good safety profile, similar to or better than that observed with standard treatments with conventional antibiotics administered once a day or even 2-4 times a day at lower doses. Dalbavancin can be administered at abnormally high doses (i.e., resulting in abnormally high and sustained serum levels) less frequently than other antibiotics and without adverse side effects, thereby enabling improved efficacy and patient compliance.

As discussed in example 1, treatment with dalbavancin resulted in a low incidence of adverse events. Serious adverse events include any adverse drug experience that occurs at any dose that results in death, life threatening, resulting in hospitalization or prolonged or persistent or significant disability or weakness of existing hospitalization. In the phase II trial described in example 1, 90% of adverse effects such as diarrhea, nausea, hyperglycemia, limb pain, vomiting, and constipation were mild to moderate in severity. The use of dalbavancin the trial of example 1 did not result in serious adverse events associated with study drug treatment.

Medicine box

The invention also provides kits for use in methods of treatment or prevention of bacterial infections. The kit includes a pharmaceutical composition of the invention (e.g., including at least one unit dose of dalbavancin) and instructions for providing information to a health care provider regarding use of the pharmaceutical composition for treating or preventing a bacterial infection. The instructions may be provided in printed form, or in the form of an electronic medium such as a floppy disk, CD or DVD, or in the form of a website where the instructions are available. A unit dose of dalbavancin often includes a dose that, when administered to an individual, results in therapeutically or prophylactically effective plasma levels of dalbavancin being maintained in the individual for at least 5 days. In certain embodiments, the kit comprises two unit doses to be administered at least 5 days apart, often about 1 week apart, often including about 1.5 to about 3 times higher than the second dose of dalbavancin for the first dose. Dalbavancin is often included as a sterile aqueous pharmaceutical composition or a dry powder (e.g., lyophilized) composition.

Suitable packaging is provided. As used herein, "package" refers to a solid substrate or material that is typically used in systems and capable of holding within a fixed range a dalbavancin composition suitable for administration to an individual. Including glass and plastic (e.g., polyethylene, polypropylene, and polycarbonate) bottles, vials, paper, plastic and plastic foil laminated envelopes, and the like. If electron beam sterilization techniques are used, the packaging should have a density low enough to allow sterilization of the contents.

The kit may also optionally include a device for administering dalbavancin, such as a syringe or device for intravenous administration; and/or for the preparation of a sterile solution of a dry powder (e.g. lyophilised) of the composition for pharmaceutical administration, e.g. such as a diluent, e.g. 5% dextrose.

The kit of the invention may include, in addition to dalbavancin, a non-dalbavancin antibiotic or a mixture of dalbavancin antibiotics for use with dalbavancin as described in the methods above.

In the following examples, the following abbreviations have the following meanings. If an abbreviation is not defined, it has a generally accepted meaning.

AcOH ═ acetic acid

Acona sodium acetate

aq. moisture content

AST-aspartate aminotransferase

ALT-alanine aminotransferase

BV is the volume of the column bed

Coefficient of Variation (CV)

d-diameter

D ═ Dalton

DCC-dicyclohexyl-carbodiamide

(dicyclohexylcarbodiammide)

DMEPA ═ 3- (dimethylamino) -propylamine

DMSO ═ dimethyl sulfonamide

eq ═ equivalent

EU ═ endotoxin unit

g is g ═ g

GC ═ gas chromatography

HCl ═ hydrochloric acid

H₂O is water

HOBT ═ hydrated 1-hydroxybenzothiazole

HPLC ═ high performance liquid chromatography

H₂SO₄Sulfuric acid

IPA ═ isopropylamine

IU is international unit

KF ═ potassium fluoride

kg is kg

L is liter

LC/MS/MS ═ liquid chromatography/mass spectrometry

LDH ═ lactate dehydrogenase

Liquid Scintillation Counting (LSC)

m³Cubic meter

MeOH ═ methanol

mg ═ mg

mL to mL

mol to mol

MW ═ molecular weight

Normal (N ═ normal)

NaOH (sodium hydroxide)

NMP ═ N-methyl-2-pyrrolidone

QTD-quantitative tissue distribution

Rt retention time

sd-standard deviation

TEA ═ triethylamine

The following examples are intended to illustrate the invention, but are not intended to limit the invention.

Examples

Example 1 efficacy and safety of once weekly dalbavancin in deep skin and soft tissue infections

This randomized, controlled study evaluated the safety and efficacy of two dalbavancin dosing regimens. Adult patients with Skin and Soft Tissue Infections (SSTI) involving deep skin structures or requiring surgical intervention were randomized into three groups: study group 1: receive 1100mg of dalbavancin by intravenous Injection (IV) on day 1; study group 2: receiving 1g of dalbavancin by IV on day 1 and 500mg of dalbavancin by IV on day 8; study group 3: receive "Standard of care". Clinical and microbiological responses and adverse events were evaluated.

Population for analysis

62 patients were randomized into the study; all patients received at least one dose of study drug. Safety and efficacy were assessed in 4 study populations and defined as follows: the intent-to-treat (ITT) population included all patients (all randomized subjects) who received at least one dose of study drug. Microbial intent-to-treat (MITT) populations are all ITT patients with culture-confirmed gram-positive pathogens at baseline. The clinically evaluable population was defined as the following: 1) meet all study entry criteria, 2) no change in antimicrobial treatment of gram-positive infections after day 4 except oral decline treatment (used in standard of care group only), 3) return to follow-up (FU) assessment visit (unless treatment failed), and 4) do not accept non-protocol approved concomitant antimicrobials (unless treatment failed). The microbiologically evaluable population is a clinically evaluable subgroup of patients with culture-confirmed gram-positive pathogens at baseline.

The study population is shown in table 16.

TABLE 16 study population for the treatment of DABAVAXIN SSTI

ITT-intent therapy

MITT-ITT population subgroup with culture-confirmed gram-positive infection

EOT-end of treatment

FU-follow-up visit

The mean age of the subjects was 50-55 years (range 18-86 years). There was no significant difference in age between treatment groups. There were gender differences between treatment groups, but an equal number of males and females were enrolled throughout the study. The patient population is mainly caucasian. These results are consistent for ITT and clinically evaluable populations.

62 patients were enrolled, 20 in study group 1 and 21 in each of study groups 2 and 3. The most common controls for standard of care are clindamycin, ceftriaxone, vancomycin and cefazolin. The mean duration of treatment for study group 3 was 15 days.

Baseline pathogen and susceptibility

Of 62 ITT patients, 66% (14 with single dose dalbavancin, 13 with two doses dalbavancin, 14 with standard of care) carried isolated pre-treatment gram-positive pathogens (MITT population). The most common pathogen is staphylococcus aureus. The distribution of baseline pathogens is shown in table 17.

TABLE 17 Baseline gram-positive pathogen and DABAVAXIN MIC Range for the MITT population

Clinical and microbiological response

The effectiveness of the three treatment regimens is determined by assessing the clinical response of the patient versus the proven or postulated microbial response. The primary efficacy endpoint was clinical response in the clinically evaluable population at the follow-up visit. The clinical response of both EOT and FU visits were classified as either successful (cure or improvement) or failed (including uncertain outcome). Patients classified as successful must not receive additional systemic antibacterial treatment for their infection. Failure is defined as the persistence of one or more local or systemic signs and symptoms of one or more SSTIs such that new or additional systemic antibacterial agents are required for use in SSTIs.

Microbial results as secondary efficacy variables were assessed in a subgroup of patients with microbiologically proven SSTI (i.e. at least one identified baseline pathogen). The microbial response (i.e., eradication, presumed eradication, persistence, presumed persistence) to each gram-positive pathogen identified at baseline is assessed. For patients who have not undergone subsequent culture, the microbial response of the baseline pathogen is presumed based on the clinical response. The microbial response of the patient at the visit of the EOT and FU is graded as success (i.e., all gram-positive organisms are eradicated or presumed to be eradicated) or failure (i.e., at least one gram-positive organism persists or presumed to persist, with partial eradication of multiple pathogens). Colonization and superinfection were assessed at both EOT and FU visits. The bacteriological response of the patient at FU revisit may also include recurrence.

Clinical efficacy

Clinical success rates are shown in table 18. In the clinically evaluable population, 61.5% of patients in the single dose dalbavancin group, 94.1% of patients in the two dose dalbavancin group, and 76.2% of patients in the standard of care group were classified as successful in FU assessment. In a heuristic sub-analysis of those patients classified as having deep or complex SSTI at baseline, two-dose dalbavancin treatment also provided higher clinical success (93.8%) compared to single-dose dalbavancin treatment and standard-of-care treatment of 58.3% and 73.7%, respectively.

Similar success rates were found in EOT and FU assessments in both supportive ITT and microbiologically assessable populations that consistently tended to have more favorable responses after receiving two doses of dalbavancin treatment (table 17). For the MITT population, patients with methicillin-resistant staphylococcus aureus (MRSA) had a single dose of dalbavancin clinical success at FU assessment of 50% (3/6), two doses of dalbavancin of 80% (4/5), and 50% of patients treated with the standard of care regimen (1/2).

TABLE 18 clinical success rates of the analyzed population and treatment groups

Microbiological efficacy

The success rates for the different treatment regimens are shown in table 19 for the different pathogens. For the microbiologically evaluable population, the eradication/presumed eradication rate for all organisms at FU evaluation was 58.3% for dalbavancin at a single dose (7/12), 92.3% for two doses (12/13), and 70.6% for patients in the standard of care group (12/17). There was no change in the MIC of dalbavancin with respect to the surviving isolates. The eradication rate of staphylococcus aureus was higher in the two dose dalbavancin group (90%) compared to the single dose dalbavancin (50%) and standard of care (60%) treatments when FU. Similar findings were observed for the MITT population; two doses of dalbavancin eradicated 80% of MRSA isolates (table 19).

TABLE 19 pathogen success rates of microbial ITT populations at FU assessment

The microbiological success rates at EOT and FU for the microbiologically evaluable and MITT populations are summarized in table 20. Comparable microbiological success rates (approximately 64% to 77%) were reported in both visits of patients treated with two doses of dalbavancin and a standard care regimen, while those given a single dose of dalbavancin had lower success rates (< 40%). Microbiologically, the microbiologic success rate at EOT/FU in the microbiologically evaluable population was found to be similar to the clinical response: 38.5%/27.3% for a single dose of dalbavancin, 72.7%/72.7% for two doses of dalbavancin, and 71.4%/64.3% for standard of care treatment. Similar findings were observed for the MITT population (data not shown).

TABLE 20 microbiological success Rate

Pharmacokinetic analysis

For patients randomized into the dalbavancin treatment group, 5ml of blood was obtained on day 8 to determine dalbavancin plasma concentration. For patients randomized to receive a 500mg dose of dalbavancin on day 8, blood was obtained immediately prior to administration of the second dose. Additional 5ml blood samples were obtained on days 10 and 24 for patients randomized into the single dose dalbavancin group, and additional 5ml blood samples were obtained on days 20 and 34 for patients receiving two doses of dalbavancin.

Plasma concentrations of dalbavancin were determined using validated liquid chromatography and mass spectrophometry. For plasma, the lower limit of quantitation was 500 ng/ml.

The mean concentrations of dalbavancin collected on study days 8, 10 and 24 in the single dose regimen were 31.1 ± 7.1, 25.2 ± 4.8 and 10.2 ± 3.5mg/l (mean ± SD), respectively. The concentration of dalbavancin receiving the two dose regimen was 30.4 ± 8.2, 21.2 ± 10.0 and 9.0 ± 4.4mg/l on study days 8 (before the second dose), 20 and 34, respectively. As expected, all patients had concentrations of dalbavancin higher than 20mg/l within the first week after receiving the first dose, and the level of higher than 20mg/l was maintained for another week at day 8 with an additional 500mg dose intravenously. The minimum bactericidal concentration is generally about 4-10 mg/l.

Security assessment

Drug safety was assessed in individual patients receiving at least one dose of study drug (ITT population) by monitoring Adverse Events (AEs) including abnormal clinical laboratory test results and vital signs. AEs were graded by investigator according to their severity (mild, moderate, severe, life threatening) and graded in relation to study drug (irrelevant, unlikely to relevant, likely to relevant, or likely to relevant).

A summary of the AE data is presented in table 21. The majority of adverse events (90%) were considered mild to moderate in severity. All severe adverse reactions (8 events out of 5 patients) were unrelated to study drug treatment. Approximately 59% of all patients reported to have had at least one treatment for an emergency AE (19 list of doses of dalbavancin, 16 two doses of dalbavancin, 21 standard of care) experienced events classified by the investigator as likely or likely to be related to the study drug. Specifically, drug-related AEs were reported in 11 (55%) single dose dalbavancin, 10 (48%) two dose dalbavancin, and 12 (57%) standard care patients. The most commonly reported drug-related AEs in the dalbavancin and standard of care treatment groups were diarrhea and nausea. A summary of the AE types observed for the different treatment groups is presented in table 22.

None of the dalbavancin-treated patients prematurely discontinued treatment due to AE. 3 of 21 patients on the standard care regimen (14%) had prematurely discontinued treatment due to AE, including 1 patient presenting with most likely drug-related urticaria on day 1, and 2 patients with study drug-independent AE (superinfection with Pseudomonas aeruginosa and elevated vancomycin trough levels).

TABLE 21 summary of Adverse Event (AE) data

TABLE 22 most common adverse events

Discussion of the related Art

Randomized phase II trials of this open label show that dalbavancin can effectively treat adults with SSTI. Most enrolled patients have deep or complex infections (> 90%) and infections requiring surgical intervention (-70%), while approximately 45% have underlying diabetes.

Two weekly doses of dalbavancin have numerically higher clinical response rates than a single dose of dalbavancin or a standard care regimen. Data from ITT and clinically evaluable populations indicate that two consecutive weekly injections of dalbavancin doses (1000 mg, 500mg weekly) are therapeutically effective at SSTI. Standard of care treatment lasted for an average of 13 days. In the follow-up, 94% of the clinically evaluable patients treated with two doses of dalbavancin were considered to be clinically successful compared to 76% of patients who provided a standard of care regimen and 61.5% of patients who received a single dose of dalbavancin.

Staphylococcus aureus is the most commonly isolated organism at baseline. In this trial, approximately 83% of patients were infected with staphylococcus aureus and 38% of all staphylococcus aureus were MRSA. Most infections (80%) are caused by a single pathogen. The MIC of dalbavancin against gram-positive isolates (including MRSA) ranged from 0.016 to 0.25 mg/L.

The microbiology success rate of the clinically evaluable population was similar to the clinical response success rate. Treatment with a two dose once weekly regimen of dalbavancin provided a higher eradication rate (92%) at an assessment 2 weeks after treatment compared to a single dose of dalbavancin treatment (58%) and standard of care treatment (71%) for all organisms combined. In summary, eradication rates of staphylococcus aureus were observed in 90%, 50% and 60% of patients, respectively. For the MITT population, the MRSA eradication rate for the two-dose dalbavancin regimen was 80% compared to 50% for the single dose dalbavancin treatment versus the standard of care treatment.

At the end of the treatment period of single and two doses once a week (respectively)Day 10 or day 20) showed little drug accumulation after receiving the second weekly dose. The observed higher clinical success rate of the two-dose regimen indicates a time-dependent killing effect, wherein sustained drug levels or drug exposure are provided at two dalbavancin doses 1 week apart. The plasma levels of dalbavancin measured at the end of the weekly dosing interval are substantially higher than the reported MIC for the pathogen causing the majority of SSTI ₉₀(< 0.03-0.5mg/L), including the pathogens found in this assay. The level is also above a minimum bactericidal concentration of 4 to 10 mg/l.

The dalbavancin dosing regimen was similar to the overall adverse reaction rate of the standard of care group. Gastrointestinal drug-related adverse events (i.e. diarrhea and nausea) were most frequently reported in the three treatment groups. Most of the events were mild and self-limiting. None of the patients treated with dalbavancin were withdrawn from the study early due to adverse effects, nor were any serious adverse events attributable to glycopeptides reported, but 14% of the standard of care groups were withdrawn due to adverse effects. The new dosing regimen thus has reduced adverse side effects compared to standard care. Data from this trial did not reveal evidence that dalbavancin induced any degree of clinically significant hepatotoxicity or nephrotoxicity.

Two-dose dalbavancin dosing regimens appear to be effective for treating patients with complex SSTIs. Dalbavancin was well tolerated in this clinical trial at both doses while having a similar spectrum of adverse events as the standard of care group.

Example 2 efficacy and safety of once weekly dalbavancin in the treatment of catheter-related bloodstream infection (CR-BSI)

The study evaluated the efficacy and safety of the once weekly dosing regimen of dalbavancin in the treatment of adult catheter-related blood flow infection (CR-BSI) due to gram-positive bacterial pathogens compared to standard treatment care vancomycin.

Method of producing a composite material

In this open-label, comparative, multicenter study, CR-BSI patients meeting inclusion/exclusion criteria due to suspected or known gram-positive pathogens were randomly assigned to one of two treatment groups. In the weekly dalbavancin therapy, dalbavancin was administered once a week, and in the vancomycin therapy, the control (vancomycin) was administered daily. Patients infected with staphylococcus aureus require removal of the catheter; care was taken by the investigator whether to treat the catheter for coagulase-negative staphylococci (CoNS) infection in patients. Efficacy is clinically based on clinical signs and improvement or resolution of CR-BSI symptoms, ensuring that there are no additional antibacterial agents, and is microbiologically based on eradication or presence of baseline pathogens or other pathogens. Safety and dalbavancin plasma concentrations were also evaluated.

Population for analysis

Approximately 80 patients were scheduled (40 in each of treatment groups 1 and 3); 67 patients (33 administered dalbavancin once weekly, 34 administered vancomycin) were analyzed. 7 (7) patients administered dalbavancin daily were included only in the safety analysis. 75 (75) patients were randomly assigned and 74 patients were treated in 13 places in the united states; 64 patients completed the study. Demographic characteristics are generally similar among the various research groups. The mean age of patients administered dalbavancin once a week was 54 years (range 20-78 years) and 57 years (range 19-85 years) with vancomycin. Male and female population mean distribution; slightly more males in the dalbavancin group and slightly more females in the vancomycin group once a week. Most patients are caucasian (> 65%), classified as likely to have CR-BSI, and have non-implanted catheters. The most common pathogens for both treatment groups were C0NS and staphylococcus aureus for the microbiological ITT population. Among the staphylococcus aureus isolates, 5/11 (45.4%) and 9/12 (75.0%) were identified as methicillin-resistant (MRSA) in study groups 1 and 3, respectively.

Diagnosis and enrollmentPrincipal standard

Patients were enrolled for a proven gram-positive bacteremia, wherein the proven gram-positive bacteremia was defined as at least one blood culture being staphylococcus aureus positive, or at least two blood cultures being positive for all other organisms (wherein at least one culture was from a transdermally drawn sample). In addition, patients who met all other inclusion criteria and also met each of the following two conditions were also included:

1. at least two of the following signs of bacteremia: core body temperature > 38.0 ℃ or < 36.0 ℃, measured rectally, orally (adding 0.5 ℃ to the measured temperature), tympanic membrane or via central catheter; WBC count > 12,000/mm³Or < 4,000/mm³Or differential white blood cell count indicates > 10% banding pattern; tachycardia (pulse rate > 100 bpm); tachypnea (respiratory rate > 20 breaths/min); transient hypotension (systolic pressure < 90mm Hg);

2. there is no obvious source of clinical manifestations of bacteremia other than the catheter (there may be local signs and symptoms at the catheter site).

Treatment of

Patients with S.aureus infections were treated for 14 days and patients with all other pathogens were treated for 7-14 days. Because of the long half-life of dalbavancin, the duration of study drug treatment was assumed to be 7 days per dose of dalbavancin given once a week. Dalbavancin was administered intravenously at a 1000mg loading dose on day 1 and a 500mg dose on day 8. Vancomycin is administered intravenously at a dose of 1000mg every 12 hours (or the dose is adjusted according to the drug level).

Evaluation criteria

Efficacy was assessed according to clinical and microbiological responses. In microbiologically intent-to-treat (ITT) populations, the primary outcome parameter is the overall response at the visit of the cure Test (TOC). Safety was assessed by collecting and analyzing Adverse Events (AEs), clinical laboratory tests, physical examinations, vital signs, and ECG data. Dalbavancin plasma concentrations were determined at up to 7 occasions (before and after the first dose on day 1, before and after the second dose on day 4 ± 2, before and after the second dose on day 8, at the end of treatment (EOT) and at TOC). Since group 2 was excluded during the study, only demographic and safety data for those patients were described; efficacy was not evaluated.

Statistical method

Efficacy, safety and dalbavancin concentration data are given using descriptive statistics. For the primary efficacy analysis, 95% confidence intervals were also determined, and for the prognostic factor analysis using the primary efficacy variable, logarithmic regression was used.

Efficacy results

Patients receiving dalbavancin once a week (87.0%, 95% CI: 73.2, 100.0) had a higher success rate for the primary efficacy analysis-overall response in the microbiology ITT population at TOC than patients receiving vancomycin (50.0%, 95% CI: 31.5, 68.5).

For secondary efficacy analysis, the overall success, clinical success, and both overall and clinical success of the dalbavancin panel were higher in the microbiology ITT and evaluable population at EOT and TOC by pathogen, by infection species, by catheter status at baseline, and by catheter type, relative to the vancomycin panel. Microbiological success according to patients (By-patient) was similar in the treatment groups at EOT, but higher in the dalbavancin study group at TOC. For CoNS, the microbiological success rate according to the pathogen (by-pathogen) is higher in the DABAVAXIN research group at EOT and TOC. By EOT, most of the signs and symptoms resolved at the catheter sites of both groups.

Safety results

There were 71 patients (95.9%) reporting Adverse Events (AE). The number of patients reporting AEs was similar in each study group, although more AEs were reported in the dalbavancin group than in the vancomycin group. The most common AEs were diarrhea, constipation, anemia, and hypotension. Researchers believe that the intensity of most AEs is mild to moderate. Adverse events associated with (likely or likely to be) study drug were considered evenly distributed among the various study groups. One of the dalbavancin study groups (1) patient (3%) had an unrelated, non-severe AE that caused dalbavancin discontinuation; no AE led to withdrawal studies. Three (3) patients in the vancomycin study group (8.8%) had AEs that caused discontinuation of the control; 2 AEs led to withdrawal studies. The distribution of SAE was similar in each treatment group; none of the SAE and vancomycin groups in the DABAVAXIN study group were considered to have SAE associated with the study drug. There were 5 deaths in this study. All AEs leading to death were unrelated to study drug. There were no clinically significant laboratory abnormalities in any of the study groups. Few laboratory values were reported as AE. None were SAE or resulted in discontinuation of study drug.

Diastolic blood pressure elevation was the most common clinically significant abnormal change in all treatment groups. 3/74 patients (4.1%, 2 patients administered dalbavancin, 1 patient administered vancomycin) reported hypertension as AE; only 1 patient (receiving dalbavancin) had elevated blood pressure as an AE considered related to the study drug. Dalbavancin did not show any effect on heart rate, atrioventricular conduction or intraventricular conduction. The average difference in effect on QTc values was that the dalbavancin group was 7 milliseconds greater than the vancomycin group. No significant difference in extremal frequency was observed between treatment groups during drug treatment.

Thus, an initial IV dose of 1000mg for dalbavancin followed by 1 week later by a second IV dose of 500mg appears to be well tolerated and very effective for the treatment of CR-BSI caused by gram-positive pathogens (higher response rate to vancomycin).

