Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
  • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
  • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention is directed to complexes of dalbavancin with endogenous proteins. These complexes can be formed in vivo, in vitro,ox ex vivo and provide for an antimicrobial effect in a treated patient.
• CVCs central venous catheters
• SSTIs Skin and soft tissue infections
• Staphylococcus aureus and Streptococcus pyogenes are the pathogens most frequently isolated from patients with deep tissue infections, although any pathogenic organism, including those found on healthy skin, may cause infection.
• Many SSTIs are mild to moderate in severity, permitting successful treatment with oral antimicrobial agents and local cleansing.
• more severe or complicated infections which frequently occur in patients with underlying risk factors (e.g., vascular compromise, diabetes) and/or infections caused by difficult-to-treat or multiply-resistant bacteria, may require potent intravenous antimicrobial therapy and aggressive surgical debridement.
• Staphylococci are a clinical and therapeutic problem and have been increasingly associated with nosocomial infections since the early 1960s.
• the coagulase-positive species methicillin-resistant Staphylococcus aureus (MRSA) has long been problematic in both community-acquired and nosocomial infections, and several coagulase-negative staphylococci have been recognized as opportunistic human pathogens, especially in the treatment of critically ill patients in intensive care units.
• MRSA methicillin-resistant Staphylococcus aureus
• Another major cause for clinical concern is the increasing isolation of penicillin-resistant Streptococcus pneumoniae strains in many parts of the world.
• Teicoplanin is at least as active as vancomycin against most Gram-positive bacteria and appears to cause fewer adverse events. Both forms of treatment require at least once daily dosing to effect complete recovery. Currently, the therapeutic options for severe infections caused by some of these pathogens is quite limited. The emerging resistance of Gram-positive pathogens to vancomycin makes the availability of new antibiotics with potential for increased effectiveness highly desirable.
• an antibiotic exhibits a suitable pharmaceutical window, less frequent dosing is possible only if the antibiotic exhibits a suitable serum half-life to maintain therapeutic effectiveness over the dosing interval desired.
• the serum half-life of an antibiotic dictates both the longevity of a drug in vivo and the length of time after administration when the serum level will reach a minimum trough level which is still bactericidally effective.
• the serum trough level over time after administration of a first dose of antibiotic dictates when a further dose must be administered to retain a minimum bactericidal level of the antibiotic in vivo.
• an antibiotic possessing activity against one or more antibiotic resistant bacterial strains, particularly MRSA, which could be administered at a dosing interval of once every 5-7 days or longer, would be of commercial value and would satisfy a long-felt need in the art.
• This invention is directed to the discovery that upon administration in vivo, dalbavancin forms a complex with endogenous proteins, e.g., human serum albumin (HSA).
• HSA human serum albumin
• the particular complex formed will, of course, be dependent upon the amoxint of dalbavancin administered, the rate of administration, and the total amount of endogenous protein available for complexation.
• This dalbavancin-protein complex formed in vivo retains dalbavancin antibacterial properties and exhibits a long serum half-life. Contrarily, many other antibiotic-protein complexes either lose potency upon complex formation and/or the complex is readily cleared from the body thereby limiting the duration of antibacterial activity.
• the formation of the complex permits systemic tissue distribution of the dalbavancin in a treated patient which is essential to the successful treatment of skin and soft tissue infections.
• this complex either exhibits antibacterial properties er se or acts as a drug depot wherein the low binding affinity of dalbavancin to the endogenous protein permits sustained release of free dalbavancin over a period of time. In either case, a prolonged antibacterial effect is achieved.
• this invention is directed to an endogenous protein-dalbavancin complex comprising both a 1:1 complex of protein to dalbavancin and a 1 :2 complex of protein to dalbavancin.
• the dalbavancin component may contain one or more of the A 0 , Ai, B 0 , B ls and MAG dalbavancin components.
• the dalbavancin component in the complex comprises at least about 80% B o; more preferably, from at least about 80% to about 98% of the B 0 component.