Example 3 Dalbavancin pharmacokinetics and renal excretion in healthy subjects

The primary objective of this study was to characterize the pharmacokinetics of dalbavancin and calculate the degree of renal excretion in healthy subjects receiving therapeutic doses of the drug. This is an open label, non-contrast study.

Study of drug treatment

A single 1000mg IV dose of dalbavancin was infused over 30 minutes into healthy male or female subjects between 18 and 65 years of age.

6 subjects (1 female and 5 male) were enrolled, received study medication and completed all aspects of the study. 3 subjects were caucasians and 3 subjects were africans of america. The mean age was 29.8 years (from 22 to 63 years). The average height was 68.6 inches (from 62 to 75 inches) and the average body weight was 179.6 pounds (from 140 to 244 pounds).

Pharmacokinetics

Blood and urine were collected on days 1, 2, 3, 4, 5, 6, 7, 14, 21, 28 and 42 of the study (24 hour collection). Blood samples were aspirated into heparinized tubes and centrifuged. Plasma was separated and stored frozen at-20 ℃ until the time of assay. Dalbavancin was assayed in plasma and urine samples using a validated LC/MS method. The lower limit of the urine and plasma quantification assay was 500 ng/mL.

Dalbavancin pharmacokinetic parameters were evaluated by a non-compartmental method using WinNonlin software (Pharsight Corporation). Peak concentration (C)_max) Values were obtained directly from the observed data. The area under the plasma concentration-time curve (AUC) was calculated using the linear trapezoidal rule. Clearance (CL) was calculated as dose/AUC. Elimination half-life (t) was assessed by linear regression of the log-linear part of the log concentration versus time curve _1/2). The estimated volume of distribution (Vz) was calculated using the regression parameters, while the volume of distribution at steady state (Vss) was calculated from the area under the curve (AUMC) at the first time instant multiplied by the dose and divided by AUC. Determined by integration of urine excretion rate (AURC)And determining the cumulative amount of dalbavancin excreted in urine. CL is calculated as the ratio AURC/AUC_ROr renal clearance: CL_R＝AURC/AUC。

The plasma concentration of dalbavancin versus time for all subjects is shown in figure 5. Pharmacokinetic parameters are presented in table 23. The concentrations were similar in all subjects. The peak plasma concentration was approximately 300mg/L and was reached immediately after the end of the infusion. Dalbavancin showed an apparent distribution volume of greater than 10L and thus was considered to be well distributed in the extracellular fluid.

T of dalbavancin at 9-12 days_1/2Slowly eliminated. The total drug clearance rate is 0.0431 + -0.0074L/hr. The fraction of unchanged drug excreted in urine was estimated to be 42% of the administered dose, and the renal clearance was estimated to be 0.018L/h. The variability observed in the subjects was low and the coefficient of variation in all pharmacokinetic parameters was less than 22%.

TABLE 23 pharmacokinetic parameters

Security assessment

Adverse events were recorded and evaluated for severity and their relationship to study drug. Laboratory data (chemical profiles, CBC and classification, urinalysis) were collected and evaluated for changes from baseline and out of range values. ECG, physical examination, and vital signs were obtained and evaluated for changes from baseline.

Dalbavancin was well tolerated in this study. No subjects died or reported severe adverse events during the study and no subjects prematurely withdrawn from the study due to AE.

All volunteers reported at least one adverse event, all mild. 3 volunteers reported the appearance of a possible study drug related disorderGood events: an object exhibiting elevated ALT (value 46IU/L, upper limit of normal 40 IU/L); a subject experienced all of the following adverse events: eosinophilia (value 0.5X 10)³μ L, upper normal limit 0.4X 10³/. mu.L), elevated LDH (value 303IU/L, upper limit of normality 90IU/L), elevated ALT (value 54IU/L, upper limit of normality 40IU/L), elevated AST (value 42IU/L, upper limit of normality 40 IU/L); and tinnitus of a subject.

No trends were found in post-baseline hematology, chemistry, vital signs and ECG results.

Discussion of the related Art

Dalbavancin was well tolerated at a single 1000mg IV dose. Plasma concentrations of dalbavancin greater than 45mg/l were maintained for at least 7 days after a single intravenous infusion of 1000 mg. This value is higher than the known bactericidal concentration (4-32 mg/l). This result supports the use of dalbavancin on a once weekly schedule. The urine elimination curve indicates that renal excretion is an important elimination pathway, with approximately 40% being excreted in urine. This finding is consistent with observations in animals. Since the kidney is not the only route of elimination, dose modulation of dalbavancin may not be necessary in kidney-impaired patients.

Example 4 dalbavancin pharmacokinetics in renal injury patients

These studies are open label studies conducted to investigate the safety and pharmacokinetics of intravenous dalbavancin when administered to patients with mild, moderate, and severe renal injury.

Study of drug treatment

Male and female patients between the ages of 18 and 80 are eligible for inclusion. The patient must be in the range of-10% to + 50% of the ideal body weight relative to gender, height and build.

Pharmacokinetics

Serial blood samples were collected from pre-dose to at least 2 weeks after the end of infusion and analyzed for dalbavancin using a validated LC-MS/MS method.

The non-compartmental method evaluates the dalbavancin pharmacokinetic parameters. The peak concentration (C) was obtained directly from the observed data_max). The area under the plasma concentration-time curve (AUC) was calculated using the linear trapezoidal method.

Pharmacokinetic parameters for patients with healthy kidney function and patients with the most severe renal impairment (severe RI) are shown in table 24.

TABLE 24 pharmacokinetic parameters

^ preliminary clinical data

Using parameter estimation by direct reference or extrapolation from curves

Based on simulation data, or inferred from another study; is not directly introduced into the abstract

AUC ═ drug exposure as estimated by area under the plasma concentration-time curve;

AUC 7-drug exposure 7 days post-dose or throughout treatment period;

AUC 14-drug exposure 14 days post-dose or throughout treatment period;

AUC 42-drug exposure 42 days post-dose or throughout treatment period;

C_maxmaximum drug concentration observed in plasma;

c7-drug concentration 7 days after administration, before administration of another possible dose;

c14-drug concentration at 14 days post-administration, before administration of another possible dose;

discussion of the related Art

For patients with severe renal injury, a single dose of 500 or 1000mg of dalbavancin is administered to the patient. As is evident from table 24, after 14 days, patients with severe renal injury who were administered a single 1000mg dose had an AUC14(mg.h/L) of approximately 22000, which is unexpectedly very similar to patients with normal renal function (auc14mg.h/L23000) who were administered a two dose regimen of 1000mg +500 mg.

Dose adjustment of dalbavancin is not required for patients with mild renal insufficiency. Dalbavancin concentrations and pharmacokinetic parameters were similar in patients with mild renal injury and normal renal function. Additionally, dalbavancin is well tolerated in patients with normal or mildly impaired renal function. See, Dowell, J.et al, "Dalbavancin dosag additions Not Requiredfor Patients with Mild Red Impatiention", 2003 ECCMID Meeting, Glasgow (Clinical microbiology and Infection, Vol.9 (suppl.1), p.291; 2003), and Stogniew, M.et al, "Pharmacokineticical attributes of Dalbavancin: well Distributed and complex Elimated with Dual Routes of Elimation "(clinical microbiology and Infection, Vol.9 (suppl.1), p.291-292; 2003), both of which are specifically incorporated herein by reference in their entirety.

Example 5 renal injury and end stage renal disease

The main route of dalbavancin elimination is the excretion of intact dalbavancin and OH-dalbavancin into the urine, and it is highly likely that the drug will be used in patients with various degrees of renal injury. For this reason, the safety and pharmacokinetics of dalbavancin in patients with various degrees of renal injury were examined in clinical studies VER001-3, VER001-11 and VER 001-13.

When the measured dose (70mg) was determined to be too low relative to the larger weekly dose recommended for the clinical study, clinical study VER001-3 ended early (5 patients enrolled; 3 patients received dalbavancin). Clinical study VER001-13 examined patients with mild and moderate renal impairment, and clinical study VER001-11 examined patients with severe renal impairment and end-stage renal disease (ESRD). Matched control patients were included in all studies. Renal injury is defined by estimated creatinine clearance: mild 51-79mL/min, moderate 31-50mL/min, and severe ≤ 30 mL/min. ESRD patients relied on dialysis (3 times per week) throughout the study. A single dose of dalbavancin IV of 1000mg was examined in patients with mild and moderate renal injury, a 500mg dose was studied in ESRD patients, and single doses of 500 and 1000mg were studied in patients with severe renal injury.

In each of these renal injury studies, dalbavancin was well tolerated. Similar percentages of patients reporting adverse events were observed in the various study groups. The adverse events reported were mostly mild or moderate in severity and were not related to study drug. There was no clinically significant laboratory confusion in any study patient receiving dalbavancin.

In the clinical study VER001-13, administration of dalbavancin to patients with normal renal function and patients with mild renal injury resulted in comparable concentration-time curves over a 60 day sampling interval. The concentration-time curves were comparable between patients with normal renal function and patients with mild or moderate renal impairment 7 days after administration throughout the relative treatment period. Over day 14, a small increase in concentration was observed in patients with moderate renal injury when the concentration was relatively low, and below 40 mg/L. After these patients were administered 1000mg dalbavancin, their plasma concentration-time profiles of dalbavancin are shown in figure 18. Dalbavancin pharmacokinetic parameters are shown in table 25. For mild or moderate renal injury (CL)_CR> 30mL/min) patients, no dose adjustment is required. The results of these studies were compared to patients studied in a population pharmacokinetic analysis (CL) _CR> 50mL/min) are negative.

In clinical study VER001-11, dalbavancin was administered to patients with normal renal function, patients with severe renal injury, and patients with ESRD. The dalbavancin concentration-time curves observed in this study are shown in figure 19. Concentrations were similar between patients with normal renal function and patients with severe renal injury or ESRD during the first 12 hours, but small and progressively larger differences were observed in patients with severe renal injury. The concentration increased by ≦ 40% for the first week after dosing, and in the remainder of the curve, the difference continued to increase. ESRD patients were divided into patients who received dalbavancin before or after the first dialysis session. No difference in concentration profiles was observed between these two subgroups. However, the concentrations in ESRD patients are similar to those in patients with normal renal function, indicating that renal insufficiency is compensated for by regular dialysis (3 times/week). The level in the dialysate corresponding to this drug dialysis level is too small to measure.

Dalbavancin pharmacokinetic parameters (VER001-11) in severe renal injury patients and ESRD patients were compared to mild or moderate renal injury patients (VER001-13) in table 25. Single doses of 500 and 1000mg were studied in patients with severe renal injury; concentrations and exposures were consistent with dose ratios (fig. 20, table 25). Individual drug exposure (AUC) over relative treatment periods _{0-7 days}) CL typically spanning individuals_CRIn agreement, only a small increase in exposure was observed in patients with severe renal injury (fig. 21). The difference in exposure is much greater when the entire concentration-time curve is examined. In patients with severe renal injury, the area under the plasma concentration-time curve extrapolated to infinity (AUCo-inf) increased by 97%. Only a 45% increase in AUCo-inf was seen in ESRD patients, which reflects a regular periodic dialysis session to compensate for renal insufficiency.

Based on pharmacokinetic parameters and data-based simulations, it is recommended to adjust the dose for patients with severe renal injury. The purpose of dose modulation is to match the concentrations and exposures of the two doses over the course of the treatment period (14 days) while minimizing overall drug exposure. A total of 9 mock dosing regimens were examined in patients with severe renal injury. The results of these simulations are summarized in fig. 22, which shows a comparison of exposure during the relative treatment period to overall exposure. A dose of 750mg of dalbavancin followed by 250mg of dalbavancin one week is for CL_cr＜30mRecommended dose adjustment for L/min patients. This dosing regimen maintains concentrations greater than 20mg/L, matching the therapeutic exposure observed for patients with normal renal function, and minimizes overall exposure. Figure 23 shows a dalbavancin concentration-time curve that simulates the following: i) patients with normal renal function who received 1000mg (day 1) +500mg (day 8), ii) patients with severe renal impairment who received 1000mg (day 1) +500mg (day 8), and iii) patients with severe renal impairment who received the recommended adjusted dose of 750mg (day 1) +250mg (day 8). Patients receiving regular dialysis therapy (2 to 3 times per week) have some compensation for drug clearance by dialysis, without the need to adjust the dose.

TABLE 25 pharmacokinetic parameters of dalbavancin in healthy and renal impaired patients following single dose administration of dalbavancin

Example 6 liver injury

Dalbavancin is eliminated by both the renal and non-renal pathways, and it would be likely that dalbavancin would be used in patients with various degrees of liver injury. Dalbavancin safety and pharmacokinetics were examined in patients with various degrees of liver injury in the clinical study VER 001-12. The study included otherwise healthy patients with mild, moderate or severe liver damage (Child-Pugh scores of 5 to 6, 7 to 9, and 10 to 15 points, respectively) and matched control patients with normal liver function. A single 1000mg IV dose of dalbavancin was administered to the patient on day 1, followed by a 500mg IV dose of dalbavancin on day 8.

Dalbavancin concentrations and exposure did not increase with increasing degree of liver injury (figure 24). Administration of dalbavancin to patients with normal liver function and to patients with mild liver injury resulted in comparable concentration-time curves over a 60 day sampling interval. Administration of dalbavancin to patients with moderate liver injury and to patients with severe liver injury resulted in a slight decrease in the concentration observed when compared to patients with normal liver function. In all groups, the drug was well tolerated.

The exposure of dalbavancin to normal liver function patients and patients with mild liver injury was comparable (table 26). The trend of decreased dalbavancin exposure and increased CL was evident for patients with moderate and severe liver injury. Overall, the variability between individuals of pharmacokinetic parameters was low, typically below 30%, although the dalbavancin pharmacokinetic parameters were statistically different between the higher and lower liver injury groups, however, there was significant overlap in the range of exposure parameters (fig. 25).

Changes in dalbavancin pharmacokinetic parameters appear to be affected by the volume of drug distribution. The volume of distribution increases in the same proportional or inverse manner as CL and AUC. The drug terminal elimination half-life was virtually unchanged between groups. Moderate and severe liver injury patients (who have significantly more ascites and edema) have a larger volume of drug distribution followed by slightly lower drug exposure.

There was an overlap in the concentration of the curves between the groups, and the average concentration remained above 20mg/L for all groups over the expected treatment period of 14 days. There was also an overlap in drug exposure between groups (figure 25). Even with the reduced mean exposure, drug exposure for severely liver injured patients exceeded the parameters required for treatment throughout the relative treatment period (14 days). For patients with any degree of liver damage, there is no need to adjust the dose of dalbavancin.

TABLE 26 pharmacokinetics of dalbavancin in healthy and hepatic patients following 1000mg dalbavancin on day 1 and 500mg dalbavancin on day 8

Example 7 measurement of protein binding of dalbavancin Using isothermal titration microcalorimetry

Binding of dalbavancin to protein was measured by isothermal titration microcalorimetry (ITC) at pH 7.4 using a Microcal VP-ITC instrument at 20mM phosphate, 150mM nacl, 25 and 37 ℃. In a standard experiment, 25X 10. mu.l protein (. about.150. mu.M) was injected into a calorimeter cell (calorimeter cell) containing a dalbavancin solution (. about.5. mu.M). The actual concentration of protein and dalbavancin was determined by measuring the absorbance at 280 nm. Control experiments included injecting protein into buffer (dalbavancin was not present) to account for the heat generated by dilution of protein under the same conditions. For comparison, similar experiments with some necessary modifications were performed using teicoplanin.

The dalbavancin experiments were performed with each of the following proteins: human albumin, dog albumin, rat albumin, bovine albumin, and human alpha-glycoprotein. Human albumin and alpha-glycoprotein were used to study teicoplanin. A comparison of binding affinities at two different temperatures is shown in table 27.

TABLE 27 comparison of apparent binding affinities (Ka,. times.10)⁵M^-1)

The ± errors quoted are the standard deviations obtained from the fitting convention.

After correction for the heat of dilution, the thermal effects were integrated by nonlinear regression analysis using a simple single site binding model with a standard microcalor software package. The raw data (μ cal/sec) for each injection was integrated and summed to give the total thermal effect, then divided by the amount of injectate to give kcal/mole injectate. The same integration was applied to the control dilution effect and subtracted from the actual titration data. This provides a differential of the binding curve, where the degree of binding is proportional to the total released (or absorbed) heat. It was then analyzed by non-linear regression methods for various standard binding models. The simplest model assumes a simple non-competitive binding equilibrium and gives three parameters:

Ka(＝1/K_diss) For binding associations (dissociation constants)

Δ H-enthalpy of binding (magnitude of signal associated with binding)

Number of binding sites (assuming the binding model is correct)

N is the molar (relative) number of injectates required to saturate all available binding sites in the sample, assuming non-competitive binding. For dalbavancin experiments, dalbavancin is the "sample" and proteins (HSA etc.) are the "injectate". The preliminary results indicated that binding was relatively weak and that it was very difficult to determine clearly the binding stoichiometry (N) due to the poor solubility of dalbavancin. However, as shown in FIG. 6, in all cases, the data fit very well to N < 1 (i.e., less than 1: 1 protein: dalbavancin). Thus, a value of 0.5 for N means that only half the moles of protein are required to bind all dalbavancin, as expected. In other words, each protein apparently binds two dalbavancin molecules. Dalbavancin may form a dimer that binds to the protein 1: 1. The binding stoichiometry modeling results showed that two molecules of dalbavancin bound to one protein molecule, whereas teicoplanin, unlike dalbavancin, exhibited a 1: 1 binding.

Human serum albumin (6X 10) was hypothesized^-4M) and alpha-glycopeptides (1.5X 10)^-5M), table 28 shows the calculated percent binding for antibiotic concentrations in the range of 1-500 μ M. To correlate this with clinical condition, peak concentrations of dalbavancin in humans were approximately 300mg/L or 165 μ M.

TABLE 28 calculated percent binding of teicoplanin and dalbavancin to plasma proteins

Not performing ND-

In the experiments described, dalbavancin bound to human serum albumin in an amount of more than 98%. The binding fraction is fairly constant over the chosen concentration range of dalbavancin, i.e., 1-500 μ M. This range encompasses therapeutic concentrations in humans. The percentage of dalbavancin binding to alpha-glycoprotein is much higher than teicoplanin. Dalbavancin demonstrated high capacity and low affinity for different sources of plasma proteins with similar Ka values in all kinds of proteins tested. The results help explain certain unique pharmacokinetic characteristics of dalbavancin. 2: 1 dalbavancin: the association and formation of protein complexes also accounts for the extended half-life and apparent volume of distribution, which is close to the extracellular water volume. The low affinity helps explain the observed in vivo activity, which greatly exceeds that expected for compounds with a free fraction close to 1%. The large capacity for plasma proteins helps explain that relatively high plasma concentrations are achieved despite the poor solubility of the compounds at physiological pH.

Example 8 pharmacokinetic Properties and tissue distribution of dalbavancin in rats

In administration of a single IV infusion of 20mg/kg³H]Two studies were performed in rats with dalbavancin. The excreta and more than 40 different tissues were collected within 70 days after administration and the tissue distribution and pharmacokinetics of the radioactivity originating from the drug were determined.

Subjecting the HPLC-purified [ alpha ], [ beta³H]Dalbavancin was used in the study. The radioisotope-labeled drug was generated via tritium exchange and purified by HPLC.

Rat mass balance study

Mass balance studies were performed to determine the mode of excretion of dalbavancin following a single Intravenous (IV) infusion of dalbavancin into male rats.

15 Male Sprague-Dawley rats received a single IV dose³H-dalbavancin (20mg/kg, 100. mu. Ci/rat). After administration, to administrationUrine and feces were collected at 24 hour intervals on the following 14, 36 and 70 days (3 rats/final collection time). Water and methanol cage washings were also collected. At the end of the collection period the carcass is analyzed. The total radioactivity content of all samples was analyzed by Liquid Scintillation Counting (LSC).

In the intravenous administration to rats³After H-dalbavancin, the radioactivity originating from the drug was eliminated in urine (-2/3 excreted radioactivity) and feces (-1/3 excreted radioactivity). Approximately half of the administered radioactivity is eliminated in urine and feces within the first week, which correlates with approximately one week of plasma t _1/2And (5) the consistency is achieved. On day 70 post-dose, only 4.5% of the dose remained in the carcass. Negligible radioactivity was recovered in the cage wash. Virtually all radioactivity administered during the study was accounted for (urine, feces, carcass, cage washes and tritium exchange).

Quantitative Tissue Distribution (QTD) study in rats

Quantitative tissue distribution studies were performed to assess the tissue distribution of dalbavancin following a single IV infusion of dalbavancin into male rats.

41 Male Sprague-Dawley rats received³A single IV infusion of H-dalbavancin (20mg/kg, 50. mu. Ci/rat). Rats (3 per time point) were euthanized at 12, 24, 48, 72, 96, 120, 144, 168, 336, 840, 1176 and 1680 hours post-dose to collect blood, plasma and tissues (including carcass). All samples were analyzed by LSC.

Concentration-time curves were determined for more than 40 tissues including kidney, liver, spleen, blood, plasma, lung and skin. Concentration of radioactivity originating from drug in tissue including skin and t_1/2Values were comparable to those observed in plasma. Dalbavancin was found to be rapidly and widely distributed in all tissues within 12 hours after injection, with a measurable concentration of drug-derived radioactivity in the tissues at the same time. Maximum concentration (C) was reached in most tissues within 24h after dosing _max). Radioactivity < dose recovered in any individual tissue after 5 days5% of the amount. By day 70 post-dose, only the carcass remained > 1% (2.34%) of the administered radioactivity. Thus, dalbavancin does not accumulate in any single tissue, organ, and blood cellular components. The radioactive concentration in the CNS in this healthy animal model is low but detectable. Dalbavancin was found to penetrate the skin with a concentration of drug-derived radioactivity equal to or higher than that in plasma. The blood to plasma ratio of radioactivity originating from the drug remains relatively constant and < 1 over time.

As part of the QTD study, bile samples from cholangiocannulated rats (4 animals) were collected at 384h (16 days) post-dose. Almost 11% of the dose was recovered in bile over 384h after administration. This represents the majority of the radioactivity derived from the drug found in the feces.

Example 9 pharmacodynamic Activity of dalbavancin

The goal of antimicrobial therapy is to provide an active concentration at the site of infection for a sufficient time to eradicate the invading pathogen. The main methods for evaluating antibacterial activity in vitro are the determination of the minimum inhibitory concentration and the minimum bactericidal concentration (MIC and MBC). However, these parameters only measure the net effect of the defined incubation period and do not characterize the time course of the antimicrobial activity. MBC does not determine whether the level and extent of bactericidal activity can be enhanced by increasing drug concentration. In addition, the MIC assay does not measure whether there is continued inhibition of the bacteria after drug withdrawal.

An increasing number of studies suggest that the rate of bactericidal activity, its dependence on concentration or exposure time, and the presence or absence of aftereffects more clearly describe the time course of antibacterial activity and are important pharmacodynamic parameters for determining an optimal dosing regimen. For example, beta-lactam antibiotics exhibit very low concentration dependence on sterilization, and the extent of sterilization is primarily due to the duration of exposure. In addition, β -lactams produce short or no aftereffects (PAE) on gram-negative bacilli. Thus, one would expect that a dosing regimen that maintains drug levels above the MIC for a sufficient time would have optimal efficacy. This has been demonstrated in a number of animal models. Fluoroquinolones, on the other hand, have a concentration-dependent bactericidal effect and produce a prolonged in vivo aftereffect. It is expected that the amount of drug, rather than the frequency of administration, will be an important determinant of the efficacy of these drugs. This has also been demonstrated in a number of animal models. More importantly, the size of the pharmacokinetic/pharmacodynamic parameters necessary for β -lactam and fluoroquinolone potency are similar in animal and human infection models.