• the complex is formed in vivo by intravenous administration of a dalbavancin composition to the patient.
• [0018] is controlled by the amount and rate of intravenous administration of the dalbavancin composition to the patient.
• the administration is conducted under conditions such that the initial plasma concentration of dalbavancin is at least 200 mg/L and at least about 90% of the complex formed has a 1 :1 ratio of dalbavancin to protein.
• this invention is directed to a dalbavancin-endogenous protein complex formed in vivo by intravenous administration of a dalbavancin composition to a mammalian patient under conditions wherein the initial plasma concentration of dalbavancin is at least 200 mg/L and further wherein that least about 90% of the complex formed has a ratio of dalbavancin to protein of 1 : 1.
• the dalbavancin-endogenous protein complex can be formed in vitro or ex vivo by admixing a solution of dalbavancin with protein under conditions wherein the complex is formed.
• this invention further provides for pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an antibacterial amount of the complex.
• Such compositions may be sterile and/ore lyophilized and/or in pharmaceutically acceptable form for administration to an individual, e.g., in a pharmaceutically acceptable aqueous formulation.
• the individual is a mammal, e.g., a human.
• the invention provides pharmaceutical compositions that include a pharmaceutically acceptable carrier, a protein-dalbavancin complex of the invention, and a non-dalbavancin antibiotic or mixture of non-dalbavancin antibiotics.
• the non- dalbavancin antibiotic or mixture of antibiotics may include at least one antibiotic that is effective against a Gram negative bacterium.
• a stabilizer is employed in the dalbavancin composition used to form the complex described herein.
• the stabilizer is employed to inhibit degradation of one or more dalbavancin components during storage whether as a lyophilized or aqueous formulation. Over time, degradation can result in the undesirable formation of less active/inactive components in the dalbavancin composition or in the formation of components which potentially could cause adverse side effects in vivo.
• Particularly preferred stabilizers are non-ionic components such as a sugar or sugar alcohol, e.g., a mono-, di-, or polysaccharide, or derivative thereof, such as, for example, mannitol, lactose, sucrose, sorbitol, glycerol, cellulose, trehalose, or maltose, or mixtures thereof.
• protein-dalbavancin complexes provided by the invention retain at least about 10% of the antibacterial of free dalbavancin.
• protein-dalbavancin complexes provided by the invention permit systemic distribution of dalbavancin in an individual when present in the individual.
• this invention is directed to a method for treating a bacterial infection in an individual in need thereof, said method comprising administering a therapeutically effective dose of dalbavancin to an individual suffering from a bacterial infection under conditions such that a protein-dalbavancin complex forms.
• a method for treating a bacterial infection in an individual in need thereof comprises administering a sufficient dose of dalbavancin to the individual to provide an initial plasma concentration of dalbavancin of at least about 200 mg/L, wherein dalbavancin forms a protein-dalbavancin complex with an endogenous protein, and further wherein that least about 90% of the complex formed has a ratio of dalbavancin to protein of 1 : 1.
• this invention is directed to a method for treating a bacterial infection in an individual in need thereof, comprising administering a therapeutically effective dose of a protein-dalbavancin complex.
• the therapeutically effective dose contains an amount of dalbavancin or protein-dalbavancin complex sufficient to provide a therapeutically effective serum level in said individual for at least 5 days.
• a first and second therapeutically effective dose of dalbavancin or protein-dalbavancin complex are administered, where the amount of the second dose is about half or less than the amount of the first dose.
• the dose of dalbavancin is about 500 mg to about 1000 mg.
• the bacterial infection comprises a Gram-positive bacterium, e.g. a penicillin-resistant or multi-drug-resistant bacterium.
• the bacterial infection comprises a skin and soft tissue infection (SSTI); in some of these embodiments, the SSTI comprises Staphylococcus aureus, in other embodiments the SSTI comprises Streptococcus pyogenes.
• the bacterial infection is reduced; in some embodiments it is eliminated.
• the individual is a mammal, e.g., a human.
• this invention is directed to a method for preventing onset of a bacterial infection in an individual, comprising administering dalbavancin in a prophylactically effective dose under conditions such that a protein-dalbavancin complex forms.
• this invention is directed to a method for preventing onset of a bacterial infection in an individual, comprising administering a protein-dalbavancin complex in a prophylactically effective dose.
• the administration of dalbavancin or protein- dalbavancin complex occurs prior to, during, or subsequent to a medical procedure; e.g., surgery, or insertion of an intravenous catheter.
• the administration of dalbavancin or protein-dalbavancin complex occurs prior to, during, or subsequent to a stay in the hospital.
• the prophylactically effective dose contains an amount of dalbavancin or protein-dalbavancin complex sufficient to provide a prophylactically effective serum level in said individual for at least 5 days.
• a first and second prophylactically effective dose of dalbavancin or protein-dalbavancin complex are administered, where the amount of the second dose is about half or less than the amount of the first dose.
• dalbavancin or protein-dalbavancin complex is administered as a single dose that comprises about 250 mg to about 1000 mg of dalbavancin.
• administration may be parenteral. e.g., intravenous, e.g., controlled intravenous administration. In some embodiments, controlled intravenous administration occurs over at least about thirty minutes.
• the methods further comprise administering a pharmaceutically acceptable carrier.
• the methods further comprise administering a non-dalbavancin antibiotic or mixture of non- dalbavancin antibiotics. In some of these embodiments, at least one of the non-dalbavancin antibiotics is effective against a Gram negative bacterium to the individual.
• the invention provides kits containing dalbavancin or a protein- dalbavancin complex, and instructions for use, where the use may be in methods of treatment or methods of prevention of a bacterial infection.
• Figure 1 depicts dalbavancin plasma concentration versus time following a single 1000 mg intravenous infusion of dalbavancin.
• Figure 2 depicts isothermal titration calorimetry data for dalbavancin binding to human serum albumin (top) and a graphical representation of the data fitted to a curve determined from a 2:1 binding model of dalbavanci protein (bottom).
• Figure 3 depicts an electrospray ionization mass spectrum of dalbavancin.
• Figure 4 is a graph of dalbavancin concentration vs. population ratio of dalbavancin multimer to monomer and depicts an increase in population ratio of dalbavancin multimer to monomer with increasing dalbavancin concentration.
• Figure 5 is a graph of pH vs. population ratio of dalbavancin multimer to monomer and depicts an increase in population ratio of dalbavancin multimer to monomer with increasing pH.
• Figure 6 depicts an electrospray ionization mass spectrum of dalbavancin in an ammonium formate 5mM pH 5 solution.
• Figure 7 depicts an electrospray ionization mass spectrum of dalbavancin in an ammonium formate 50 mM pH 5 solution.
• Figure 8 depicts an electrospray ionization mass spectrum of dalbavancin in an ammonium formate 100 mM pH 5 solution.
• Figure 9 depicts an electrospray ionization mass spectrum of teicoplanin (50 ⁇ g/mL) in water.
• Figure 10 depicts an electrospray ionization mass spectrum of teicoplanin (100 ⁇ g/mL) in water.
• Figure 11 depicts the effect of HSA on the apparent dissociation constant for dalbavancin/tri-peptide binding at 26°C (pH 7.4).
• Figure 12 depicts a comparison of isothermal calorimetry (ITC) data for binding of tri-peptide to vancomycin and dalbavancin under identical conditions, using the same tri-peptide solution.
• ITC isothermal calorimetry
• Figure 13 (A and B) depicts the possible interaction of dalbavancin monomers and multimers (including dimers) with tri-peptide ligand and HSA.
• Figure 13(A) depicts dalbavancin in monomer-dimer equilibrium in solution, binding as monomer to two separate sites on HSA;
• Figure 13(B) depicts ligand binding to dalbavancin dimer in solution and more weakly to dalbavancin monomers attached to HSA.