The following study was designed to characterize the in vivo pharmacodynamics of dalbavancin. The effect of dosing regimens on the in vivo efficacy of dalbavancin was determined in an experimental femoral infection model in neutropenic mice. Studies were also conducted to investigate: [1] which pharmacokinetic parameters (peak serum level, area under the concentration versus time curve (AUC), duration of serum level over MIC) best predict the potency of dalbavancin, and [2] whether the PK/PD parameter magnitude required for potency is similar among common gram-positive bacteria including penicillin-resistant pneumococci and methicillin-resistant staphylococcus aureus. Finally, the effect of the site of infection on the activity of dalbavancin against streptococcus pneumoniae and staphylococcus aureus was measured in the femoral and pneumonia infection models.

Study of MICs of organisms and dalbavancin

The study organisms and their MICs for dalbavancin are listed in table 29. MIC was determined in MHB using standard NCCLS microdilution techniques. MHB was supplemented with 3% lysed horse blood for MIC determination against streptococcus pneumoniae. All MICs were performed in at least duplicate.

TABLE 29 in vitro Activity of dalbavancin against Streptococcus pneumoniae and Staphylococcus aureus strains

5 pneumococcal and 6 staphylococcal organisms were used. The DABAVAXIN MIC range for pneumococcus is 0.004-0.03 mg/L. The MIC range for S.aureus isolates was narrower and higher than that for pneumococcus, 0.06-0.12 mg/L.

Pharmacokinetics

Plasma pharmacokinetics of dalbavancin in thigh-infected, neutropenic Swiss ICR mice are shown in figure 44. Cyclophosphamide injection: 150mg/kg 4 days before the study, 100mg/kg 1 day before the study and 100mg/kg every 48h after the start of infection until the end of the study, thereby producing neutropenia in this and all other studies. The neutrophil count remained below 100/mm for the duration of the study³. Doses of 2.5, 5, 10, 20, 40 and 80mg/kg were studied. The drug was administered by intraperitoneal injection in a volume of 0.2 ml. Blood was drawn from each group of three mice by retro-orbital aspiration into capillary tubes for heparin anticoagulation assay at 0.5, 1, 2, 4, 6, 24, 48, 72 and 96 hours post-dose. Plasma was isolated and plasma concentrations of dalbavancin were measured using microbiological assays using bacillus subtilis as the test organism. Peaks were observed at 4-6 h. The dalbavancin half-life was determined by linear least squares regression. AUC was calculated from the mean concentration using the trapezoidal method. AUC were estimated at 24, 36, 48, 72, 96h and extrapolated to infinity. The 24h AUC was calculated by dividing the 6-day AUC by 6. Dalbavancin has linear pharmacokinetics. The half-life is extended, varying from 7.6 to 13.1 hours.

Protein binding

The effect of drug binding to serum and serum proteins was studied by comparing the MICs of dalbavancin in broth, infected mouse serum, human serum, and albumin against two staphylococcus aureus strains (table 30). The MICs for both strains in broth were 0.12 mg/L. MIC (arithmetic) in 95% mouse serum increased to 32 mg/L. The MIC in the mouse serum ultrafiltrate increased to only 0.5mg/L, which means that the majority of the MIC differences were in protein binding. Similar studies with human serum and albumin resulted in an increase of only 8 mg/L. Based on MIC differences between broth and mouse serum, the degree of protein binding was estimated to be 99.6%. This degree of binding is taken into account in the subsequent pharmacodynamic analysis. The degree of binding in human serum was estimated to be 96%.

TABLE 30 Effect of serum, serum ultrafiltrate and human albumin on the in vitro Activity of dalbavancin against selected Staphylococcus aureus strains

Infection model

The murine femoral infection model was used for all organisms throughout the different studies. In this well established model, 2 hours before starting treatment, will be about 10⁶cfu of the study organism was injected into one or two strands (at 0.1 ml). In subsequent studies, the number of organisms in the thigh at the start of treatment varied by 10 ^7.15-7.59cfu/strand. The murine lung infection model was used for only a single isolate of streptococcus pneumoniae or staphylococcus aureus. In this model, 50 μ l (approximately 10 μ l) was inoculated intranasally via anesthetizing the nostrils of mice^8.5cfu/ml) infected mice. Treatment was started 2 hours after inoculation, at which time the mice had 10^7.4-7.6cfu/lung.

Time of in vivo sterilization

The effect of a single dose of dalbavancin on killing streptococcus pneumoniae and staphylococcus aureus strains in vivo over time is shown in figures 45 and 46. Each point represents the average of 4 strands. 5 dose levels ranging 16 fold were used. The dose level against Staphylococcus aureus used in this study ranged from 5-80 mg/kg. The dosage level against Streptococcus pneumoniae used in this study ranged from 0.625-10 mg/kg. The extent of kill was very extensive for both species (> 2 log, with the highest dose studied). However, the degree and rate of killing pneumococcal isolates is greater than staphylococcal strains. Studies with the two highest doses of dalbavancin resulted in killing of staphylococcus aureus. 3 of the 5 dose levels used in this study against Streptococcus pneumoniae reduced the bioburden in the thighs of infected mice by nearly 4 log.

Study of dosing regimen

In these studies, groups of mice were given 144h (6 days) intraperitoneally in 0.2ml volumes on multiple dosing schedules varying both dose and dosing interval. The dosing intervals chosen were 12, 24, 36 and 72 hours. 5 different doses (2 fold increase) were used. Studies against Staphylococcus aureus utilize total dose (mg/kg/6 days) levels ranging from 30-480mg/kg/6 days. Studies against Streptococcus pneumoniae utilized total dose (mg/kg/6 day) levels ranging from 0.6125-10mg/kg/6 day. FIGS. 47 and 48 illustrate dose response curves of dalbavancin to S.pneumoniae and S.aureus strains at different dosing intervals in the femur of neutropenic mice. Each point represents the average of 4 strands. In general, increased dosing intervals resulted in a slight shift of the dose response curve to the left, indicating that dosing regimens with less frequent administration of large doses were more effective.

The respective dose-response curves were also mathematically characterized using a maximum effect model. The method uses the hill equation to estimate the maximum effect (Emax), the dose required to obtain 50% Emax (P50), and the dose response slope by nonlinear regression. From these parameters we can then calculate the dose required to produce a net bacteriostatic effect over the course of 144 hours of treatment, and the dose necessary to produce 1 and 2 log reductions in organism load. The bacteriostatic dose and the dose associated with 1 and 2 log kill for each drug organism combination and different dosing regimens are shown in table 31.

Table 31. dalbavancin doses required to achieve net bacteriostatic effect, 1 and 2 log kill, for four different dosing intervals in the streptococcus pneumoniae and staphylococcus aureus infection model.

The dose is expressed as the total dose over a 6 day period (mg/kg)

In studies against streptococcus pneumoniae, increasing the dosage regimen from 12 to 36 hours did not result in significant changes in the dosage associated with the three microbiological endpoints. However, less drug is required at the efficacy of the 72h dosing interval. A similar relationship was observed in studies against staphylococcus aureus. The only dosing regimen to produce 1 and 2 log kill on staphylococcus aureus was at 36 and 72h intervals.

Pharmacodynamic parameters associated with efficacy

For each dosing regimen studied, we determined which PK/PD parameter was most correlated with efficacy by correlating the number of bacteria in the thigh at the end of 144 hours of treatment with the following parameters: [1]peak/MIC ratio, [2 ]]AUC/MIC ratio, and [3]Percent dosing interval where serum levels exceeded MIC. The PK/PD parameter values for those doses not specifically studied were extrapolated from the values of the closest study dose. The relationship between log cfu per strand and peak/MIC ratio, 24-hour AUC/MIC ratio and percent time that serum levels exceeded MIC for streptococcus pneumoniae is illustrated in figure 49 and figure 50 for staphylococcus aureus. Each point represents the average of four strands. For both organisms, a strong correlation was observed for the 24-hr AUC/MIC and peak/MIC ratios. However, regression of the 24hAUC/MIC ratio data resulted in the strongest correlation. The data presented in these figures were also analyzed by the same maximal dose response model described above, except that different PK/PD parameters were used instead of dose. R ²Representing the determinant observed for the relationship between potency and individual PK/PD parameters. Determining the coefficient (or R)²) Represents the percent variation in bacterial numbers, which can be attributed to individual PK/PD parameters, and for AUC/MIC and C_maxthe/MIC is high.

PK/PD parameter size or target

AUC/MIC versus multiple pathogens required to determine bacteriostatic actionIn vivo, we investigated the in vivo activity of dalbavancin against the 24-and 72-hour dosing regimen of 5 streptococcus pneumoniae and 6 staphylococcus aureus strains 6 days after treatment. The dose response curves of dalbavancin against these different strains are shown in figures 51 and 52. In FIGS. 51 and 52, the dose is expressed as the ratio of the mean 24h AUC/MIC of free drug over the 6 day study period. In general, the shape of the dose response curves for all strains is similar. The location of the dose-response curve is related to the MIC of the organism. However, the dose response curve for pneumococcal organisms is shifted slightly to the left. The shift in this curve indicates that less drug is required for efficacy against pneumococci than against staphylococci. Bacteriostatic dose, 1 log and 2 log kill, and associated free drug 24-hr AUC/MIC and C_maxthe/MIC is shown in Table 32. The degree of bacterial kill is relatively similar for most strains. All strains showed more than 4 log in the 6 day study ₁₀Decrease in cfu.

TABLE 32 efficacy of dalbavancin against Streptococcus pneumoniae and Staphylococcus aureus.

For the dalbavancin regimen with every 24h administration, the 24h AUC/MIC values of the free drug associated with bacteriostatic effects against streptococcus pneumoniae and staphylococcus aureus were 17.6 ± 6.9 and 265 ± 143, respectively. The dose response curve is steep and the 24h AUC/MIC values associated with 1 and 2 log kill are not significantly higher. As shown by the dose response curves, treatment against pneumococci required 12-23 times less drug as per 24h AUC/MIC. For Staphylococcus aureus, MBC is 2-4 times higher than MIC. If the AUC/MBC of Staphylococcus aureus is taken into account, the value is only 4-8 times higher than that of Streptococcus pneumoniae.

For a dosing regimen with a lower dosing frequency (every 72h), the free drug 24hAUC/MIC values associated with bacteriostatic effects against Streptococcus pneumoniae and Staphylococcus aureus were 7.2 + -4.5 and 160 + -67, respectively. For more spaced dosing regimens, the PK/PD size necessary to reach the three in vivo microbiological endpoints (bacteriostatic dose, 1 and 2 log kill) was lower. When dalbavancin was administered every 72h, the 24h AUC/MIC values associated with the different endpoints were 1.3-2.4 fold lower than at each 24h dose.

Penicillin resistance in streptococcus pneumoniae and methicillin resistance in staphylococcus aureus did not affect the 24h AUC/MIC required for dalbavancin potency.

Effect of neutrophils on the Activity of dalbavancin

To determine the effect of neutrophils on dalbavancin activity, we compared the dose response curves for 24 hour dosing in normal and neutropenic mice infected with streptococcus pneumoniae. The subsequent dose response curves are shown in figure 53. Each point represents the average of 4 strands. Using the hill equation, the bacteriostatic dose and the doses associated with 1 and 2 log kill were calculated from the parameters estimated by nonlinear regression. The doses required to reach these endpoints (mg/kg/6 days) in normal and neutropenic mice are shown in table 33. The presence of neutrophils results in a 1.7 to 2.1 fold reduction in the dose required for efficacy. However, these differences were not statistically significant.

TABLE 33 influence of neutrophils on the in vivo efficacy of dalbavancin against Streptococcus pneumoniae.

Effect of infection site on the Activity of dalbavancin

To determine the effect of the site of infection on dalbavancin activity, we compared the dose response curves for 24 hour dosing in the femoral and pulmonary infection models (fig. 54). Streptococcus pneumoniae and Staphylococcus aureus were used in both models. The dose response curves were almost identical in both models using streptococcus pneumoniae. In a similar study against staphylococcus aureus, the dose response curve in the pulmonary model was shifted to the left, which means that less drug was required for efficacy at the site of infection. However, in staphylococcal studies, the confidence intervals were large and these differences were not significant.

Conclusion

The above studies have characterized the in vivo pharmacodynamic activity of dalbavancin against a variety of pathogens. These can be summarized as follows:

in both femoral and pulmonary infection models, dalbavancin produced bactericidal activity in vivo against both streptococcus pneumoniae and staphylococcus aureus.

The efficacy of dalbavancin is dose-dependent.

Dose-dependent PK/PD parameters-24 h AUC/MIC and C_maxthe/MICs are all strongly linked to the in vivo activity of dalbavancin. Regression of the dose response data with the 24h AUC/MIC parameters resulted in the highest coefficient of determination. In these models, the maximum dosing interval therapy was more effective.

The 24hAUC/MIC values associated with microbiological effects were similar between strains within the species studied. Beta-lactam resistance in streptococcus pneumoniae and staphylococcus aureus did not affect the in vivo activity of dalbavancin.

Lower 24h AUC/MIC values are required for efficacy against streptococcus pneumoniae than against staphylococcus aureus. This can be explained in part by the species difference between MIC and MBC.

Efficacy against streptococcus pneumoniae, whether or not based on net bacteriostatic effect, 1 and 2 log kill required a free drug AUC/MIC of approximately 100 over 6 days of treatment in these infection models. Efficacy against staphylococcus aureus, based on the same effect, required a free drug AUC/MIC of approximately 1000 over 6 days.

Neutropenia has minimal effect on the activity of dalbavancin in vivo.

Example 10 quantitative determination of dalbavancin in plasma by HPLC-MS/MS

As described below, an HPLC-MS/MS method was developed to quantitatively measure dalbavancin in plasma.

Preparation of dalbavancin calibration and quality control standards

After preparing a stock solution of dalbavancin by dissolving dalbavancin in deionized water to prepare a 1000 μ g/ml solution, serial dilutions were made in deionized water to prepare 500, 50 and 10 μ g/ml solutions.

Calibration standards for 100, 60 and 40 μ g/ml dalbavancin concentrations were prepared by peaking human plasma at 1000 μ g/ml dalbavancin stock solutions prepared as described above. Calibration standards at concentrations of 20 and 10 μ g/ml were prepared by peaking human plasma in the appropriate volume of 500 μ g/ml stock solution of dalbavancin, and 0.5 μ g/ml by peaking human plasma in the appropriate volume of 10 μ g/ml stock solution.

Quality control standards for 90 and 30 μ g/ml dalbavancin were prepared by peaking human plasma with a stock solution of 1000 μ g/ml dalbavancin prepared as described above in the appropriate volume. A quality control standard of 1.5. mu.g/ml was prepared by peaking human plasma in the appropriate volume of 50. mu.g/ml solution.

Preparation of internal standard working solution

The 30 μ g/ml internal standard working solution BI-K0098 is a diethyl-amino-propyl-amino derivative of a-40926, prepared by the following procedure. Approximately 10mgBI-K0098 was dissolved in approximately 10ml mobile phase A (80% 10mM ammonium formate/formic acid, pH3(v/v), 10% acetonitrile (v/v) and 10% 2-propanol (v/v)) to give a 1000. mu.g/ml internal standard stock solution. The stock solution (300. mu.l) was then diluted to a volume of 10ml with mobile phase A to give 30. mu.g/ml internal standard solution.

Preparation of analytical samples

Samples for quantitative determination of dalbavancin concentration in plasma were prepared as follows. 100 μ l of internal standard working standard solution was added to 50 μ l of calibration or quality control standard solution prepared as above and mixed. The mixture was allowed to equilibrate at room temperature for 5 minutes, followed by the addition of 250 μ l acetonitrile. The mixture was then vortexed for 10 seconds and subsequently centrifuged at approximately 10,000rpm for 1 minute on ALCmicro-centrifugette 4214. The supernatant was transferred to a clean tube and evaporated to dryness at about 40 ℃ in a Savant Speed-Vac system. The sample was then suspended in 150. mu.l of mobile phase A.

Analytical method

A50. mu.l sample prepared for analysis as described above was injected into a Phenomenex Jupiter C18 column (50X 2mm, C185. mu.m 300A) and analyzed under gradient HPLC conditions at a flow rate of 0.3 ml/min. The gradient conditions were: initially, 80% mobile phase a/20% mobile phase B (20% 10mM ammonium formate/formic acid, pH3(v/v), 40% acetonitrile (v/v), 40% 2-propanol (v/v)); 1 minute, 20% mobile phase a/80% mobile phase B; 2 min, 20% mobile phase a/80% mobile phase B; 2.5 minutes, return to the original conditions.

The HPLC system was coupled to a PE SCIEX API-2000 triple quadrupole mass spectrometer operating the turbine ion spray in positive ionization mode. Air is used to generate a spray of the ion source. The probe temperature was set at 500 ℃ and nitrogen was used as the gas curtain gas. Multiple Reaction Monitoring (MRM) is utilized using nitrogen as the collision gas. Analytes were detected by ion migration: dalbavancin is 909.3Da → 1429.3Da, and internal standard is 923.3Da → 1457.3Da (BI-K0098). To avoid contamination of the mass spectrometer, post column split was performed at 1 and 2.5 minutes from the start of the chromatography operation.

Sample Control Software (Software Sample Control)1.4 was used for acquisition of data analysis and MacQuan 1.6 Software was used for synthesis of chromatogram peaks and statistical data evaluation.

Calibration curve

The linearity of the assay method is evaluated by assaying the calibration standard to generate a calibration curve. The concentration of dalbavancin in plasma samples was determined by calculating the peak area ratio between dalbavancin and the internal standard.

A calibration curve for dalbavancin concentration over the analytical range of 0.5-100 μ g dalbavancin/ml human plasma was constructed using the equation y ═ a + Bx (1/x weighted), where a represents the curve intercept, B represents the curve slope, x represents the dalbavancin concentration (μ g/ml) of the calibration standard, and y represents the peak area ratio of dalbavancin to the internal standard. Three separate calibration curves were constructed. The results show that the ratio of dalbavancin/internal standard area and dalbavancin concentration vary linearly over the analysis range. The lower limit of quantitation (LLOQ) was 0.5. mu.g dalbavancin/ml human plasma. The slope of the calibration curve is reproducible and its correlation coefficient is greater than 0.9995.

Stability of dalbavancin in plasma

The stability of dalbavancin in plasma samples was determined by analyzing three replicate quality control standards of human plasma samples prepared as above at two different concentrations, 1.5 and 90ug/ml respectively. The detectable concentration of dalbavancin was stable after three cycles of freeze-thaw treatment. The concentration of dalbavancin in the treated samples was stable after 24 hours at room temperature. No decrease in dalbavancin concentration was observed for the time zero sample.

Example 11 Dalbavancin Mass Spectrometry

The properties of the dalbavancin multimers in solution were investigated and the conditions affecting the overall ratio of dalbavancin multimers to dalbavancin monomers were determined by electrospray ion mass spectrometry (ESI-MS).

Experiments were performed using an Applied Biosystem API III + mass spectrometer equipped with a TurboIonSpray source, Triple Quadrupole analyzer and operating in cationic mode. Preferred conditions are listed in table 34 below.

Table 34: instrument conditions for dalbavancin analysis on an Applied biosystems API 111+ mass spectrometer.

Dalbavancin solution

The instrument parameters were adjusted for a dalbavancin solution containing 0.1mg/ml dalbavancin in a 8: 2 water to isopropanol solution. Dalbavancin with a spectral range of 500-. The resulting spectrum, as shown in figure 7, indicates the presence of dalbavancin multimers. As a non-limiting example, one trace of the spectrum may be attributed to B ₀As a homopolymer of (2nM + y: (a))⁺3) In the presence of an ionic species, where n is a positive integer, representing the multiplicity of the homopolymer, for example, when the multimer is a homodimer, n ═ 1; and when the multimer is a homotetramer, n is 2, M represents the mass of the monomer, y is n and⁺and 3 represents the three ionic charges added. For example, when n is 1, y is 1 and M is B₀When is of quality, provide B₀A homodimer of (a). Attribution of this dimer species to mass spectra (2M)⁺3) Ion traces.

Effect of dalbavancin concentration on the Overall ratio of dalbavancin multimers to monomers

The effect of dalbavancin concentration on the overall ratio of multimers to monomers was evaluated by mass spectroscopy using the conditions described above. Spectra were obtained by direct injection of dalbavancin solutions at concentrations of 20, 40, 60 and 80 μ g/mL. The main peak intensity was reported as a function of dalbavancin concentration and the overall ratio of dalbavancin multimers to monomers was determined as shown in figure 8.

The data indicate that the overall ratio of dalbavancin multimers to dalbavancin monomers increases with increasing concentration. This may help explain the large loading capacity of drugs that may be administered to an individual. The action of the multimer as a monomer depot may reduce the tendency of high concentration samples to form precipitates and increase the concentration that can be administered to an individual. The presence of multimers may also allow doses of dalbavancin to be administered rapidly to an individual.

For example, as shown in FIG. 7, a non-limiting example of a method of determining the overall ratio of dalbavancin multimers to monomers is provided by determining the ratio between the ionic A and B peak intensities. Dividing the peak a intensity by the peak B intensity provides a measure of the overall ratio of dalbavancin multimers to monomers.

Effect of pH on the Overall ratio of Dalbavancin multimers to monomers

Under the above-mentioned instrument conditions and at the following solution pH values: the effect of solution pH on the overall ratio of dalbavancin multimer to monomer was evaluated at 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5. The overall ratio of dalbavancin multimers to monomers was determined at each pH value as shown in figure 9 and the pH values were plotted as lines. The overall ratio of dalbavancin multimer to monomer was determined to increase with increasing pH.

Without being limited by theory, it is believed that the ionic group (e.g., a carboxylic acid group on a first dalbavancin monomer) contributes to the stabilization of the dalbavancin multimer by forming an ionic interaction with an oppositely charged ion (e.g., a tertiary nitrogen group) on a second dalbavancin monomer. The ionic interaction may be influenced by pH. It is believed that the increased tendency of dalbavancin to exist as a polymer at higher pH values indicates that ionic interactions are important in polymer stability. In particular, it is believed that the dalbavancin multimers are stable at lower pH values, presumably due to interference from ionic interactions that contribute to the stability of the multimers, since specific functional groups (e.g., carboxylic acid groups) can be protonated at lower pH values.

Effect of solution Ionic Strength on the Overall ratio of Dalbavancin multimers to monomers

The effect of solution ionic strength on the overall ratio of dalbavancin multimers to monomers was determined by mass spectroscopy. Finnigan LCQ previously adjusted and calibrated in electrospray mode using Ultramark 1621, caffeine and MRFA (L-methionyl-arginyl-phenylalanyl-arginine)^DecaMass spectra were acquired on an ion trap instrument in electrospray anode mode. All mass spectra were recorded using the conditions listed in table 35. The sample parameters to be investigated are listed in table 36.

TABLE 35 MS conditions

TABLE 36 sample parameters

The sample aqueous solution was injected via a Harward syringe pump at a rate of 10. mu.L/min and a mass spectrum as shown in FIGS. 10-12 was obtained.

The mass spectra obtained indicate that the overall ratio of dalbavancin multimers to monomers is affected by ionic strength. An increase in buffer concentration was found to correspond to a decrease in the mass trace of the multimer and thus a decrease in the ratio of the dalbavancin multimer to the monomer population.

As described herein, ionic interactions are believed to be important in the stability of the dalbavancin multimers. The fact that increased ionic strength is associated with decreased polymer mass trace strength confirms the role of ionic interactions in polymer stability. However, multimer stabilization may involve another second interaction due to the still existing multimer mass trace at higher ionic strength.

Without being bound by any theory, it is believed that hydrophobic interactions are important in stabilizing dalbavancin multimeric species. If the stability of the non-covalent dalbavancin multimer is due solely to ionic interactions, it is expected that an increase in ionic strength will result in a total loss of the multimeric mass species. In other words, it is expected that as the ionic strength of the solution increases, the ionic interactions that stabilize the multimers will be destroyed as the increased total number of ions in solution will be more readily associated with their monomers. Thus, the solution ionic strength will cause the multimer to break down into monomer components and the resulting mass spectrum will not contain any multimer mass traces. However, even at high solution ionic strength (e.g., 100mM ammonium formate), the presence of dalbavancin multimers can be detected in the mass spectrum. Thus, it is believed that the dalbavancin multimers are stabilized, at least in part, by hydrophobic interactions.

Compounds of similar structure

It is believed that the improved efficacy of dalbavancin is due, at least in part, to its ability to form multimers. Even compounds with very similar structures are not considered to have the unique features described, and the ability of compounds with chemical structures similar to dalbavancin to form multimers was investigated by mass spectrometry. Mass spectra of electrospray anode mode were obtained using Ultramark 1621, caffeine and MRFA (L-methionyl-arginyl-phenylalanyl-arginine) on a finnigan lcqdeca ion trap instrument previously tuned and calibrated in electrospray mode. All mass spectra were recorded using the conditions listed in table 37. The sample parameters investigated are listed in table 38. The sample aqueous solution was injected via a Harward syringe pump at a rate of 10. mu.L/min and a mass spectrum as shown in FIGS. 13 and 14 was obtained.

TABLE 37 Mass Spectrometry conditions

TABLE 16 sample parameters

n.a. ═ unregulated

Similar glycopeptide antibiotics (teicoplanin) did not show multimeric complexes in solutions of various concentrations. This result supports an indication that structurally similar compounds are not able to form multimeric species in solution, and that this phenomenon may play an important role in dalbavancin activity.

Example 12 matrix-assisted laser Desorption/ionization time of flight (MALDI-TOF) Mass Spectroscopy of protein-dalbavancin complexes

Mu.l HSA, 0.150mM and 10. mu.l dalbavancin solution (from 0.075mM, 0.15mM, 0.3mM to 1.5mM) were mixed and incubated at 37 ℃ for 60 minutes. Samples for analysis were prepared using the dry droplet technique. Mass spectra were obtained on a BRUKER FLEX III, time-of-flight mass spectrometer previously adjusted and calibrated using standard bovine serum albumin, and 200 laser emission generated mass spectra were acquired and averaged. Matrix: 9 parts of DHB-9(2, 5-dihydroxy-benzoic acid) are saturated in acetonitrile/H2O (50: 50) and 1 part of sinapic acid is saturated in acetonitrile/H2O (50: 50). 0.5. mu.l of the sample solution was mixed with 0.5. mu.l of the matrix solution and placed on the laser target.

Dalbavancin binds to protein (1HSA +1 dalbavancin) as a monomer. At very high ratios of dalbavancin protein (1: 2, 1: 10), the presence of a complex containing 2 molecules of dalbavancin/molecular protein was observed.

Example 13 isothermal titration calorimetry of dalbavancin binding to N, N' -diacetyl-Lys-D-Ala-D-Ala in the Presence of human serum Albumin

Binding of dalbavancin to N, N' -diacetyl-Lys-D-Ala was investigated by Isothermal Titration Calorimetry (ITC) in the presence of a range of concentrations of HSA (up to 600 μ M) at 25 ℃, which is a peptide analogue of the cell wall target of dalbavancin, and some additional measurements were performed at 37 ℃. HSA increases the solubility of dalbavancin and decreases its binding affinity to tripeptide ligands. The results were compared to those of vancomycin. The observed effect remains stable at relatively low HSA concentrations, consistent with an uncompetitive binding model that allows ligand binding to dalbavancin (both in solution in free state) and (weaker) to the dalbavancin-HSA complex.

Preliminary experiments demonstrated that dalbavancin and vancomycin show similar binding curves in the presence of serum proteins: both exothermically after binding to N, N' -diacetyl-Lys-D-Ala-D-Ala, but there is no evidence of binding to the dipeptide (D-Ala-D-Ala) or Lys-D-Ala-D-lactate. Data for dalbavancin/tripeptide interaction with K _dissBinding was consistent from 1-10 μ M (depending on temperature), similar to vancomycin under the same conditions. In the presence of HSA, the solubility of dalbavancin is significantly increased and the binding affinity to tripeptides is reduced in a manner consistent with competitive or non-competitive binding of antibiotics to HSA. The experiments described in this example were designed to: (a) comparing the measured values of dalbavancin/tripeptide at different temperatures (25 and 37 ℃) and different HSA concentrations; (b) the data is used to build a binding model to compare with observed numbers.

Dalbavancin was provided by Biosearch Italia. Other reagents were from Sigma: vancomycin hydrochloride (Sigma V-2002, fw 1485.7), N' -diacetyl-Lys-D-Ala-D-Ala (Sigma D-9904, fw 372.4), human albumin (HSA; SigmaA-3782; mw 69,366).

The antibiotics and peptides were dissolved in an aqueous buffer containing HSA (20mM sodium phosphate, 150mM NaCl, pH 7.4) with gentle stirring immediately before the experiment. Peptide concentration was determined gravimetrically. Using Moore mattingCoefficient ∈ 12430 (dalbavancin, a)₂₈₀ ^1％68.42), epsilon 280-6690 (vancomycin) the concentration of dalbavancin was determined by weight or by UV absorbance. By UV absorption (HSA,. epsilon.) ₂₈₀＝37,700；A₂₈₀ ^1％5.44) HSA concentration was measured. Spectra were recorded at room temperature using a Shimadzu UV-160A or UV-1601 spectrophotometer in a 1cm pathlength quartz cuvette, given as requiredRange of absorbance, the sample is quantitatively diluted with buffer.

Isothermal titration calorimetry was performed using a Microcal VP-ITC instrument at 25 ℃ and 37 ℃ using standard operating procedures. See, e.g., Wiseman et al, anal. biochem. (1989)179, 131-; cooper, et al, philios. trans. r.soc. lond.ser.a-Math phys. eng Sci (1993)345, 23-35; cooper, A, Isothermal tracking Microcalorimetry in C.Jones, B.Mulloyand A.H.Thomas (Eds.), Microcopy, Optical Spectroscopy, and Macro techniques, human a Press, Totowa, NJ, (1994) p.137-; cooper, a., Microcalorimetry of Protein-Protein interactions in j.e.ladburg and b.z.chowdry (Eds.); biocalcometry: the Applications of calibration in The biological sciences.Wiley, (1998) page 103-111; and Cooper, a., curr, opin, chem, biol. (1999)3, 557-563. The samples were carefully degassed prior to loading to avoid the formation of air bubbles in the calorimeter cell. Each experiment typically involved injecting 25X 10. mu.l peptide solution (. apprxeq.1 mM) into a calorimeter cell (volume. apprxeq.1.4 ml) containing antibiotic solution (. apprxeq.20-100. mu.M). Control experiments involved injecting ligand into buffer under the same conditions to determine the heat of peptide dilution, and the values were used for the correction of the raw binding data prior to analysis. The dalbavancin/tripeptide binding experiments were repeated several times at each temperature. ITC binding data were analyzed using standard Microcal ORIGIN software to determine the apparent number of binding sites (N), binding affinity (K) _ass＝1/K_diss) And enthalpy of binding (Δ H).

Watch 39

Thermodynamic data for tripeptide binding to dalbavancin and vancomycin were determined by ITC postulated a simple non-synergistic binding model: temperature and HAS effects.

The binding ITC experiment of tripeptides to dalbavancin in the absence of HSA gave consistent data in terms of binding affinity, with mean Kdiss in the interval of 1.4, 3.1 and 8.4 μ M at 10, 25 and 37 ℃ respectively (table 39). The bonding is exothermic. The concentration calculations here indicate that N is closer to 0.5 under the conditions described. This is consistent with the binding of one tripeptide molecule per dalbavancin dimer under the conditions described. The binding affinities and enthalpies of binding were comparable to those observed for vancomycin under the same conditions (Table 39, and D.McPhail, A.Cooper, J.chem.Soc-Faraday Trans. (1997)93, 2283-. It was also noted that vancomycin undergoes ligand-induced dimerization at higher concentrations.

Although the binding is clearly more exothermic, the addition of HSA to the dalbavancin mixture decreased the apparent binding affinity of the tripeptide to dalbavancin (table 39). The concentration dependence of HSA on this at 25 ℃ is shown in FIG. 15. Apparent K_dissInitially elevated (faded down) until 35 μ MHSA, remained relatively constant for higher HSA concentrations (600 μ M) approaching physiological levels. Plateau at high HSA concentrations (K) _diss35 μ M) corresponds to a binding affinity that is about 10-12 times weaker than in the absence of HSA. Similar reductions were observed at 37 ℃.

The HSA effect is not due to interaction with tripeptides. Binding of vancomycin to tripeptides in the presence of HSA the control ITC experiment gave results comparable to those observed in the absence of HSA (see table 39). This indicates that neither the peptide nor the closely related antibiotic vancomycin interacts with HSA in solution. It can be concluded that any effect of HSA on the dalbavancin/tripeptide interaction must be attributed to the interaction of HSA with dalbavancin.

While not wishing to be bound by theory, the above data are consistent with a non-competitive binding model. This model assumes that the tripeptide ligand (L) can bind to dalbavancin (D) in the free state and in the dalbavancin-HSA complex (possibly with different affinities).

K_L＝[D][L]/[DL]

K_HSA＝[D][HSA]/[D.HSA]

K_LDHSA＝[D.HSA][L]/[LD.HSA]

Apparent (observed) ligand binding dissociation constant (non-competitive)

K_app，LIs [ total D ]][L]/[ Total DL Complex]

＝([D]+[D.HSA])[L]/([DL]+[LD.HSA])

＝K_L{1+[HSA]/K_HSA}/{1+[HSA]·K_L/K_HSA·K_LDHSA}

This shows K_app，LThe hyperbolic dependence on the free HSA concentration, which corresponds well with the observed data (fig. 15). At high [ HSA ]]This then reaches an asymptotic (plateau) value.

K_appL＝K_LDHSA(for large [ HSA)])

This indicates that the binding affinity of dalbavancin to tripeptides when bound to HSA is about 35 μ M compared to 3 μ M for free dalbavancin (25 ℃ panel).

Other mechanisms may be suggested depending on whether dalbavancin acts as a monomer or dimer in the interaction with a peptide or protein. Direct comparison of dalbavancin with vancomycin (which showed clear 1: 1 binding at the low concentration) showed complete binding of dalbavancin at the lower molar ratio (lower N) (figure 16). This is in contrast to 2: 1 dalbavancin: the peptide complexes were identical.

However, the apparent N value increased in the presence of HSA (table 39) and could be more consistent with 1: 1 complexation. While not wishing to be bound by theory, the model shown in fig. 17, which shows the possible interaction of dalbavancin monomers and dimers with tripeptide ligands and HSA, is consistent with this observation. Fig. 17A depicts dalbavancin in solution in monomer-dimer equilibrium (mainly as a dimer), but bound as a monomer to two separate sites on HSA. This is consistent with the 0.5 value observed for binding of dalbavancin by ITC to serum protein (example 3). Figure 17B depicts ligand binding to dalbavancin dimer in solution and (more weakly) to dalbavancin monomer linked to HSA. This is consistent with the non-competitive binding of dalbavancin to tripeptides and HSA at variable apparent stoichiometry.

In summary, this example shows that HSA reduces the binding affinity of dalbavancin to the tripeptide ligand in a manner consistent with a non-competitive mechanism, and that dalbavancin binding to HSA retains its ability to bind the tripeptide ligand, albeit with reduced affinity. The results are also consistent with a model in which dalbavancin is in monomer-multimer equilibrium (mainly multimers) in solution, where the multimers have strong affinity for the peptide ligand. Monomers of dalbavancin, both free and bound to serum albumin, may also reduce affinity binding to peptides.

Example 13 preparation of A-40926 with dalbavancin

Preparation of A-40926

The native glycopeptide A-40926 produced by fermentation is the starting material for the preparation of dalbavancin. It was produced as a mixture of A-40926 and its acetyl derivatives by Nonomurasp ATCC 397727 (see U.S. Pat. No. 4,935,238 and B. Golstatin et al, Antimicrobial Agent and Chemotherapy, Dec.1987, pp.1961-1966). The acetyl derivative is first deacetylated to A-40926. The deacetylation is followed by purification of A-40926 by polyamide column chromatography as described below. The following description represents the current production process. The amounts reported here are about 1/4 of the amounts typically operated in commercial preparations.

Deacylation of A-40926

While stirring, the mixture was adjusted to 10m with NaOH 30%³The fermentation broth (23 ℃) containing a total of about 1g/LA-40926 and its acetyl derivatives is at pH 11.4. Stirring was continued for 6 hours, then the temperature was reduced to 15 ℃ and the broth (0.12 m with 0.1 μ) was microfiltered³Koch Protosep IV microfilter for ceramic membranes). Water is continuously added to the retentate during microfiltration to obtain 20-25m at the end of the process³The percolate and 4.5-5m³The retentate (half of the starting value).

The exudate solution containing A-40926 was analyzed by HPLC. When the deacetylation was complete, the pH of the exudate solution was adjusted at pH 7 (stored at 20 ℃) with 30% sulphuric acid. In this example, 25m was obtained³A filtered broth containing 6.62kg of A-40926(268 mg/L). The deacetylation yield was 66.2%. If the microfiltration process is carried out for a longer time and the extraction volume used in the process is higher, the yield can be increased to 90%.

Purification of A-40926 on a Polyamide column

After extraction, the A-40926 contained in the filtered broth is purified on a polyamide column as described below. The amounts reported in this description are about 1/10 of the amounts typically operated in commercial production and represent current production methods.

500L of polyamide resin SC6 (from Macherey Nagel) was suspended in demineralized water and loaded into the column. The resin was then adjusted to pH 6-6.5 by eluting the column with at least 2BV (bed volume) of a buffer solution prepared by dissolving 4kg of sodium carbonate in 800L of water and adjusting the pH of the resulting solution with acetic acid.

A-40926 filtered broth (9000L; assay rate 0.275 mg/L; A-409262475 g; pH 6. + -. 0.2; temperature 10. + -. 3 ℃ C.) was loaded into the column at a rate of about 5g activity per liter of resin (typically an activity/resin ratio of 5-8g/L was used). The column was washed with the following solutions: 3BV (1500L) of a solution at pH 6 prepared by dissolving 7.5kg of sodium carbonate in 1500L of demineralized water and adjusting the pH with acetic acid; 4BV (2000L) of a solution at pH 8, prepared by dissolving 10kg of sodium carbonate in 2000L of demineralized water and adjusting the pH with acetic acid; 1.5BV (750L) of a solution at pH 9, prepared by dissolving 4kg of sodium carbonate in 750L of demineralized water and adjusting the pH with acetic acid.

A-40926 was recovered from the column by eluting with 4BV (2000L) of a buffer solution at pH 10 prepared by dissolving 10kg of sodium carbonate in 2000L of demineralized water and adjusting the pH with acetic acid. Collecting the fraction containing purified A-40926 (A-40926 concentration is greater than 0.5g/L and main component (B) ₀+B₁) HPLC area% of greater than 80%), neutralized with 1N HCl and analyzed by HPLC. About 2000L of final clear solution was obtained.

The resin used for purification was regenerated with a 1: 1 mixture of isopropanol/5% Na OH at 1.5BV, followed by washing with 5BV of demineralized water.

A-40926 concentration

The solution from the column is subjected to several rounds of dilution/concentration steps to eliminate most of the inorganic salts of the solution. The solution was concentrated to 80L by nanofiltration using a membrane cut to 250D, diluted with 80L of demineralized water, and re-concentrated by nanofiltration at the starting column (80L). This operation was repeated at least 5 times. The pH of the final solution (80L, pH 7.5) was adjusted to 6.3 with 23% HCl. The solution was then diluted with 80L of acetone and its pH was readjusted to pH 2.6 with 23% HCl.

Decolorization of

680g of charcoal PA 200C (. about.0.3 g/g A-40926) was added to the solution (160L) obtained in the above step while stirring. Stirring was continued at room temperature for at least 30 minutes, followed by the addition of about 0.5 to 0.6Kg of filter aid (DIF-BO). The mixture was filtered through a filter cartridge. The clear solution obtained was concentrated in vacuo (45 ℃) to bring the acetone below 10%. The final volume was about 100L. The pH was then adjusted to 6.7 with aqueous NaOH and the concentration step was continued using conventional nanofiltration until the concentration of A-40926 was about 100 g/L. 20L of concentrated solution (A-409261884g, 94.2g/L) was obtained.

Precipitating and drying

The previous solution was diluted with 20L of acetone while stirring and its pH was maintained at 5.1 adjusted with 10% HCl. To this solution was added an additional 5 volumes of acetone (100L) to complete the A-40926 precipitation. If the water content is < 15% at this point, additional acetone is added. After 2 hours the suspension was centrifuged and the solid was washed with 3X 10L of fresh acetone. The mother liquor was analyzed and drained after confirming the absence of product.

The solid A-40926 was dried in a static dryer at 30-35 deg.C and under reduced pressure until the residual acetone was less than 2% and the water was less than 10%. The product was then sieved through a 50 mesh screen to obtain 2.08kg of purified A-40926(HPLC assay 81.4%, water 6.2%, sulfated ash 4.8%). The yield starting from activity on the column was 68.4%.

Synthetic dalbavancin

By Malabarba and Donadio (1999), Drugs of the future, 24 (8): 839-846 Synthesis of dalbavancin (BI-397) from the native glycopeptide A-40926. In particular, A-40926 is first subjected to an esterification step to produce MA, which is then subjected to an amidation step to produce MA-A-1. The final hydrolysis step is followed by conversion of MA-A-1 to dalbavancin.

Esterification step (step 1)

The following description represents the current approach in use.

Preparation H₂SO₄96%/MeOH (solution A)

In a 15L round bottom flask equipped with a mechanical stirrer and thermometer, 2.28L H was placed₂SO₄96% (per kg of A-40926 powder-300 mL H₂SO₄96%) was added dropwise to 7.9L MeOH. The temperature was maintained between 0 and 5 ℃ using an external ice bath.

Reaction process

Starting material A-40926(7.6 kg; batch No. 019, detection rate 85.09%; activity 6.46 kg; 3.73mol) was suspended in MeOH (46L) in a 140L glass-lined reactor and the resulting suspension was cooled at 0 ℃. + -. 2 ℃. The suspension was treated at this temperature with solution A (H2SO4/MeOH) previously prepared. The resulting solution was stirred at 0 ℃ for 22-26 hours while monitoring the reaction by HPLC analysis every two hours (reaction aliquots were diluted 100-fold in a 1: 1 acetonitrile/water mixture). Esterification is considered complete when the residual a-40926 is less than 5% and the diester does not exceed 10% calculated as HPLC area%.

Ester (MA) isolation

When the reaction was complete, the mixture was cooled at-5 ℃ (+/-2 ℃) and diluted with the same volume of cold water (54L) while keeping the temperature below 5 ℃. The product (MA) was precipitated by adjusting the pH of the solution at 5.5(+/-0.2) by slow addition of 10.2L Triethylamine (TEA). Stirring was continued for an additional 1 hour at 0-2 ℃ followed by centrifugation of the solid obtained, washing with water (10L per kg of A-40926) and finally with MeOH previously cooled at 10-15 ℃ (3L per kg of starting A-40926). Water was washed before removing sulfate from MA.

The mother liquor and wash solutions were analyzed separately and drained if they contained less than 1-2% activity. The product was dried under vacuum (50mmHg) at 35-40 ℃ (external temperature) until residual water was less than 10%. 7.6kg of MA were obtained as a light brown powder (5.63kg activity, 3.23 mol).

Analysis showed the following values of HPLC area%: MA 89.8.

A-409263.2, diester derivative 5.9. The HPLC detection rate is 74.2 percent, and the activity is 5.637 Kg; 3.23 mol; the yield was 86.5%. This material was used in the next step without further purification.

Amidation step (step 2)

[004421 the following description represents the current production method.

Preparation of a DMSO/HCl mixture (solution B)

DMSO (1.6L) was placed in a 10L round bottom flask equipped with a mechanical stirrer and thermometer and cooled in an ice bath below 10 ℃. HCl 37% (1L) was then added slowly while stirring, keeping the temperature of the mixture below 25 ℃.

Amidation Process (production MA-A-1)

5.95kg of the starting material MA (assay rate 76.3%, KF 8.9%; 2.68mol) were slowly dissolved in 19.2L of a 1: 1DMSO/MeOH mixture (1.6L DMSO vs. 1.6L MeOH per kg MA powder) at room temperature while stirring. After stirring for 1 hour 709mL of 3- (dimethylamino) -propylamine (DMEPA, MW 102.1; density 0.812 g/mL; 5.63 mol; 1.96mol per mol of starting MA) and 325g of 1-hydroxybenzotriazole hydrate (HOBTH 20; MW 153.1; 2.04 mol; 0.71mol per mol of starting MA) were added to the reaction mixture. Stirring was continued until a complete solution was obtained, then the mixture was adjusted at pH3-3.1 by slowly adding about 2.0L of solution B (DMSO/HCl) (measured after diluting the reaction aliquot 10-fold with water).

Dicyclohexylcarbodiimide (DCC) solution (prepared by dissolving 1.03kg of DCC (4.99 mol; MW 206.3; 1.74mol per mol MA) in 4.1L of a 1: 1DMSO/MeOH mixture) was added to the stirred reaction mixture over 10 minutes. Stirring was continued for 5 hours, then an additional 51.5g of solid DCC (0.25mol) was added to the reaction mixture until the residual MA was reduced to less than 5%, while maintaining the isourea level below 4-5%. Isoureas are a group of by-products resulting from the further reaction of dalbavancin with excess DCC.

The reaction is generally complete after an additional 2 hours (7 hours total). At the end the mixture was diluted with water (60L) to reduce the DMSO concentration to 15% (v/v) and adjusted to pH 2.3 with HCl 1N (0.85L) to remove any residual DCC.

Hydrolysis of MA-A-1 into dalbavancin (step 3)

After 30 minutes the mixture was adjusted to pH with 15% NaOH (8L)

12.0-12.1. Stirring was continued for 4 hours while maintaining the mixture at this pH by adding a small amount of NaOH 15%. Thereafter the residual MA-A-1, calculated as HPLC area%, was less than 0.2%.

The mixture was then acidified with 1N HCl (19L) at pH 3.0 and the suspension was filtered to remove the dicyclohexylurea formed. The solid filter cake was washed on the filter with demineralized water (2X 20L). The washing liquid was collected together with the filtrate to obtain a clear solution, which was analyzed by HPLC. 152.8L of a solution containing 21.74g/L of dalbavancin were obtained (total activity 3322 g; 1.828mol, yield 68.2%).

Purified dalbavancin

The following description represents the current production process.

The 152.8L of solution obtained from the hydrolysis step and containing 3322g of dalbavancin activity was divided into two portions and each portion was purified separately on the same column containing 400L of polyamide. The activity/resin ratio in the two purification runs was 4.3 and 4.0g/L, respectively.

Polyamide column preparation

Glass-lined columns (internal diameter 40cm, h 320cm) containing 400L of polyamide resin were cleaned according to the resin regeneration procedure (see below) and conditioned at 2BV (800L) with demineralized water acidified with 4L AcOH (pH 3.2).

Purifying the first fraction

With H₂O (56L) diluted the first 76.4L of the starting solution to reduce the DMSO content below 5% (V/V) and acidified to pH 2.78 with 1N HCl (3.4L). This solution was then loaded onto the column at a flow rate of 150L/h. After loading the resin was washed with the following solutions: 4BV (1600L) H acidified with AcOH (8L)₂O, pH 3.2; 5BV (2000L) AcONa 0.1M, pH 8.2; 1BV (400L) H acidified with AcOH (1L)₂O, pH 3.2. H acidified at 4BV (2400L) with AcOH (6L) and pH 3.4₂O/MeOH (8: 2) eluted dalbavancin.

22 portions of each 50-60L were collected in the elution step and analyzed by HPLC. 9 to 25 parts (80% HPLC area of dalbavancin concentration higher than 0.5g/L and (B0+ B1)) were collected together, thus obtaining 969L of a solution with 1.56kg dalbavancin (yield 93.9%). This solution was then concentrated by nanofiltration, thus obtaining 125.7L of a solution with 1.38kg of dalbavancin. 850L of exudate solution containing 145g of impure dalbavancin (8.7%) was neutralized and discharged.

Resin regeneration

The resin was washed with the following solution before re-use: 2.5BV (1000L) of 1: 1 MeOH/water acidified with acetic acid (2.5 mL/L); 2.5BV (1000L) 1: 10.5% NaOH/isopropanol; 10BV (4000L) of demineralized water. The resin was then re-equilibrated with 2BV (800L) of water acidified with acetic acid (2.5 mL/L).

Purifying the second fraction

With H₂O (56L) diluted the second portion of the starting solution (76.5L) from the hydrolysis step to reduce the DMSO contentLess than 5% (v/v) and acidified to pH 2.87 with 3.0L 1N HCl. The second fraction is then purified as described in the purification of the first fraction. The collected fractions (volume 972L, 1.54kg dalbavancin, yield 92.7%) were concentrated by nanofiltration to obtain 133L of a solution with 1.46kg activity. 850L of exudate solution containing 73g of dalbavancin (4.3%) was discharged.

The concentrated solutions from both purification steps were reanalyzed and collected together to yield 258L of a solution containing 2840g of purified dalbavancin. The purification yield was 86%. The overall yield starting from MA was 58.3%.

Final polyamide regeneration

After the second purification run, the polyamide was regenerated with 2.5BV of 1: 1 MeOH-water acidified with AcOH (2.5L) pH 3.4, 2.5BV of 1: 10.5% NaOH-isopropanol, 10BV of demineralized water.

Decolorization and precipitation of dalbavancin

1.5mol 1N HCl per mole of dalbavancin and 0.3g charcoal CG1(0.85kg from NORIT) per gram of dalbavancin were added to the 258L solution obtained above. The mixture was stirred at room temperature for at least 45 minutes. The pH was 3.1. The suspension was then filtered over a SUPRA DISC cartridge. SDP-EK1 from SEITZ-SCHENK was washed with the filter cake with 50L H2O/MeOH 8: 2. The filtrate was analyzed and re-concentrated by nanofiltration using MPS 44 membrane with a cut-off value of 250D. 21.3L of a concentrated solution (pH 4.1; MeOH 1.9%, GC) containing 119g/L of dalbavancin were obtained. 909mL of 1N HCl was finally added to adjust the pH at 2.63, which corresponds to a salification ratio of 1.65mol HCl/mol dalbavancin.

The solution (22.2L) was poured into 200L of acetone while stirring. The solid obtained after decantation was centrifuged and washed with 14L of fresh acetone. The product was then dried under reduced pressure (50mmHg) at 35 ℃ while maintaining the internal temperature below 30 ℃ for 17 hours. During the drying process, 1L of pyrogen-free water (< 250EU/mL) (divided into two portions of 0.5L each) was sprayed onto the solids after 3 and 5 hours to remove residual acetone that would otherwise be difficult to eliminate. The product was then sieved (50 mesh) to obtain 2592g of dalbavancin (HPLC assay rate 82.4%; water (KF) 14%; C1-3.0%).

Example 14 alternative Process for the preparation of A-40926 with dalbavancin

The following method is an alternative method that can be used in the preparation of A-40926 and dalbavancin.

Preparation of A-40926 on XAD-7HP

Deacetylation and mycelium microfiltration

150L of a fermentation broth containing A-40926 (pH7) are stirred at room temperature (24 ℃) in a suitable reactor and brought to pH11.5 with 2.5N NaOH solution (2.5L). After stirring for 4 hours, the broth was adjusted to pH 10.6 with 15% HC1 and microfiltered through a 0.2 micron membrane. 439L of clear permeate was collected and then concentrated by nanofiltration using MPS 44 membranes with a critical value of 250D. The concentrated solution of A-40926 obtained (58.6L; 3.89g/L) was adjusted to pH 6.4 and stored at 4 ℃ until use.

Column preparation and purification

XAD-7HP resin (8L) was suspended in a 1: 1 water/methanol solution, filtered and loaded into an appropriate glass column (12 cm internal diameter) with a peristaltic pump.

The resin was then washed with water and equilibrated with 6BV of aqueous sodium carbonate solution buffered at pH 6, prepared by dissolving 5g of sodium carbonate per liter of water and adjusting the pH with acetic acid.

A portion of the concentrated broth containing 194g A-40926 was loaded into an XAD-7HP column. The resin was then washed with the following two buffered solutions at a flow rate of 1/2 BV/hr to eliminate a portion of the hydrophilic and colored species present: 3BV (24L) of 0.5% aqueous acetic acid, adjusted to pH 5 with 30% sodium hydroxide; a5 BV (40L) 8: 2 water/acetone mixture, acidified with 5mL of acetic acid/L water.

A-40926 was finally eluted at 8BV (64L) with a 1: 1 water/acetone mixture acidified with 5mL of acetic acid/L water. 16 portions of 4L each were collected. The concentrated fractions (5 to 15) in which the concentration of A-40926 was greater than 0.5g/L were pooled together to obtain a solution (43L, 3.8g/L) containing 163.4g of A-40926. The column yield was 81.3%. The other fraction (200L) containing A-40926 of 0.23g/L (45.3 g; 22.2%) having poor purity was discharged.

The resin was regenerated after elution with a 6BV (55L) NaOH 0.5%/isopropanol (1: 1) mixture and finally washed to neutrality with 10BV water.

Carbon treatment

The collected fractions were adjusted to pH 2.5 with HCl 37% (70mL) and then decolorized with 50g of charcoal PA 200 form (0.3g/g A-40926). The resulting suspension was stirred at room temperature for 2 hours and then filtered through a KS 50 filter (d 25cm, time 2.5 hours) to obtain 45.6L of a yellowish a-40926 solution (3.5 g/L; yield 96.4%).

Concentrating

The decolorized solution was adjusted at pH 7 with NaOH 30% (230mL) and concentrated by nanofiltration and ultrafiltration. The use of this technique is important for eliminating hydrophilic species detected on HPLC chromatograms at Rt 2-4 minutes. When the retentate was concentrated to the starting volume of 1/10(4L), the same volume of water was added and the resulting solution was concentrated again. This concentration/dilution step was repeated three times to reduce the residual acetone to 0.25%. The final solution was analyzed by HPLC (2.2L, 146.3g A-40926, 66.5g/L, 91.5% yield). The purification yield was 75.4%.

A-40926 crystal

300mL portions of A-40926 solution (19.9g A-40926) were further concentrated to 100mL by using a laboratory scale ultrafilter and then heated at 60-65 ℃. The pH of this solution was adjusted (30% Na0H) to 7 and 1.2mL of a 5: 1 acetone/isopropanol mixture was added dropwise to each mL of the concentrated solution at this temperature. The resulting mixture was allowed to cool at 20 ℃. After 1.5 hours, the solid obtained is filtered, washed on the filter with acetone and dried at 40 ℃ for 15 hours. 20.6g of product were obtained (HPLC assay rate 82.0%; A-4092616.9 g). The precipitation yield was 84.9%. The overall yield starting from the filtered broth was about 64%.

Preparation of A-40926 on CG-71

Column preparation

CG-71 resin (350mL) was poured into a glass column (inner diameter ═ 4cm) and washed with water. The resin was equilibrated with 3BV of sodium carbonate solution prepared by dissolving 5g of sodium carbonate in water with acetic acid to pH 6. 250mL of fermentation broth (pH 7) containing 14.7g A-40926 was loaded onto a column (42g/L resin). The resin was washed with the following three solutions: 1050mL (3BV) of aqueous sodium carbonate solution (5g/L) adjusted to pH 6 with acetic acid; 1750mL (5BV) of aqueous sodium carbonate solution (5g/L) adjusted to pH 8 with acetic acid; 3150mL (9BV) of aqueous sodium carbonate solution (5g/L) adjusted to pH 9 with acetic acid.

Followed by extraction of the activity with 10BV of demineralized water. 2O portions, each 500mL, were collected. From 12 to 15 parts were collected together to obtain 2.2L of a purified solution containing 11.7g A-40926 (yield 79.6%). This solution was then concentrated by ultrafiltration and the concentrated solution was further diluted with demineralized water and ultrafiltered again. The obtained solution was further concentrated to 50mL under reduced pressure.

A-40926 crystal

The concentrated solution was heated at 60 ℃ and treated with a 5: 1 acetone/IPA mixture (60mL) while stirring. The mixture was then slowly cooled at room temperature. The solid obtained was filtered, washed on the filter with acetone and dried in vacuo at 35 ℃ for 80 hours. 8.9g of purified A-40926 was obtained (HPLC assay 84.2%). The overall yield was 51%.

Alternative amidation step in the synthesis of dalbavancin using N-methyl-2-pyrrolidine (NMP) as solvent

The MA mixture was added portionwise to a 1: 1NMP/MeOH mixture (64mL) while stirring. Stirring was continued at 20-25 ℃ until complete solution, followed by addition of DMEPA (2.42 mL; 1.96mol/eq MA) and HOBT (1.06 g; 0.71mol/eq MA). The pH of the reaction mixture (examined for samples diluted 1: 10 with water) was adjusted to 3.0 with 9.37mL of 15% HCl in NMP (previously prepared from 34.0mL of HCl 37% dissolved in 57.7mL of NMP). A solution of DCC (3.17 g; 1.57mol/eq MA) in NMP/MeOH 1: 1(12.7mL) was then added while stirring. The reaction was monitored by HPLC. The reaction was complete after about 6 hours (MA-A-188.9%, MA 7.3%, ISO 3.7%). This experiment shows that NMP can be a convenient alternative to DMSO for amidation reactions. The whole process is not affected by this solvent change and the final dalbavancin obtained is chemically equivalent to other batches.

An alternative method for the preparation of dalbavancin: single pot process

10g A-40926 complex (HPLC titer 80.66%, 4.6mmole) was suspended in 24mL MeOH while stirring at room temperature in a 100mL glass reactor. The mixture was cooled at 0 ℃ and a solution of 4g HCl (g) in 16.4mL MeOH was added to complete the product dissolution. The temperature was then raised to 20 ℃ while stirring was continued for an additional 24 hours.

Thereafter 40mL of DMSO and 0.4g of HOBT were added to the reaction mixture.

Followed by the addition of 1, 1-dimethylaminopropylamine, thereby adjusting the pH of the resulting reaction mixture to between 3-3.1 (measured after diluting the sample to 9: 1 with water). Then 1.8g of solid DCC was added and stirring was continued for an additional 15 hours. At this point the reaction mixture was transferred to a 1L glass reactor and diluted with 80mL of water. The pH was then brought to 12 by adding 240mL of 15% NaOH. Stirring was continued for an additional 60 minutes and the mixture was acidified at pH 2.8 with 260mL of 15% aqueous HCl. About 800mL of a final clear solution containing 6.4g of dalbavancin was obtained (yield 76%).

HPLC analysis showed that the profile of the product obtained was comparable to that obtained with other manufacturing methods.

N¹⁵，N¹⁵-dialkyl antibiotic compounds

The invention provides N for use, e.g., in the prevention and/or treatment of microbial infections in mammals¹⁵，N¹⁵-a dialkyl antibiotic compound. For convenience, in the description herein, the dalbavancin compounds are numbered according to U.S. patent No. 5,750,509 and are depicted in fig. 26.

In certain embodiments, the present invention provides N of formula (III)¹⁵，N¹⁵-a dialkyl antibiotic compound, or a pharmaceutically acceptable salt or solvate thereof:

in certain embodiments, the present invention provides N of formula (IV)¹⁵，N¹⁵-a dialkyl antibiotic compound, or a pharmaceutically acceptable salt or solvate thereof:

formulas (III) and (IV) provide the N of the present invention¹⁵，N¹⁵-a peptide core of a dialkyl antibiotic compound. This peptide comprises 7 amino acids with the amino terminus on the right, as shown in formulas (III) and (IV), and the carboxy terminus on the left. According to this aspect of the invention, N¹⁵，N¹⁵The dialkyl antibiotic compound being at the N or N terminus¹⁵The above contains two alkyl substituents. In other words, in the formulae (III) and (IV), R¹And R¹' is an alkyl group.

In a preferred embodiment, R¹And R¹Is' a C_1-4An alkyl group. In certain embodiments, R¹And R¹' are each independently selected from propyl, ethyl and methyl. In further embodiments, R ¹And R¹' are each independently selected from ethyl and methyl. In a more preferred embodiment, R¹And R¹' at least one is methyl. In the most preferred embodiment, R¹And R¹' is methyl.

The remainder of the molecule can be modified in the same manner as dalbavancin compounds known to those skilled in the art. Accordingly, X is an aminoalkylamino group as defined in the previous section. Typical aminoalkylamino groups are described in U.S. patent No. 5,750,509. In certain embodiments, X is N, N-dimethylaminopropylamino.

Accordingly, in certain embodiments, the present invention provides a compound of formula (V) or a pharmaceutically acceptable salt or solvate thereof:

n of the invention¹⁵，N¹⁵The dialkylantibiotic compounds may be glycosylated at one or more positions, as known to those skilled in the art. In a preferred embodiment, N is¹⁵，N¹⁵-the dialkyl antibiotic compound is glycosylated at the G and M positions of formula (III) or (IV). In particular embodiments, M is hydrogen or a sugar moiety. For example, M may be hydrogen, α -D-mannopyranosyl or 6-O-acetyl- α -D-mannopyranosyl. In particular embodiments, G is hydrogen or a sugar moiety. For example, G may be hydrogen or glucuronamine.

In certain embodiments, one or both of the sugar moieties may be acylated or acetylated, or both. For example, when G is glucuronamide, the glucuronamide moiety may be acylated with a fatty acid. The fatty acid may be any fatty acid known to those skilled in the art. In particular embodiments, the fatty acid is selected from 8-methylnonanoic acid, n-decanoic acid, 9-methyl-decanoic acid, n-undecaneAcids, 10-methyl-undecanoic acid, n-dodecanoic acid, 11-methyl-dodecanoic acid, n-tridecanoic acid, 12-methyl-tridecanoic acid and n-tetradecanoic acid. In a preferred embodiment, the fatty acid is C₁₂A fatty acid. In a particular embodiment, the fatty acid is 10-methyl-undecanoic acid.

In certain embodiments, the present invention provides a compound of formula (VI), or a pharmaceutically acceptable salt or solvate thereof:

in the formula (VI), R²Is C_10-14An acyl group. In certain embodiments, R²Selected from the group consisting of 8-methylnonanoic acid, n-decanoic acid, 9-methyl-decanoic acid, n-undecanoic acid, 10-methyl-undecanoic acid, n-dodecanoic acid, 11-methyl-dodecanoic acid, n-tridecanoic acid, 12-methyl-tridecanoic acid, and n-tetradecanoic acid. In a preferred embodiment, R²Is C¹²A fatty acid. In certain embodiments, R ²Is 10-methyl-undecanoic acid.

As described in the synthetic section below, N¹⁵，N¹⁵The dimethyl antibiotic compound may be synthesized from a dalbavancin compound. Accordingly, some of N of the present invention¹⁵，N¹⁵The-dimethyl antibiotic compound has dalbavancin A₀、A₁、B₀、B₁、C₀And C₁Sugar moieties and fatty acids.

In certain embodiments, the invention provides N¹⁵，N¹⁵-a dimethyl antibiotic compound. N is a radical of¹⁵，N¹⁵The dimethyl antibiotic compound is included in N¹⁵Formula (I) or (II) having two methyl groups. As described above, N¹⁵，N¹⁵The dimethyl antibiotic compound may be glycosylated with one or more sugar moieties. In certain embodiments, N¹⁵，N¹⁵-dimethyl antibiotic combinationThe compound is acylated on one or more of these sugar moieties. In a preferred embodiment, the acyl group is 10-methyl-undecanoic acid.

In a preferred embodiment, the present invention provides N having the following structure (VII)¹⁵，N¹⁵-a dimethyl antibiotic compound, or a pharmaceutically acceptable salt or solvate thereof:

in certain embodiments, the invention N¹⁵，N¹⁵The dialkyl antibiotic compound is purified. As used herein, the term purified refers to N¹⁵，N¹⁵-the dialkyl antibiotic compound is enriched relative to the other dalbavancin compounds in the composition. E.g. N ¹⁵，N¹⁵Dialkyl antibiotic compounds versus production of N¹⁵，N¹⁵The mixture of dialkyl antibiotic compounds may be enriched, for example, as derived from the fermentation broth described in the examples below. In certain embodiments, N¹⁵，N¹⁵-the dialkyl antibiotic compound is enriched two, three, five, ten, one hundred, one thousand or ten thousand fold.

In a further embodiment, N¹⁵，N¹⁵The dialkyl antibiotic compound is isolated. As used herein, the term isolated refers to N¹⁵，N¹⁵-the dialkyl antibiotic compound is enriched relative to the non-dalbavancin compound in the composition. E.g. N¹⁵，N¹⁵Dialkyl antibiotic compounds versus production of N¹⁵，N¹⁵The mixture of dialkyl antibiotic compounds may be enriched, for example, as derived from the fermentation broth described in the examples below. In certain embodiments, N¹⁵，N¹⁵Two, three, five, ten, one hundred fold enrichment of-dialkyl antibiotic compoundsOne thousand times or ten thousand times.

In still further embodiments, N¹⁵，N¹⁵The dialkyl antibiotic compound is purified and isolated. As used herein, the terms purified and isolated refer to N¹⁵，N¹⁵The dialkyl antibiotic compound is enriched relative to the non-bavancin compound and relative to the other dalbavancin compounds in the composition. E.g. N ¹⁵，N¹⁵Dialkyl antibiotic compounds versus production of N¹⁵，N¹⁵The mixture of dialkyl antibiotic compounds may be enriched, for example, as derived from the fermentation broth described in the examples below. In certain embodiments, N¹⁵，N¹⁵-the dialkyl antibiotic compound is enriched two, three, five, ten, one hundred, one thousand or ten thousand fold.

N¹⁵，N¹⁵-dialkyl antibiotic compounds

The invention also provides N having a carboxylic acid group at carbonyl 63¹⁵，N¹⁵-a dialkyl antibiotic compound. These compounds are useful, for example, in the preparation of N of the invention¹⁵，N¹⁵-a dialkyl antibiotic compound. In certain embodiments, these are¹⁵，N¹⁵Dialkyl antibiotic compounds are also useful for the prevention and/or treatment of microbial infections in mammals.

In certain embodiments, the present invention provides N of formula (VIII)¹⁵，N¹⁵-a dialkyl antibiotic compound, or a pharmaceutically acceptable salt or solvate thereof:

in certain embodiments, the present invention provides N of formula (IX)¹⁵，N¹⁵-a dialkyl antibiotic compound, or a pharmaceutically acceptable salt or solvate thereof:

formulae (VIII) and (IX) provide N in this aspect of the invention¹⁵，N¹⁵-a peptide core of a dialkyl antibiotic compound. The peptide comprises 7 amino acids with the amino terminus on the right, as described in formulas (III) and (IV), and the carboxy terminus on the left. According to this aspect of the invention, N ¹⁵，N¹⁵The dialkyl antibiotic compound being at the N or N terminus¹⁵The above contains two alkyl substituents. In other words, in the formulae (I) and (II), R¹And R¹' is an alkyl group. Furthermore, in formulae (VIII) and (IX), X is OH, as in the compounds of the antibiotic A40926 known to the person skilled in the art.

In a preferred embodiment, R¹And R¹Is' a C_1-4An alkyl group. In certain embodiments, R¹And R¹' are each independently selected from propyl, ethyl and methyl. In further embodiments, R¹And R¹' are each independently selected from ethyl and methyl. In a more preferred embodiment, R¹And R¹' at least one is methyl. In the most preferred embodiment, R¹And R¹' is methyl.

N of the invention¹⁵，N¹⁵The dialkylantibiotic compounds may be glycosylated at one or more positions, as known to those skilled in the art. In a preferred embodiment, N is¹⁵，N¹⁵-the dialkyl antibiotic compound is glycosylated at the G and M positions of formula (VIII) or (IX). In particular embodiments, M is hydrogen or a sugar moiety. For example, M may be hydrogen, α -D-mannopyranosyl or 6-O-acetyl- α -D-mannopyranosyl. In particular embodiments, G is hydrogen or a sugar moiety. For example, G may be hydrogen or glucuronamine.

In certain embodiments, one or two sugarsThe moieties may be acylated or acetylated, or both. For example, when G is glucuronamide, the glucuronamide moiety may be acylated with a fatty acid. The fatty acid may be any fatty acid known to those skilled in the art. In particular embodiments, the fatty acid is selected from the group consisting of 8-methylnonanoic acid, n-decanoic acid, 9-methyl-decanoic acid, n-undecanoic acid, 10-methyl-undecanoic acid, n-dodecanoic acid, 11-methyl-dodecanoic acid, n-tridecanoic acid, 12-methyl-tridecanoic acid, and n-tetradecanoic acid. In a preferred embodiment, the fatty acid is C₁₂A fatty acid. In a particular embodiment, the fatty acid is 10-methyl-undecanoic acid.

In certain embodiments, the present invention provides a compound of formula (X), or a pharmaceutically acceptable salt or solvate thereof:

in the formula (X), R²Is C_10-14An acyl group. In certain embodiments, R²Selected from the group consisting of 8-methylnonanoic acid, n-decanoic acid, 9-methyl-decanoic acid, n-undecanoic acid, 10-methyl-undecanoic acid, n-dodecanoic acid, 11-methyl-dodecanoic acid, n-tridecanoic acid, 12-methyl-tridecanoic acid, and n-tetradecanoic acid. In a preferred embodiment, R²Is C₁₂A fatty acid. In certain embodiments, R ²Is 10-methyl-undecanoic acid.

As described in the synthetic section below, N¹⁵，N¹⁵The-dimethyl antibiotic compound can be synthesized from the antibiotic A40926 compound. Accordingly, some of N of the present invention¹⁵，N¹⁵The dimethyl antibiotic compound has the antibiotic A40926 factor A₀、A₁、B₀、B₁、C₀And C₁Sugar moieties and fatty acids.

In certain embodiments, the invention provides N¹⁵，N¹⁵-a dimethyl antibiotic compound. N is a radical of¹⁵，N¹⁵The dimethyl antibiotic compound is included in N¹⁵Formula (VIII) or (IX) having two methyl groups. As described above, N¹⁵，N¹⁵The dimethyl antibiotic compound may be glycosylated with one or more sugar moieties. In certain embodiments, N¹⁵，N¹⁵-the dimethyl antibiotic compound is acylated on one or more of these sugar moieties. In a preferred embodiment, the acyl group is 10-methyl-undecanoic acid.

In a preferred embodiment, the present invention provides N having the following structure (XI)¹⁵，N¹⁵-a dimethyl antibiotic compound, or a pharmaceutically acceptable salt or solvate thereof:

in certain embodiments, the invention N¹⁵，N¹⁵The dialkyl antibiotic compound is purified. As used herein, the term purified refers to N¹⁵，N¹⁵The dialkyl antibiotic compound is enriched relative to the other antibiotic compounds in the composition. E.g. N ¹⁵，N¹⁵Dialkyl antibiotic compounds versus production of N¹⁵，N¹⁵The mixture of dialkyl antibiotic compounds may be enriched, for example, as derived from the fermentation broth described in the examples below. In certain embodiments, N¹⁵，N¹⁵-the dialkyl antibiotic compound is enriched two, three, five, ten, one hundred, one thousand or ten thousand fold.

In a further embodiment, N¹⁵，N¹⁵The dialkyl antibiotic compound is isolated. As used herein, the term isolated refers to N¹⁵，N¹⁵-the dialkyl antibiotic compound is enriched relative to the non-antibiotic compounds in the composition. E.g. N¹⁵，N¹⁵Dialkyl antibiotic compounds relative toProduction of N¹⁵，N¹⁵The mixture of dialkyl antibiotic compounds may be enriched, for example, as derived from the fermentation broth described in the examples below. In certain embodiments, N¹⁵，N¹⁵-the dialkyl antibiotic compound is enriched two, three, five, ten, one hundred, one thousand or ten thousand fold.

In still further embodiments, N¹⁵，N¹⁵The dialkyl antibiotic compound is purified and isolated. As used herein, the terms purified and isolated refer to N¹⁵，N¹⁵The dialkyl antibiotic compound is enriched with respect to the non-antibiotic compounds in the composition and with respect to the other dalbavancin compounds. E.g. N ¹⁵，N¹⁵Dialkyl antibiotic compounds versus production of N¹⁵，N¹⁵The mixture of dialkyl antibiotic compounds may be enriched, for example, as derived from the fermentation broth described in the examples below. In certain embodiments, N¹⁵，N¹⁵-the dialkyl antibiotic compound is enriched two, three, five, ten, one hundred, one thousand or ten thousand fold.

Process for preparing the Compounds of the invention

Such N may be prepared according to one skilled in the art¹⁵，N¹⁵Dialkyl antibiotic compounds any method of preparation of the compounds of the invention is obvious.

For example, in certain embodiments, N¹⁵，N¹⁵The corresponding N may be converted by any technique apparent to those skilled in the art to the corresponding antibiotic compound or composition of the invention¹⁵-alkylation of monomethyl compounds or compositions. For example, as shown in scheme 1, by contacting a compound or composition with HCHO, NaBH, at room temperature₃CN、DMF、H₂O and NaHCO₃May be contacted with the mixture of (A) to thereby obtain N¹⁵Methylation of monomethyl compounds or compositions. In particular embodiments, N is¹⁵-monomethyl dabigatranDissolving the star compound or composition in water and DMF, adding formaldehyde and sodium bicarbonate, and adding NaBH₃CN, generation of N ¹⁵，N¹⁵-a dimethyl antibiotic compound or composition.

Route scheme 1

Corresponding to N¹⁵Monomethyl compounds or compositions can be prepared according to known techniques, such as those described extensively in U.S. patent No. 5,750,509 and U.S. patent application publication No. 2004/0142883, the contents of which are incorporated by reference in their entirety. If the compound or composition is in its N¹⁵Protecting groups are present on the nitrogen and they can be removed by any technique known to those skilled in the art.

Suitable starting materials include dalbavancin A₀、A₁、B₀、B₁、C₀And C₁And antibiotic A40926 factor A₀、A₁、B₀、B₁、C₀And C₁. According to this method, as shown in the following examples, from dalbavancin B₀Preparation of N¹⁵，N¹⁵-dimethyl dalbavancin B₀. The preparation of these dalbavancin compounds is extensively described in U.S. patent application publication No. 2004/0142883. These dalbavancin compounds have the following structure:

wherein R is as follows

In a further embodiment, N of the invention may be prepared from the antibiotic A40926 fermentation broth¹⁵，N¹⁵-a dialkyl antibiotic compound. Fermentation broths comprising the antibiotic A40926 and related compounds may be prepared according to the described techniques, for example, in U.S. Pat. No. 5,750,509 and U.S. patent application publication No. US 2004/0142883. The broth can be purified to produce the desired antibiotic A40926 compound, which can be esterified, amidated and hydrolyzed according to U.S. Pat. No. 5,750,509 and U.S. patent application publication No. US 2004/0142883 to produce a mixture of dalbavancin compounds. N can be purified and/or separated from the mixture according to any technique apparent to those skilled in the art ¹⁵，N¹⁵-a dialkyl antibiotic compound. In the following examples, HPLC techniques were used to purify N of the present invention¹⁵，N¹⁵-a dialkyl antibiotic compound.

Composition comprising a metal oxide and a metal oxide

In another aspect, the invention provides compositions comprising one or more compounds of the invention. Generally, the compositions of the present invention comprise a compound of the present invention and one or more other compounds. The other compounds may be compounds of the present invention, compounds known to those skilled in the art, compounds not yet discovered or disclosed, or other compounds.

In certain embodiments, the composition is a pharmaceutical composition described in more detail in the sections below.

In particular embodiments, the compositions comprise N of the invention¹⁵，N¹⁵A dialkyl antibiotic compound, and dalbavancin or an antibiotic a 40926 compound. The dalbavancin or antibiotic a 40926 compound may be any dalbavancin or antibiotic a 40926 compound known to those skilled in the art. Typical dalbavancin compounds include those described in U.S. patent No. 5,750,509 and U.S. patent application publication No. US 2004/0142883, the entire contents of which are hereby incorporated by reference. Typical antibiotic A40926 Compounds Such as those described in U.S. patent nos. 4,935,238, 4,868,171, and 4,782,042, the entire contents of which are hereby incorporated by reference.

Typical dalbavancin compounds include dalbavancin A₀、A₁、B₀、B₁、C₀And C₁. The preparation of these dalbavancin compounds is extensively described in U.S. patent application publication No. US 2004/0142883. These dalbavancin compounds are further described in the above section. Of course, the compositions of the present invention may include additional dalbavancin compounds not listed in the above exemplary dalbavancin compounds.

In certain embodiments, the composition comprises a multimer of a compound of the invention. The multimer may be a dimer, trimer or larger multimer. The polymer may be a homopolymer, a heteropolymer or a mixture of homopolymer and heteropolymers. For example, the multimer may comprise a combination of any of the factors present in the dalbavancin composition, including any of the dalbavancin factors a₀、A₁、B₀、B₁、B₂、C₀、C₁、D₀、D₁MAG or hetero B₀. For example, the multimer may include N¹⁵-alkyl dalbavancin compounds and N¹⁵，N¹⁵-dialkyl dalbavancin compounds. In certain embodiments, the invention also provides homodimers of the compounds of the invention. In a further embodiment, the present invention provides heterodimers of a compound of the present invention and a dalbavancin compound. The dalbavancin compound may be the dalbavancin of the invention or known to those skilled in the art.

In certain embodiments, the compositions of the present invention comprise a significant amount of a N of the present invention relative to a dalbavancin or antibiotic a 40926 compound¹⁵，N¹⁵-a dialkyl antibiotic compound. In certain embodiments, N of the present invention is relative to a dalbavancin or antibiotic a 40926 compound¹⁵，N¹⁵The dialkyl antibiotic compound is at least 0.05, 0.1, 0.15, 0.2, 0.25, g,0.5, 0.75, 1.0, 1.25, 1.5, 1.6, 1.7, 1.75, 1.8, 1.9, 2.0, 2.5, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98 or 99%. In a preferred embodiment, the compound is a compound of formula (V).

In certain embodiments, the compositions of the present invention comprise a significant amount of a N of the present invention relative to the dalbavancin compound¹⁵，N¹⁵-a dialkyl antibiotic compound. In certain embodiments, N of the present invention is relative to the dalbavancin compounds¹⁵，N¹⁵Dialkyl antibiotics the compounds of the invention are at least 0.05, 0.1, 0.15, 0.2, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.6, 1.7, 1.75, 1.8, 1.9, 2.0, 2.5, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98 or 99% of the composition. In a preferred embodiment, the compound is a compound of formula (V).

In certain embodiments, the compositions of the present invention comprise a significant amount of a N of the present invention relative to the antibiotic a 40926 compound¹⁵，N¹⁵-a dialkyl antibiotic compound. In certain embodiments, N of the present invention is relative to the antibiotic A40926 compound¹⁵，N¹⁵-the dialkyl antibiotic compound is at least 0.05, 0.1, 0.15, 0.2, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.6, 1.7, 1.75, 1.8, 1.9, 2.0, 2.5, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98 or 99% of the composition. In a preferred embodiment, the compound is a compound of formula (V).

In certain embodiments, the compositions of the invention comprise a significant amount of the N of the invention relative to other compounds¹⁵，N¹⁵-a dialkyl antibiotic compound. In certain embodiments, N of the invention is relative to other compounds¹⁵，N¹⁵-the dialkyl antibiotic compound is at least 0.05, 0.1, 0.15, 0.2, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.6, 1.7, 1.75, 1.8, 1.9, 2.0, 2.5, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98 or 99% of the composition. Excellence inIn an alternative embodiment, the compound is a compound of formula (V).

In a further embodiment, the present invention provides a pharmaceutical composition comprising dalbavancin a ₀、A₁、B₀、B₁、C₀And C₁In connection with the invention N¹⁵，N¹⁵-a combination of dialkyl antibiotic compounds, wherein the amount of the compound of the invention is relative to one or more or all of dalbavancin a₀、A₁、B₀、B₁、C₀And C₁Is enriched. The composition may be enriched, for example, such that N is¹⁵，N¹⁵-a dialkyl antibiotic compound relative to one or more or all of dalbavancin a₀、A₁、B₀、B₁、C₀And C₁At least 0.05, 0.1, 0.15, 0.2, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.6, 1.7, 1.75, 1.8, 1.9, 2.0, 2.5, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the composition. In a preferred embodiment, the compound is a compound of formula (V).

In certain embodiments, the invention provides compositions comprising dalbavancin B₀And the invention N¹⁵，N¹⁵-a combination of dialkyl antibiotic compounds. In a further embodiment, the present invention provides a pharmaceutical composition comprising dalbavancin B₁And the invention N¹⁵，N¹⁵-a combination of dialkyl antibiotic compounds. In a further embodiment, the present invention provides compositions comprising dalbavancin B₀Dalbavancin B₁And the invention N¹⁵，N¹⁵-a combination of dialkyl antibiotic compounds. In a preferred embodiment, the invention N¹⁵，N¹⁵The dialkyl antibiotic compound is a compound of formula (V). In a particularly preferred embodiment, the present invention provides a pharmaceutical composition comprising a compound of formula (V), dalbavancin B ₀And dalbavancin B₁The composition of (1). These compositions may be purified, isolated, or both purified and isolated. In certain embodiments, the composition is 90, 91, 92, 93, 94, 95, 96, a,97. 98, 98.5, 99 or 99.5% pure. "pure" refers to the amount of a compound comprising the composition. For example, comprising a compound of formula (V), dalbavancin B₀And dalbavancin B₁The composition of (a) will comprise at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 98.5, 99 or 99.5% of these three compounds relative to the other compounds in the composition. Purity can be assessed by any method known to those skilled in the art, such as HPLC, for example by area under the% curve.

In the compositions of the present invention, the amount of each component can be calculated by weight, molar amount, or other techniques known to those skilled in the art.

Application method

Provides administration of the invention N to an individual in need of treatment of a bacterial infection¹⁵，N¹⁵-a dialkyl antibiotic compound or composition. In one embodiment, N is¹⁵，N¹⁵The dialkyl antibiotic compound is a compound of formula (V). Treatment may include prophylaxis, treatment or cure. The method comprises administering N in a therapeutically or prophylactically effective amount¹⁵，N¹⁵-a dialkyl antibiotic compound or composition.

As used herein, a "therapeutically effective amount" refers to the amount of a compound or composition that will achieve the desired therapeutic result (e.g., reduce or eliminate bacterial infection) when administered to a patient for treatment of a disease. The "therapeutically effective amount" may vary depending on, inter alia, the compound, the disease and its severity, and the age, weight, etc. of the patient to be treated, and may be administered in one or more doses. A "prophylactically effective amount" refers to an amount of N that, when administered to an individual susceptible to and/or suffering from a bacterial infection, is sufficient to prevent or reduce the severity of future bacterial infections¹⁵，N¹⁵-the amount of the dialkyl antibiotic compound or composition, the predisposition, for example, due to a medical procedure or hospitalization, or exposure to a subject infected with bacteria. N is a radical of¹⁵，N¹⁵The dialkyl antibiotic compound or composition may be administered in a pharmaceutically acceptable carrier.

N¹⁵，N¹⁵The dialkyl antibiotic compound or composition may be N¹⁵，N¹⁵-administration of a "unit dose" in a formulation of a dialkyl antibiotic comprising an amount of the compound sufficient to provide therapeutically or prophylactically effective plasma levels of the compound for a plurality of days, at least about 5 days, a week, or 10 days when administered to a subject. In some embodiments, the compound is a compound of formula (V). In some embodiments, the compound is a component of a formulation that includes an amount of the compound sufficient to provide therapeutically or prophylactically effective levels of the compound for a plurality of days, often at least about 5 days, a week, or 10 days. The dosing interval, or time between doses, can be, for example, about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more days.

As used herein, the terms "individual" or "subject" or "patient" are used interchangeably to refer to a vertebrate, typically a mammal, often a human.

In addition, a compound or composition of the invention may be used in combination with one or more other N's that are present in the inhibiting compound, composition and/or composition¹⁵，N¹⁵-dialkyl antibiotics or stabilizers of other antibiotic components. In some embodiments, the stabilizing agent is selected from the group consisting of mannitol, lactose, sucrose, sorbitol, glycerol, cellulose, trehalose, maltose, raffinose, and mixtures thereof. In one embodiment, the formulation comprises mannitol. In another embodiment, the formulation further comprises lactose. In one embodiment, the composition may be prepared with a 1: 2 weight ratio of mannitol: n is a radical of¹⁵，N¹⁵-dialkyl antibiotic compounds. In another embodiment, the composition may have a 1: 4 weight ratio of mannitol to lactose to N¹⁵，N¹⁵-dialkyl antibiotic compounds.

In some embodiments, the composition or other formulation comprises N¹⁵，N¹⁵-dimethyl antibioticsA compound, a dalbavancin compound, or a combination thereof. The composition or other formulation may be administered at a dose that results in a therapeutically effective (i.e., bactericidal) plasma level of the drug for several days, often at least about 5 to about 10 days, often at least about one week. For example, N in plasma ¹⁵，N¹⁵The dialkyl antibiotic compound or composition may be maintained at or above a minimum bactericidal concentration of about 4mg/l for at least 5 days. N is a radical of¹⁵，N¹⁵The dialkyl antibiotic compound or composition may maintain plasma levels of at least about 5mg/l, at least about 10mg/l, at least about 20mg/l, at least about 30mg/l, at least about 40mg/l for at least 5 days, at least about one week or more. N can be measured by methods known in the art¹⁵，N¹⁵-plasma concentrations of the dialkyl antibiotic compound or composition, such as liquid chromatography, mass spectrometry, or microbiological bioassays.

N¹⁵，N¹⁵The upper limit of the plasma concentration level of the dialkyl antibiotic compound or composition may be determined by the dose that inhibits unacceptable adverse effects in the patient population being treated.

N¹⁵，N¹⁵The dialkyl antibiotic compound or composition may be administered in a single dose or in multiple doses. When administered as a single dose, N may be¹⁵，N¹⁵-dialkyl antibiotic compounds or compositions are made to contain a sufficient amount of N¹⁵，N¹⁵-a dialkyl antibiotic compound to achieve in vivo antibacterial properties for at least 5 days, optionally for at least 6 days, optionally for at least 7 days, optionally for at least 8 days, optionally for at least 9 days, optionally for at least 10 days, optionally for at least 11 days, optionally for at least 12 days, optionally for at least 13 days, optionally for at least 14 days, optionally for at least 15 days.

When multiple doses are employed, N¹⁵，N¹⁵The dialkyl antibiotic compound or composition may be administered once a week for two or more weeks. In one embodiment, N is¹⁵，N¹⁵The dialkylantibiotic compound or composition may be administered in at least two doses, often two doses separatedFrom about 5 to about 10 days, more often once a week for two weeks. In certain embodiments, this regimen provides significant advantages over traditional antibiotic treatment regimens.

N¹⁵，N¹⁵The dialkylantibiotic compound or composition may also be administered in multiple doses, separated by two or more days or at least one week, or in one or more once-every-two-week doses. In some embodiments, N is¹⁵，N¹⁵The dialkyl antibiotic compound or composition may be administered once a week, followed by once every two weeks, or once a month. In some embodiments, N is¹⁵，N¹⁵The dialkyl antibiotic compound or composition may be administered at weekly intervals for 2, 3, 4, 5, 6 or more weeks.

Most advantageously, N is not required because a higher, lower frequency dose is used¹⁵，N¹⁵-the dialkyl antibiotic compound or composition is administered daily. For example, a single or multiple dose may be about 0.1 to about 5 grams. A single dose of about 0.1 to about 4 grams, e.g., about 3 grams, can be administered for the treatment of various infections. When multiple doses are administered, for example once per week, each dose may be in the range of about 0.25 to about 5.0 grams.

For embodiments in which a single dose is administered to treat an infection, the dose may be about 0.1 to about 5 grams, or about 0.5 to about 4 grams, or about 1 to about 3.5 grams, or about 2 to about 3 grams, for example about 3 grams. In some embodiments, a single dose of about 1, 1.5, 2, 2.5, or 3 grams may be administered to treat a bacterial infection. For embodiments in which a single dose is administered for prophylaxis, the dose may be, for example, from about 0.1 to about 3 grams, or from about 0.1 to about 1 gram, e.g., about 0.5 or about 0.25 grams.

In a dosage regimen comprising multiple doses, the individual doses may be the same or different. In some embodiments, a first dose that is higher than one or more subsequent doses may be administered, which may be, for example, about 1.5-3 times higher. For example, the first dose may be about 0.5 grams to about 5 grams and the second dose about 0.25 grams to about 2.5 grams, the first dose may be about 0.8 to about 2 grams and the second dose about 0.4 to about 1 gram, or the first dose may be about 0.4 to about 3 grams and the second dose about 0.2 to 1.5 grams.

In some embodiments, at least 2 doses are administered, wherein the first dose comprises about twice the N of the subsequent dose¹⁵，N¹⁵-a dialkyl antibiotic compound or composition. In one embodiment, the first dose comprises about 1 gram of N ¹⁵，N¹⁵A dialkyl antibiotic compound or composition, and a subsequent dose comprises about 0.5 grams. In another embodiment, the first dose comprises about 0.5 grams and the subsequent dose comprises about 0.25 grams.

In some embodiments, N may be administered in two doses of the same or different amounts, two or more days apart or at least about one week apart¹⁵，N¹⁵-a dialkyl antibiotic compound or composition. For example, two about 0.2 to about 1.5 gram doses of N may be administered about 5 to about 10 days or about 1 week apart¹⁵，N¹⁵-a dialkyl antibiotic compound or composition. In one embodiment, about 1 gram of the first dose and about 0.5 gram of the second dose of N may be administered about 1 week apart¹⁵，N¹⁵-a dialkyl antibiotic compound or composition.

In a multiple dose regimen, the time between doses may range, for example, from about 5 to about 10 days, often about one week. The frequency of administration may be, for example, two weekly doses or multiple weekly doses. The dosing interval or time between doses can be, for example, any of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more days. The number of doses administered may be, for example, one, two, three, four, five, six or more doses, each dose after the initial dose being administered after a selected dose interval.

In certain embodiments in a multi-dose regimen, the "trough level" or N in the plasma after the first dose and just prior to administration of the second dose¹⁵，N¹⁵The level of the dialkyl antibiotic compound or composition may be at least about 4 mg/l.At the end of a dosing interval, such as about one week, N¹⁵，N¹⁵-the trough level of the dialkyl antibiotic compound or composition is preferably at least about 20mg/l, at least about 30mg/l, or at least about 40 mg/l.

N¹⁵，N¹⁵The dialkyl antibiotic compound or composition may be administered parenterally, for example intramuscularly (i.m.), intravenously (i.v.), subcutaneously (s.c), intraperitoneally (i.p.) or intrathecally (i.t). The dosing schedule and actual dosage administered may depend on factors such as the nature and severity of the infection, the age, weight, general health of the patient, and the particular patient to N¹⁵，N¹⁵The tolerability of the dialkyl antibiotic compound or composition varies, but this is determinable by a health professional. In one embodiment, at N¹⁵，N¹⁵One week after a 1 gram intravenous dose of the dialkyl antibiotic compound or composition, followed by a 0.5 gram intravenous dose.

The drug may be administered and delivered to the patient at a controlled rate, e.g., intravenously, so that the concentration in the blood does not increase too quickly or precipitate. In some embodiments, N is administered at an appropriate rate ¹⁵，N¹⁵-a dialkyl antibiotic compound or composition, such that the drug forms a complex with endogenous proteins in the bloodstream. Without being bound by a particular theory, it is believed that endogenous proteins such as human serum albumin can interact with one or two molecules of N in vivo¹⁵，N¹⁵-a dialkyl antibiotic compound to form a complex. When a sufficient amount of N is present¹⁵，N¹⁵Dialkyl antibiotic compositions, up to two N are believed¹⁵，N¹⁵The molecule of the dialkylantibiotic compound can bind to the endogenous protein and bind to the independent N at two different binding sites¹⁵，N¹⁵Molecules of a dialkyl antibiotic compound to form this complex. Alternatively, dimer N¹⁵，N¹⁵It is also possible that the dialkyl antibiotic can bind to a single binding site on the endogenous protein.

N¹⁵，N¹⁵-dialkyl antibiotic compoundsOr the duration of infusion of the composition may be, for example, from about 1 minute to about 2 hours. E.g. N¹⁵，N¹⁵The dialkyl antibiotic compound or composition may be administered for an infusion duration of about 30 minutes, wherein the dosage may be about 0.5 to about 1 gram. Intravenous administration at controlled rates produces a far greater excess of N in vivo than is achievable in solution phase at physiological pH in vitro¹⁵，N¹⁵-dialkyl antibiotic compound or composition concentration. While not wishing to be bound by theory, this may be attributed to N ¹⁵，N¹⁵Dialkylantibiotics forming complexes with endogenous proteins such as serum albumin, which increase the plasma uptake of N¹⁵，N¹⁵-the capacity of the dialkyl antibiotic compound or composition.

In certain embodiments, N is formed in vitro or ex vivo¹⁵，N¹⁵The dialkyl antibiotic complex may allow for faster administration, for example at least about 1 minute, at least about 10 minutes, or at least about 20 minutes. By reacting human serum albumin and/or another endogenous protein with N¹⁵，N¹⁵-the complex may be formed by mixing a dialkyl antibiotic compound or composition, thereby forming the complex in vitro or ex vivo, and then administering this complex to the patient to be treated. Alternatively, human serum albumin or other endogenous proteins may be obtained from autologous sources, or by expression from microorganisms modified to contain genes for the proteins.

N applied¹⁵，N¹⁵The amount of the-dialkyl antibiotic compound or composition may be any of the dosages disclosed herein. The dosage may be selected so that one or more of N¹⁵，N¹⁵The dialkyl antibiotic compound is maintained at therapeutically or prophylactically effective (i.e., bactericidal) plasma levels for an extended period of time, such as at least 5 days, or about one week or more. Preferably, the N is applied to produce and maintain a germicidal concentration for at least about one week (or about 5 to about 10 days) ¹⁵，N¹⁵-a dose of a dialkyl antibiotic compound or composition. The bactericidal concentration may be defined as killing bacteria initially present in the in vitro experiment over a 24 hour period toAbout 80%, at least about 85%, at least about 90%, or at least about 95%, 96%, 97%, 98%, or 99% less of any of the required N¹⁵，N¹⁵-the concentration of the dialkyl antibiotic compound or composition. N in plasma¹⁵，N¹⁵The minimum bactericidal concentration of the dialkyl antibiotic compound or composition may be about 4 mg/l.

N may be used¹⁵，N¹⁵Examples of indications for which the dialkyl antibiotic compounds or compositions and methods of the invention prevent or treat include complex and uncomplicated SSTIs, bloodstream infections (BSIs), catheter-associated bloodstream infections (CRBSIs), osteomyelitis, prosthetic joint infections, surgical prophylaxis, endocarditis, nosocomial or community-acquired pneumonia, pneumococcal pneumonia, empirical therapy for febrile neutropenia, joint cavity infections, and device infections (e.g., pacemakers and internal cardiac defibrillators). Can be used for preventing or treating gram-positive or antibiotic-resistant bacterial infections, such as Bacillus, Corynebacterium, Listeria, enterococcus, Staphylococcus, Streptococcus, Neisseria, or Clostridium infections, especially Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus pyogenes, Streptococcus pneumoniae, group A and group C streptococci, enterococcus faecalis, Bacillus subtilis, Neisseria gonorrhoeae, or Clostridium difficile. Can use the present invention N ¹⁵，N¹⁵Other infections prevented or treated by the dialkyl antibiotic compounds, compositions and methods include gram negative bacteria such as bartonella, brucella, campylobacter, enterobacter, escherichia (and other Proteobacteria), franciscella, helicobacter, haemophilus, klebsiella, legionella, leptospira, morganella, moraxella, proteus, providencia, pseudomonas, salmonella, serratia, shigella, stenotrophomonas, vibrio and yersinia infections, especially escherichia coli, proteus vulgaris, pseudomonas aeruginosa and yeasts such as candida albicans.

The invention also includes the use of the compounds, compositions and methods of the invention for the prevention or treatment of other infectious bacteria, such as helicobacter pylori, Borelia burgdorferi, Legionella pneumophila, Mycobacterium sporozoites (genus) (e.g., Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare (Mycobacterium avium), Mycobacterium subclinicum, Mycobacterium kansaii, Mycobacterium gordonii), Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (group A Streptococcus), Streptococcus agalactiae (group B Streptococcus), Streptococcus viridis, Streptococcus bovis, Streptococcus anaerobis, Campylobacter pathopoiesii, enterococcus, influenza, Bacillus antracis, Corynebacterium diphtheriae, Corynebacterium, Salvia suis, Clostridium capsulatum, Clostridium tetani, Bacillus aerogenes, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, and the like, Fusobacterium nucleatum, Streptococcus moniliforme, Treponema pallidium, Leptospira, and Actinomyces israeli.

The prevention and treatment of infections and diseases described herein can be accomplished with the compounds, compositions, and methods of the present invention. In one embodiment, N is¹⁵，N¹⁵The dialkyl antibiotic compound is a compound of formula (V). In some embodiments, the patient has not been previously treated with one or more antibiotics, such as vancomycin or teicoplanin. In other embodiments, the patient has not previously used N¹⁵，N¹⁵A dialkyl antibiotic compound or composition has been treated. In some embodiments, the patient has previously tested the bacteria for resistance to antibiotics. In other embodiments, the patient has not previously been tested for antibiotic resistance by the bacteria.

The invention also includes methods of preventing or treating SSTI. Patients who may benefit from such prevention or treatment may have deep or superficial infections. SSTIs can include deeper soft tissue and/or require significant surgical intervention, such as severe abscesses, infected ulcers, severe burns, or deep and extensive cellulitis. Can also be used for preventing or treating surgical wound infection.

The clinical manifestations of skin and skin structure infections can vary from mild folliculitis to severe necrotizing fasciitis. Acquisition patterns may also vary with community acquired skin and skin structure infections, which are preceded by injury caused by occupational exposure or recreational activities and are often associated with a large diversity of pathogens. Hospital acquired skin and skin tissue infections are commonly associated with surgery, pressure sores and catheterization. Post-operative infection is the third most common nosocomial infection and accounts for 17% of all nosocomial infections reported to the national nosocomial infection monitoring system (NNIS). The most common source of infection is the endogenous flora of the patient. Staphylococcus aureus, coagulase-negative staphylococcus, and enterococcus are the most frequently isolated pathogens from SSTI.

Symptoms of SSTI infection can include erythema, tenderness or pain, heat or localized warmth, fluid drainage or drooling, swelling or hardening, redness or wave movement. Patients that may benefit from treatment with the methods of the invention include patients with deep or complex infections or infections requiring surgical intervention, or patients with underlying diabetes or peripheral vascular disease. The infection is often caused by gram-positive bacteria, such as staphylococci or streptococcal species, such as staphylococcus aureus or streptococcus pyogenes. The method of treating a bacterial infection of the skin or soft tissue comprises administering a therapeutically effective amount of N in an amount and according to the above-described dosage regimen¹⁵，N¹⁵-administering to a subject in need of treatment a dialkyl antibiotic compound or composition. In one embodiment, N is¹⁵，N¹⁵The dialkyl antibiotic compound is a compound of formula (V). In one embodiment, N is administered intravenously in two doses¹⁵，N¹⁵A dialkyl antibiotic compound or composition, often spaced about 5 to about 10 days apart, more often spaced about 1 week apart. In some embodiments, the first dose comprises at least twice the second dose of N¹⁵，N¹⁵-a dialkyl antibiotic compound or composition. In one embodiment, the first dose is about 1000mg and the second dose is about 500 mg.

As will be appreciated by those skilled in the art, the method of administration described herein will vary depending upon, inter alia, the compound, the disease and its severity, the age, weight, etc. of the patient being treated, but can also be determined by a physician without undue experimentation.

The invention also provides methods for prophylactically preventing the onset of a bacterial infection, such as an infection caused by staphylococcus aureus, or by neisseria or clostridium bacteria. In the prophylactic methods of the invention, a prophylactically effective amount of N will be¹⁵，N¹⁵-a dialkyl antibiotic compound or composition is administered to an individual who may be susceptible to developing a bacterial infection, for example by a medical procedure. N is a radical of¹⁵，N¹⁵The dialkyl antibiotic compound or composition may be administered in an amount sufficient to provide a prophylactically effective plasma level for at least about 1 day, at least about 3 days, at least about 5 days, or at least about one week or more. N is a radical of¹⁵，N¹⁵The dialkyl antibiotic compound or composition may be administered e.g. parenterally, e.g. via i.m., i.v., i.p., s.c or i.t. injection, before or after surgery or simultaneously with surgery as a prophylactic step against infection. The N may be administered prior to, immediately after, one or more days before or after, or about one week or during an invasive medical procedure such as surgery or hospitalization in a medical care facility such as a hospital ¹⁵，N¹⁵-a dialkyl antibiotic compound or composition to prevent infection. The prophylactic method may be used in any situation in which an individual is likely or likely to be infected with a bacterial infection, including situations in which an individual has been exposed or is likely to be exposed to a bacterial infection. For prophylactic methods, N¹⁵，N¹⁵The dialkylantibiotic compound or composition may be administered as a single dose, or as two or more doses of the same or different amounts spaced apart by a period of days to about one week. In one embodiment, N is¹⁵，N¹⁵The dialkyl antibiotic compound or composition may be administered prior to or simultaneously with insertion of the intravenous catheter to prevent blood flow related infections. In one embodiment, N is¹⁵，N¹⁵The dialkyl antibiotic compound is a compound of formula (V).

For prophylactic methods, N¹⁵，N¹⁵The dialkyl antibiotic compound or composition may be administered in single or multiple doses according to any of the dosing regimens described above. N is a radical of¹⁵，N¹⁵The dialkyl antibiotic compound or composition may be administered as a single dose comprising about 0.1 to about 3 grams, or about 0.1 to about 1 gram, for example about 0.25 grams or about 0.5 grams. In one embodiment, a single dose of about 0.25 grams is administered intravenously over a time period of about 2 minutes to about 1 hour (e.g., about 30 minutes). In another embodiment, N may be administered intravenously concurrently with the administration of another drug (e.g., antibiotic) therapy ¹⁵，N¹⁵-a dialkyl antibiotic compound or composition.

In any of the foregoing methods of treatment or prevention, N¹⁵，N¹⁵The dialkyl antibiotic compound or composition may be administered simultaneously or sequentially with at least one other antibiotic. In some embodiments, except N¹⁵，N¹⁵At least one other antibiotic, besides the dialkylantibiotic, can be administered, which is directed against one or more N¹⁵，N¹⁵Dialkyl antibiotic compounds are not effective against gram-negative and/or gram-positive bacterial species (e.g. bactericidal). In some embodiments, N is¹⁵，N¹⁵The dialkyl antibiotic compound and the at least one antibiotic that may be effective against (e.g. bactericidal) the at least one gram-negative bacterial species are administered as a mixture in a dalbavancin composition.

Pharmaceutical composition

The invention provides for the application of N according to the above method¹⁵，N¹⁵-a dialkyl antibiotic compound or composition. The pharmaceutical composition of the invention may be N¹⁵，N¹⁵-a unit dosage form of a dialkylantibiotic compound or composition comprising N sufficient to provide a therapeutic or prophylactic effect when the compound or composition is administered to an individual¹⁵，N¹⁵Plasma levels of the dialkyl antibiotic compound or composition last for several days (at least about 3 days, At least about 5 days, or at least about one week or more) of N¹⁵，N¹⁵-a dialkyl antibiotic compound or composition, and a pharmaceutically acceptable carrier. Generally, N is therapeutically or prophylactically effective¹⁵，N¹⁵The plasma level of the dialkyl antibiotic compound or composition may be at least about 4mg per liter of plasma. N is a radical of¹⁵，N¹⁵The plasma levels of the dialkyl antibiotic compound or composition can be measured by methods well known in the art, such as liquid chromatography, mass spectrometry, or microbiological assays. Other N in serum, as is well known to those skilled in the art¹⁵，N¹⁵The level of the-dialkyl antibiotic compound or composition can be quantified in a similar manner.

N¹⁵，N¹⁵The dialkyl antibiotic compound or composition may optionally be in a pharmaceutically acceptable form for administration to the individual, e.g. as a pharmaceutically acceptable non-toxic salt.

N¹⁵，N¹⁵Examples of suitable salts of the dialkyl antibiotic compounds or compositions include salts formed by standard reactions with organic and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, succinic acid, citric acid, ascorbic acid, lactic acid, maleic acid, glutamic acid, camphoric acid, glutaric acid, glycolic acid, phthalic acid, tartaric acid, lauric acid, stearic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, sorbic acid, picric acid, benzoic acid, cinnamic acid and the like. Representative examples of bases which can form salts with dalbavancin include alkali metal or alkaline earth metal hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and barium hydroxide; ammonia and aliphatic, alicyclic, or aromatic amines such as methylamine, dimethylamine, diethylamine, ethanolamine, and picoline (see, e.g., U.S. patent No. 5,606,036).

In some embodiments, pharmaceutically acceptable N suitable for parenteral administration, e.g., intravenous injection, is provided¹⁵，N¹⁵-an aqueous formulation of a dialkyl antibiotic compound or composition.In one embodiment, N is¹⁵，N¹⁵The dialkyl antibiotic compound is a compound of formula (V). To prepare the N¹⁵，N¹⁵Aqueous formulations of the dialkylantibiotic compounds or compositions may be prepared using methods well known in the art, and any pharmaceutically acceptable carrier, diluent, excipient or other additive commonly used in the art may be used. In one embodiment, a pharmaceutically acceptable aqueous formulation for intravenous injection comprises 5% dextrose.

Pharmaceutical compositions for parenteral administration comprising N¹⁵，N¹⁵A dialkyl antibiotic compound or composition and a physiologically acceptable diluent, such as deionized water, physiological saline, 5% dextrose, water miscible solvents (e.g., ethanol, polyethylene glycol, propylene glycol, and the like), non-aqueous vehicles (e.g., oils such as corn, cottonseed, peanut, and sesame oils), or other common diluents. The formulation may further include a solubilizing agent such as polyethylene glycol, polypropylene glycol, or other known solubilizing agents, a buffer for stabilizing the solvent (e.g., citrate, acetate, and phosphate), and/or an antioxidant (e.g., ascorbic acid or sodium bisulfite) (see, e.g., U.S. patent No. 6,143,739). Other suitable Pharmaceutical carriers and formulations thereof are described in "Remington's Pharmaceutical Sciences" of e.w. martin. The pharmaceutical formulations of the present invention may also be prepared to contain acceptable levels of particles (e.g., no particles) and to be pyrogen-free (e.g., to meet injectable requirements in the united states pharmacopeia), as is known in the art.

In one embodiment, by drying (e.g., lyophilizing) N¹⁵，N¹⁵A dose of a dialkyl antibiotic compound or composition (which often contains a stabilizer or mixture of stabilizers) is dissolved in an amount of water of sufficient volume to dissolve and preferably in deionized water to provide a pharmaceutical composition. For example, the amount of water sufficient to effect dissolution may be about 10mL, and the resulting pH may be above 3.0, about 3.5 to 4.5. Diluting the solution by adding it to a second quantity of aqueous diluent, said aqueous diluent comprising 5%Dextrose, e.g., the amount contained in a drip bag for intravenous administration, to raise the pH of the solution to about 5-5.5. In another embodiment, the pH of the solution in the drip bag may be about 4.5. The second quantity of aqueous diluent may be deionized or sterilized, or both. In one embodiment, the aqueous diluent is 5% dextrose. Other dissolution methods and N thereof¹⁵，N¹⁵Formulations of-dialkyl antibiotics will be apparent to those skilled in the art.

Can hold N therein¹⁵，N¹⁵-the pharmaceutical composition for parenteral use is constituted in sterile vials containing a therapeutically or prophylactically effective amount of one or more unit doses of N as described above, under conditions of bactericidal effectiveness of the dialkyl antibiotic compound or composition ¹⁵，N¹⁵-a dialkyl antibiotic compound or composition, optionally including an excipient. The compound or composition may or may not be in the form of a dry (e.g., lyophilized) powder. Before use, a physiologically acceptable diluent may be added and the solution withdrawn by syringe for administration to the patient. The above pharmaceutical formulations may be sterilized by any acceptable means including, for example, electron beam or gamma sterilization or by sterile filtration.

Formulations for parenteral administration may include N¹⁵，N¹⁵A dialkyl antibiotic compound or composition, for example, at a concentration of about 0.1 to about 100mg, about 0.5 to about 50mg, about 1 to about 10mg, about 1 to about 5mg, or about 2 to about 4mg N per ml of the final formulation¹⁵，N¹⁵-a dialkyl antibiotic compound or composition. In one embodiment, N is¹⁵，N¹⁵The dialkyl antibiotic compound is a compound of formula (V).

In some embodiments, the pharmaceutical compositions of the invention comprise N¹⁵，N¹⁵A mixture of a dialkyl antibiotic compound or composition and one or more additional antibiotics. Preferably, at least one non-dalbavancin antibiotic in the mixture is effective against (e.g., bactericidal against) one or more gram-negative bacterial species, such asThreonamide) and/or effective against (e.g., bactericidal) N ¹⁵，N¹⁵One or more gram-positive strains against which the dialkyl antibiotic compound or composition is not effective (such as linezolid or daptomycin). The mixture may also include a pharmaceutically acceptable carrier as described above. In some embodiments, the pharmaceutical composition comprises N¹⁵，N¹⁵A dialkyl antibiotic compound or composition and one or more additional antibiotics, in some embodiments, N¹⁵，N¹⁵The dialkyl antibiotic composition comprises N of formula (V)¹⁵，N¹⁵-a dialkyl antibiotic compound.

In some embodiments, the pharmaceutical compositions of the present invention comprise one or more inhibitors of one or more N¹⁵，N¹⁵Degradation of the dialkyl antibiotic compound into a stabilizing substance of less active or inactive material. As used herein, "stabilizing material" or "stabilizer" refers to stabilizing one or more N present in a composition¹⁵，N¹⁵-a substance at the level of a dialkyl antibiotic compound. "stabilizing effective amount" means an amount sufficient to increase one or more of the N that may be present in the composition¹⁵，N¹⁵-the amount of stabilizer of the long-term stability of the dialkyl antibiotic compound. In some embodiments, a stabilizing effective amount may be provided by a mixture of two or more stabilizing materials, wherein each material alone is not present in an amount sufficient to provide a stabilizing effect.

Examples of stabilizers include, for example, nonionic substances, such as sugars, e.g. mono-, di-or polysaccharides or derivatives thereof; a sugar alcohol; or a polyol. The stabilizing substance includes, for example, mannitol, lactose, sucrose, sorbitol, glycerol, cellulose, trehalose, maltose, raffinose, or mixtures thereof.

In one embodiment, N is¹⁵，N¹⁵-the dialkyl antibiotic formulation comprises mannitol. In another embodiment, N is¹⁵，N¹⁵The dialkyl antibiotic formulation further comprises lactose. In one embodiment, N is¹⁵，N¹⁵-a dialkyl antibiotic composition in a 1: 2 weight ratio of mannitol: n is a radical of¹⁵，N¹⁵-dialkyl antibiotic compounds. In another embodiment, N is¹⁵，N¹⁵-the dialkyl antibiotic composition mannitol lactose N in a 1: 4 weight ratio¹⁵，N¹⁵-dialkyl antibiotic compounds. The combination of mannitol and lactose can provide greater stabilization than either substance alone. The pH of the pharmaceutical compositions of the invention may be, for example, from about 2 to about 9, alternatively from about 3 to about 8, alternatively from about 4 to about 7, alternatively from about 5 to about 6, alternatively from about 3.5 to about 4.5. The pH of the pharmaceutical compositions of the invention may also be, for example, less than about 9, alternatively less than about 8, alternatively less than about 7, alternatively less than about 6, alternatively less than about 5, or alternatively less than about 4. The pH of the pharmaceutical compositions of the invention may also be, for example, greater than about 2, greater than about 3, alternatively greater than about 3.5, alternatively greater than about 4, alternatively greater than about 4.5, alternatively greater than about 5.0, alternatively greater than about 5.5, alternatively greater than about 6.0, alternatively greater than about 6.5, or alternatively greater than about 7.0.

In some embodiments, one or more methods may be employed to reduce MAG (invention N)¹⁵，N¹⁵A dialkyl antibiotic compound or a derivative of dalbavancin compound that does not contain an acyl glucamine moiety) and/or other degradation products. For example, lyophilization of N in the presence of a stabilizing material such as mannitol¹⁵，N¹⁵A dialkyl antibiotic compound or composition may be used to reduce the amount of MAG formed.

Can be substituted by N¹⁵，N¹⁵The dialkylantibiotic compounds and compositions are kept at a temperature below ambient, such as about 5 ℃, to improve stability.

As demonstrated in the examples herein, N is administered once weekly at high dose levels (i.e., resulting in abnormally high and sustained serum levels)¹⁵，N¹⁵The dialkylantibiotic compounds or compositions may show an exceptionally good safety profile, similar to or better than that of lower doses administered once a day or even 2-4 times a dayThe safety observed with standard treatments of conventional antibiotics. High doses (i.e., resulting in abnormally high and sustained serum levels, such as 200-¹⁵，N¹⁵A dialkyl antibiotic compound or composition without adverse side effects, enabling improved efficacy and patient compliance.

In certain embodiments, with N¹⁵，N¹⁵Treatment with a dialkyl antibiotic compound or composition results in a low incidence of adverse events. Serious adverse events include any adverse drug experience that occurs at any dose that results in death, life threatening, resulting in hospitalization or prolonged existing hospitalization, or permanent or significant disability or disability.

Medicine box

The invention also includes kits for use in methods of treatment or prevention of bacterial infections. Kits may include a pharmaceutical compound or composition of the invention (e.g., including at least one unit dose of N¹⁵，N¹⁵-a dialkyl antibiotic compound or composition) and instructions for providing information to a health care provider regarding use of the compound or composition for treating or preventing a bacterial infection. The instructions may be provided in printed form, or in the form of an electronic medium such as a floppy disk, CD or DVD, or in the form of a website where the instructions are available. Unit dose of N¹⁵，N¹⁵The dialkyl antibiotic compound or composition may include N that, when administered to an individual, renders the treatment or prevention effective¹⁵，N¹⁵The plasma levels of the dialkyl antibiotic compound or composition may be maintained in the individual at a dose for at least 5 days. In certain embodiments, the kit comprises two unit doses to be administered at least 5 days apart, about 1 week apart, or comprises N in a first dose that can be about 1.5 to about 3 times higher than a second dose ¹⁵，N¹⁵-a dialkyl antibiotic compound or composition. In some embodiments, N may be included as a sterile aqueous pharmaceutical composition or a dry powder (e.g., lyophilized) composition¹⁵，N¹⁵-a dialkyl antibiotic compound or composition. In a fruitIn the embodiment, N¹⁵，N¹⁵The dialkyl antibiotic compound is a compound of formula (V).

In some embodiments, suitable packaging is provided. As used herein, "packaging" refers to N that is typically used in a system and can be adapted for administration to an individual¹⁵，N¹⁵-a solid matrix or material in which the dialkyl antibiotic compound or composition is held within a fixed range. Including glass and plastic (e.g., polyethylene, polypropylene, and polycarbonate) bottles, vials, paper, plastic and plastic foil laminated envelopes, and the like. If electron beam sterilization techniques are used, the packaging should have a density low enough to allow sterilization of the contents.

The kit may also optionally include a pharmaceutical composition for administering N¹⁵，N¹⁵Devices for dialkyl antibiotic compounds or compositions, such as syringes or devices for intravenous administration; and/or for the preparation of sterile solutions of dry powder (e.g. lyophilized) compositions for pharmaceutical administration, e.g. diluents such as 5% dextrose.

The medicine box of the invention is N¹⁵，N¹⁵The dialkyl antibiotic compound or composition may also include, in addition to the non-dalbavancin antibiotic or a mixture of non-dalbavancin antibiotics to react with N as described in the above method¹⁵，N¹⁵-a dialkyl antibiotic compound or composition is used together.

The following synthetic and biological examples are provided to illustrate the invention and are not to be construed in any way as limiting the scope of the invention.

Examples

Example 15: purification of N¹⁵，N¹⁵-dimethyl dalbavancin B₀

The preparation of A-40926 and subsequent synthesis of dalbavancin is described above. This example describes the purification of a compound of formula (la) from a mixture of dalbavancin compounds prepared according to the inventionN of (V)¹⁵，N¹⁵Dimethyl antibiotic compounds, i.e. N¹⁵，N¹⁵-dimethyl dalbavancin B₀. It will be apparent to those skilled in the art that N is¹⁵，N¹⁵-dimethyl dalbavancin B₀Is at N¹⁵Modified dalbavancin B with two methyl groups₀. For the purposes of this example and the following examples, N is in the moiety where the compound is yet to be fully characterized¹⁵，N¹⁵-dimethyl antibiotic B₀Referred to as "Compound A". And, for convenience in the following sections, N will be referred to¹⁵，N¹⁵-dimethyl dalbavancin B₀Called dalbavancin B₂。N¹⁵，N¹⁵-dimethyl antibiotic B ₀Dalbavancin B₂And compound a is the same molecule.

Sample preparation

About 2L of the organic-aqueous solution from the purification of dalbavancin prepared according to the process herein was concentrated by evaporation of acetonitrile and water under vacuum. Diethyl ether was added to the residue to give 6g of crude solid. After dissolution in a water/acetonitrile 90/10v/v mixture, the solid was purified on silanized silica gel (column volume 1L; column id. about.5 cm) eluting with a discontinuous gradient of water (pH3 acetic acid)/acetonitrile. Fractions containing compound a were collected, concentrated to a smaller volume (several ml) and subjected to preparative HPLC purification.

The enriched fraction was collected. After evaporation of acetonitrile in vacuo and freeze-drying, a few μ g of compound a was recovered.

Example 16: n of Compound A¹⁵Identification of amine substituents

Compound A has a molecular weight of 1828Da, comparable to that of the Dabawan component B₀And B₁(m.w.1814) 14 units more. HPLC-ESI-MS analysis confirmed that, unlike the other components of the complex, the change occurred at the N of the terminal amino group¹⁵The above.

Having a methylamine group (R-NHCH)₃) Other dalbavancin compounds in their fragmented formsThe spectrum (ESI-MS/MS) showed 31 units (-NH)₂CH₃) The neutrality of (a) is lost. Fragment mass spectra of compound a showed a loss of 45 units instead of 31 units. This type of neutral loss can be explained by two structural hypotheses: n is a radical of ¹⁵Possibly N, N-dimethylamino (R-N (CH)₃)₂Tertiary amine) or N-ethylamine group (R-NHCH)₂CH₃Secondary amine).

HPLC-ESI-MS analysis of hydrolyzed and derivatized Compound A can distinguish between these two hypotheses. FIG. 29 shows the source B after acid hydrolysis₀Three peptide fragments of (a). Two dipeptides are formed, containing amino groups N¹⁵And chlorine atom "AA 1+ AA 3", "AA 5+ AA 7", and chlorine atom-containing tripeptide "AA 2+ AA4+ AA 6". Other hydrolysis products are fatty acid and DMEPA4 (dimethylamino-propylamino) chains.

For compound a, the dipeptide "AA 1+ AA 3" must be different, as shown in figure 30. The correct hypothesis can be verified by reacting the hydrolysate with a derivatizing agent (derivitizing agent) which reacts only with primary and secondary amines (see fig. 31).

Component B₀The dipeptide "AA 1+ AA 3" should be reacted with two sets of derivatizing reagents (see FIG. 31 a). According to amino group N for compound A¹⁵Two different derivatized peptides "AA 1+ AA 3" were obtained (FIG. 31 b). It is theoretically expected from the proposed hypothesis that these two molecules will have molecular weights that are readily distinguishable by mass spectrometry.

Material

Compound A

Dalbavancin

37 percent of hydrochloric acid, reagent grade, Rudi Pont cod.750-11

Methanol HPLC grade, j.t.baker cod.8402

AccQ TagTM Chemistry Package，Waters cod.WAT052880

Hydrolysis

Hydrolysis of samples and standards was performed in the PICO-TAG Work Station (Waters, Mildoford, MA, USA).

All 360. mu.g of Compound A and about 1mg of dalbavancin standard were hydrolyzed in the presence of 1% (w/v) phenol in 6N HCl at 105 ℃ for 24 hours. The reaction mixture was cooled, evaporated to dryness and adjusted to a final volume of 500L with methanol.

Derivatization

The Waters AccQ-Fluor kit was chosen for peptide derivatization. AccQ-Fluor reagent is an N-hydroxysuccinimide-activated heterocyclic carbamate amine derivative compound. The structure and reaction scheme for this derivatizing reagent is depicted in scheme 2.

Route map 2

1mL of "reagent diluent" was added to the vial containing "reagent powder". The vial was vortexed for 10 seconds and heated to 55 ℃ until completely dissolved. The reconstituted AccQ-Fluor reagent was approximately 10mM acetonitrile. Mu.l of the hydrolyzed sample or standard was placed in a vial with a conical stopper and 20. mu.l of "AccQ-Fluor borate buffer" was added and vortexed briefly. At this point, 40. mu.l of reconstituted AccQ-Fluor reagent was added and the sample vortexed for 30 sec. The vial was heated at 55 ℃ for 10 min. Under these conditions, the amino group should react and possible by-products should be minimized. After the reaction, the sample was ready for HPLC analysis.

HPLC-UV-MS analysis

A Thermo Finnigan Surveyor MS pump, a diode array detector and an autosampler,

ThermoQuest Finnigan LCQ Deca quality detector equipped with ESI interface.

And (3) chromatography: column: AccQ-TagTM (Waters C18 NovoPak 4_ m 3.9X 150 mm); column temperature: 37 ℃; flow rate: 1 mL/min; phase A: ammonium acetate 140mM pH 5 (acetic acid); phase B: water/acetonitrile 60/40 v/v; UV detector: 254 nm; injection volume: 20 μ l. The eluent obtained from the column was split and UV and quality detection was performed simultaneously.

Mass spectrometry

Sample input conditions: capillary temperature: 200 deg.C

Casing gas: n is a radical of₂40 (arbitrary unit)

Auxiliary gas: n is a radical of₂20 (arbitrary unit)

Sample input voltage setting: polarity: positive for

Power supply voltage: 4.7kV

Capillary voltage: 10V

Tube lens offset distance: 40V

Scanning conditions are as follows: the scanning mode is as follows: complete ms

Scanning range: 100-700amu

The number of micro-scans: 3

Maximum ion time: 50ms

Results

Derivatized and hydrolyzed dalbavancin fragments were separated by HPLC and evaluated by ESI-MS (data not shown). Fragment AA1+ AA3 was 737(m/z) in size, fragment AA5+ AA7 was 689(m/z) in size, and fragment AA2+ AA4+ AA6 was 1086(m/z) in size.

The derivatized and hydrolyzed fragments of Compound A were separated by HPLC and evaluated by ESI-MS. Fragment AA1+ AA3 was 737(m/z) in size, fragment AA5+ AA7 was 581(m/z) in size, and fragment AA2+ AA4+ AA6 was 1086(m/z) in size.

The size of AA5+ AA7 demonstrates that the compoundsA is N¹⁵，N¹⁵-a dimethyl compound.

Example 17: dalbavancin B₀Methylation of

Dalbavancin B was prepared according to the procedure outlined above₀And purified by HPLC according to the method described in U.S. patent application publication No. 2004/0142883. Will dalbavancin B₀Selective N-methylation of secondary amines to N¹⁵，N¹⁵-dimethyl antibiotic B₀. The reaction mixture was then analyzed by HPLC-UV and HPLC-UV-MS in the following examples.

Material

Dalbavancin B₀

Water, MmilliQ grade

NaBH₃CN, sodium cyanoborohydride, Fluka cod.71435

DMF, N, N-dimethylformamide, Carlo Erba cod.444926

HCHO, formaldehyde solution, 36.5% aqueous solution, Reidel de Haen cod.33220.

NaHCO₃Sodium bicarbonate, reagent grade

Method of producing a composite material

49.5mg of dalbavancin were dissolved in 12.5mL of water and 1.5mL of DMF and charged into a round-bottomed flask (pH 3.5). Three 200. mu.l aliquots were removed and diluted with 800. mu.l of water (t 0). 76. mu.l of a 36% (V/V) aqueous solution of formaldehyde and 2.5mg of sodium bicarbonate (pH5.8) were added. After the sodium bicarbonate was completely dissolved, 8mg of NaCNBH was added with stirring₃. Three aliquots of 200. mu.1 of the reaction mixture were immediately removed and washed with 800. mu.l of water and 300. mu. lCH₃CN dilution to give a clear solution (t 0'). The reaction mixture was left at room temperature for 30 minutes. After 10 and 30 minutes, the reaction medium is sampled by extracting 3 aliquots of 200. mu.l, each with 800. mu.l of water and 300. mu.l of CH ₃CN dilution (t10 and t 30).

After 30 minutes, the reaction was stopped by cooling the flask to-20 ℃. The reaction was monitored by HPLC-UV and HPLC/UV/MS analysis.

Example 18: n is a radical of¹⁵，N¹⁵-dimethyl antibiotic B₀Structural confirmation of

This example demonstrates compounds a and N from the above example¹⁵，N¹⁵-dimethyl antibiotic B₀Are identical.

Reaction samples t0, t 0', t10 and t30 were analyzed by HPLC-UV and compared to reference standards. the chromatograms for t0, t10 and t30 were very similar, demonstrating that methylation occurred immediately.

The chromatogram of t 0' versus t0 is shown in fig. 33. Methylation of B₀The same retention time as compound a from example 15 above is shown. Thus, the hypothesis was demonstrated that Compound A is B₀So that the fatty acid side chain of compound a is 10-methylundecanoic acid.

The HPLC-UV-MS results confirm the structural conclusions of HPLC-UV as reported in the previous paragraph. N, N' -dimethyl B₀Completely overlapping with the peak of compound a.

Thus demonstrating N of Compound A¹⁵The group at (a) is a dimethylamino group instead of ethylamine. An illustrative structure of component compound a is reported in figure 28.

The structure of Compound A was further confirmed by NMR, ESI-MS and IR. NMR spectra at 313K were recorded on Bruker AMX 600. Samples were dissolved in DMSO-d6 and proton and carbon NMR assignments were determined using COSY-DFTP, ROESY and HMQC spectra. A mixing time of 350msec was chosen for the ROESY experiment. Ionization by electrospray in anodic mode at Thermoquest Finnigan LCQ ^decaThe mass spectrum was determined at 250 ℃. The infrared spectra were recorded on a KBron aBruker IFS 48 instrument.

The NMR peak assignments are provided in fig. 34. Table 40 reports₀Comparative B₂1H allocation of (1). B is₂The NMR spectrum of the compound shows that the compound is related to dalbavancin B₀There are many similarities. The fatty acid side chains contain an isopropyl end group. Most protons and carbon are chemically displaced and coupled to B₀The components were found to be approximately the same. The major chemical shift deviations are observed for signals belonging to amino acid 1. In particular, 2.31ppm (¹³C δ 40.06ppm) gives NOEs corresponding exactly to six protons and associated with protons x1, 1f and 1 e. Its chemical shift and dipolar correlation suggest that this signal is due to the dimethylamino group belonging to the first amino acid spin system.

Watch 40

The infrared spectra are provided in fig. 35. ESI-MS is provided in FIG. 36. The data prove that the structure of the compound A is the dalbavancin B₂(N¹⁵，N¹⁵-dimethyl dalbavancin B₀)。

Example 19: dalbavancin B₂(N¹⁵，N¹⁵-dimethyl dalbavancin B₀) Activity of (2)

Separation of N¹⁵，N¹⁵-dimethyl dalbavancin B₀N, N¹⁵Monomethyl dalbavancin compounds and several known antibiotics, and tested their antibacterial activity in vitro. The compounds were prepared according to the methods described in the above examples and U.S. patent application publication No. 2004/0142883. Each dalbavancin compound was purified by HPLC.

Sample preparation

Separating out N¹⁵-monomethyl dalbavancin compounds and N¹⁵，N¹⁵The-dimethyl antibiotic compound was dissolved in 0.01N HCl: DMSO: 95: 5 v/v. Prior to dissolution, individual samples were analyzed by HPLC and quantified. Preparation of 256. mu.g/mL N¹⁵-monomethyl dabigatranBavancin compound (A)₀、A₁、B₀And B₁Dalbavancin) and 128. mu.g/mL of isolated N¹⁵，N¹⁵-dimethyl antibiotic B₀(referred to as "B" in the following table)₂Dalbavancin ").

As reported in table 41, the chromatographic purity of the main peak was evaluated in area% relative to the total chromatographic area:

table 41: different N¹⁵-monomethyl and N¹⁵，N¹⁵Chromatographic purity of the main peak of the dimethyl antibiotic compound

Microbiological characterization

The compounds tested included the above isolated N¹⁵-monomethyl dalbavancin compounds and N¹⁵，N¹⁵-dimethyl antibiotic compound, and comprising N¹⁵，N¹⁵Dalbavancin compositions of dimethyl antibiotic compounds ("DA 025/a"), Vancomycin (VA) (Sigma Chemical co., st.louis, M0), Gentamicin (GE) (Sigma Chemical co., st.louis, MO), penicillin G (pen.g) (Sigma Chemical co., st.louis, M0), and amphotericin B (amph.b) (Sigma Chemical co., st.louis, MO).

Microorganisms

The microorganisms used were reference strains obtained from the American Type Culture Collection (ATCC) (Rockville, Md.), SmithKline and French Laboratories (SKF) and Upjohn Company (UC) (Kalamazoo, Ml.).

Minimum Inhibitory Concentration (MIC) determination

Standard NCCLS procedures were followed by broth microdilution (NCCLSDcument M7-A6, Vol.23, No. 2, "Methods for differential solubility tests for bacteria which grow aerobically, "Approved guiding. January 2003, which is incorporated by reference in its entirety), with or without 30% adult Bovine Serum (BS) (PAA laboratories GmBH, Haidmann paging Austria), using approximately 5X 10⁵CFU/ml of bacteria were inoculated and MIC was determined. The culture medium comprises cation-regulatedHinton broth (Difco Laboratories, Detroit, MI) with CaCl in each case₂And MgCl₂Adjusted to final concentrations of 20mg/L and 10 mg/L. After incubation at 35 ℃ for 20-24 hours, the assay results were read.

Results

MIC results are summarized in table 42 below. Against a group of gram-positive bacteria, various isolated N¹⁵-monomethyl dalbavancin compounds and N¹⁵，N¹⁵-dimethyl antibiotic compound ("B)₂") is comparable or slightly more active than the DA composition, wherein the DA composition comprises N¹⁵，N¹⁵-mixtures of dialkyl antibiotic compounds. The blank solution (HCL 0.01N/DMSO 95: 5) used to dissolve the test compounds was inactive.

Significantly, N is¹⁵，N¹⁵-dimethyl antibiotic compound ("B)₂") shows activity against gram-positive bacteria comparable to or higher than N¹⁵-the activity of monomethyl dalbavancin compounds. For example, in the gram-positive range, N¹⁵，N¹⁵-dimethyl antibiotic compound ("B)₂") is comparable or higher than B₀Activity of (2).

Quite remarkably, N¹⁵，N¹⁵-dimethyl antibiotic compound ("B)₂") also showed activity against the range of gram-negative bacteria tested. In fact, the only compound active against the representative gram-negative bacteria in this test is the isolated N¹⁵，N¹⁵-dimethyl antibiotic compound ("B)₂") has an activity in the range of 16-32mg/L MIC.

The MIC values of VA and GE reference compounds against ATCC reference strain were within the range of NCCLS values.

TABLE 42 different N¹⁵，N¹⁵In vitro Activity of Dialkylantibiotic Compounds and reference strains

In the above table:

staphylococcus aureus/s.aureus: the culture medium is characterized in that the culture medium comprises staphylococcus aureus,

s. epidermidis: staphylococcus epidermidis Streptococcus pyrogenes: the streptococcus pyogenes is used as a raw material,

s. pneumoniae: streptococcus pneumoniae, Enterococcus faecalis/E.faecalis: the bacteria of the enterococcus faecalis are,

bacillus subtilis: bacillus subtilis, Escherichia coli/e.coli: (ii) an Escherichia coli bacterium, wherein,

Proteus vulgaris: proteus vulgaris, Pseudomonas aeruginosa: the pseudomonas aeruginosa is a microorganism which is a microorganism belonging to the genus Pseudomonas,

candida albicans: candida albicans, ref. strain: reference strains

No lowest dilution of activity.

Quality control Range for MIC of reference Strain (NCCLS M100S-12, Vol.22, No.1, Jan.2002):

¹VA 0.5-2mg/L

²VA 1-4mg/L

³GE 0.25-1mg/L

example 20: preparation of a pharmaceutical composition comprising dalbavancin B₂Dalbavancin compositions

Preparation of a pharmaceutical composition comprising purified dalbavancin B by preparative HPLC₀Dalbavancin B₁And dalbavancin B₂The composition of (1).

The dalbavancin mixture prepared according to the above method was purified by HPLC. Purified in Kromasil C18, 16 μm,is carried out on the aperture. The buffer system was 50mmol NH adjusted to pH 5.5₄H₂PO₄An aqueous solution. The mobile phase eluted was 66/34 buffer/acetonitrile mixture. The method produces a composition comprising 97% dalbavancin B₀Dalbavancin B₁And dalbavancin B₂The dalbavancin composition of (1) (analyzed by HPLC). A typical analytical HPLC chromatogram is in FIG. 37, where dalbavancin B₀At about 29.901, dalbavancin B₁At about 30.79 (unlabeled) and dalbavancin B₂At about 33.282.

Hetero B₀Is characterized by

Hetero B₀The mass spectrum of (FIG. 38) shows that the ion of m/z 1817 corresponds to a singly protonated molecule, with the other ions being attributed to cationic adducts or partial source fragments. 1816Da molecular weight and fragmentation pattern were the same as those observed for the B component, supporting the exchange of iso-B ₀Identified as B₀Diastereoisomers of (a) the same molecule having one or more stereogenic centers of inverted chirality.

It was found that in each preparation step, iso B increased too much each time the pH increased too much₀The level of (c) increases. Thus, the preparation of iso-B by alkali treatment of dalbavancin₀。

Sample preparation (BI-K0096)

At room temperature, by applying an aqueous NaOH solution (p) for a prolonged period of time (about 165 hours)H12.7) treatment of 10g of batch 027 of dalbavancin, obtaining about 300mg of iso B₀. After neutralization, the reaction product is recovered as a brown solid crude product which is purified by double chromatography on a silanized silica gel column eluted with water (DH 3.5; acetic acid)/acetonitrile in a discontinuous gradient.

The entire 79.98% area of BI-K0096 was separated from the enriched fraction. The HPLC profile is reproduced in fig. 39.

In order to verify the impurity of BI-K0096 and dalbavancin is different B₀The prepared compound BI-K0096 was analyzed by HPLC using three different methods. BI-K0096 is chromatographically related to iso-B under all conditions tested₀The impurities are identical.

Structural analysis

MS and MS/MS spectra are reported in FIGS. 40A-C. These spectra and component B₀The mass spectra of (A) are identical. From this mass analysis it is not possible to confirm the stereochemical differences.

In DMSO-d on a 600MHz spectrometer⁶In (B) is₀Is/are as follows¹H and¹³the CNMR spectra reveal much resemblance to the dalbavancin spectra, but also differ significantly (see fig. 41 and 42, respectively). Hetero B₀And B₀The assignment of (a) is reported in table 43 and the corresponding position of the proton is identified in figure 43.

TABLE 43 dalbavancin iso B₀NMR distribution of

Chemical shifts of protons and carbon and B for certain parts of the molecule₀The composition is approximately the same. The main chemical shift shifts are observed for signals belonging to amino acid sequences 1 to 4. The protons of these spin systems show negligible positive or negative Δ δ (chemical shift difference). Amino acid 3 in particular shows a largeRelative changes in the number (x3, w3, 3f, 3 d); x3 is also the only resonance that appears to be³J_HHWith a significant modification, the coupling constant CH (x3) -NH (w3) is now 6.54Hz, relative to B₀Medium is about 10.4. Furthermore, ROESY experiments showed differences in bipolar relationships between x3 and most of the protons adjacent to x 3. The reported NMR results and published literature data on teicoplanin epimers clearly indicate that x3 is the epimerization center and that epimerization induces a conformational change in at least a portion of the molecule.

Microbiological characterization

The compounds used were:

the aforementioned hetero B₀

Reference standard for Dalbavancin (DA) (BI-K397 batch 025/A/AS 1)

Vancomycin (VA) (121K 1140 batch Sigma Chemical Co.St Louis.MO, USA)

Gentamicin (GE) (57H 1099 batch Sigma Chemical Co.St Louis.MO, USA)

Penicillin G (Pen.G) (batch 43H1134 Sigma Chemical Co.StLouis, M0, USA)

Amphotericin B (amph. B) (batch 61H4039 Sigma Chemical co. Stlouis, MO, USA).

Dissolving vancomycin, dalbavancin and amphotericin B at 10mg/mL in dimethyl sulfoxide (DMSO) and diluting in distilled water; gentamicin and penicillin G were dissolved in distilled water.

Culture medium

Will be provided withHinton broth (Difco Laboratories, Detroit, MI, USA) was treated with CaCl separately₂And MgCl₂Adjusted to final concentrations of 20mg/L and 10 mg/L.

Serum

Adult Bovine Serum (BS) (batch A05123-159 PAA laboratories GmBH Haidmann weg Pasching Austria).

Microorganisms

The microorganisms used were reference standard strains from the American type culture Collection (ATCC, Rockville, USA), Smith Kline and French Laboratories (SKF) and Upjohn Company (UC, Kalamazoo, Michigan, USA).

Minimum Inhibitory Concentration (MIC)

By broth microdilution, following standard NCCLS procedures [13 ]]With or without 30% bovine serum, using approximately 5X 10⁵CFU/mL of bacterial inoculum, MIC was determined. The results were read after incubation at 35 ℃ for 20-24 h.

Results

Against gram-positive bacteria, hetero B₀The activity of (D) was very similar to that of dalbavancin (see Table 44)

TABLE 44 EXCITATION B₀And in vitro Activity of reference Compounds

*****

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art that certain changes and modifications may be made without departing from the spirit and scope of the invention. Accordingly, the description should not be construed as limiting the scope of the invention, which is delineated by the appended claims.

All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.

Claims (1)

1. A pharmaceutical composition comprising:

dalbavancin factor B₀And at least one additional factor A selected from dalbavancin factor A₀、A₁、B₁、B₂、C₀、C₁Iso B₀And the dalbavancin factor of MAG; and is

Wherein, factor B₀A content of not less than 75% of the HPLC distribution of all dalbavancin components present, and wherein the acetone content is not more than 2.5%;

and, when the composition comprises dalbavancin factorB₀And MAG, the MAG content is less than the 3.0% HPLC distribution of all dalbavancin components present.