• the present invention provides protein-dalbavancin complexes, pharmaceutical compositions of protein-dalbavancin complexes, and improved methods of treatment of antibiotic-resistant bacterial infections.
• this invention provides for dalbavancin compositions having activity against one or more antibiotic resistant strains of bacteria and particularly MRSA, which compositions can be administered on a dosing regimen of once every
• Dalbavancin which is also referred to in the scientific literature as BI 397 or
• NER001 is a semi-synthetic glycopeptide mixture, the properties of which have been reported in U.S. Pat. ⁇ os. 5,606,036, 5,750,509, 5,843,679, and 5,935,238.
• dibavancin refers to compositions comprising one or more, preferably two or more, closely related homologs, termed “Ao,” “A l5 “ “B 0 ,” “Bi,” “C 0 ,” and “Ci,” as described below, or monomers, multimers (i.e., dimer or higher order multimer), tautomers, esters, solvates, or pharmaceutically acceptable salts thereof.
• Ao a vancin
• dimer or “multimer” refers to either a homodimer or homomultimer, i.e., a dimer or multimer composed of monomers of the same dalbavancin homolog, or a heterodimer or heteromultimer, i.e., a dimer or multimer composed of monomers of at least two different dalbavancin homologs.
• Dalbavancin often includes "MAG," a non-homolog variant described below. Individually, dalbavancin homologs and MAG are sometimes referred to herein as “dalbavancin components.”
• Dalbavancin is prepared by chemical modification of the natural glycopeptide complex A-40,926 as described in Malabarba and Donadio (1999) Drugs of the Future
• amoxint of each of the components present in a dalbavancin composition is dictated by a variety of factors, including, for example, the fermentation conditions employed in the preparation of the natural glycopeptide complex A-40926, which is the precursor to dalbavancin (see, e.g., U.S. Pat. No.
• dalbavancin compositions comprise at least about 80 to about 98% by weight of the Bo component.
• dalbavancin comprises the following amounts of B 0 : Table 1. Preferred Amounts of Bo Component in Dalbavancin Composition
• each range represents the mole % of B 0 relative to the total dalbavancin_components present in the dalbavancin composition including MAG
• dalbavancin components are bactericidally active against a number of Gram-positive bacteria.
• MAG non-homologous dalbavancin component
• MAG is thought to be a decomposition product of one or more of the other dalbavancin components. Accordingly, in a preferred embodiment, the amount of MAG in 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.
• Dalbavancin is thought to inhibit the biosynthesis of the bacterial cell wall by binding to D-alanyl-D-alanine-terminating precursors of peptidoglycans.
• Dimeric or higher order multimers of dalbavancin may possess further antibacterial properties by interaction of the lipophilic side chains with the cytoplasmic membrane of bacteria. See, for example, Malabarba and Ciabatti, et al. (2001) Current Medicinal Chemistry 8:1759-1773.
• a further elaboration on dalbavancin multimers may be found in U.S. Serial No. 10/ , entitled "DALBAVANCIN
• 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 in vitro and in vivo against a number of Gram-positive bacteria, including most drug resistant strains.
• Dalbavancin is typically administered to an individual as a dalbavancin composition.
• the term "dalbavancin composition” or “dalbavancin formulation” refers to a composition, typically a pharmaceutical composition comprising dalbavancin, as defined above, and one or more other non-dalbavancin components such as, for example, a pharmaceutically acceptable carrier, a stabilizer, a buffers, or other similar components.
• dalbavancin is effective at dose intervals of one week.
• an advantage of dalbavancin versus other treatment options is the ability to administer this antibiotic on a once- eekly basis, thereby maximizing patient compliance and potentially minimizing the need for or decreasing the length of a hospital stay for parenteral antibiotic administration. Less frequent dosing often permits treatment on an out-patient basis, thus decreasing treatment costs.
• a second dose of dalbavancin approximately one week after administration of the first dosage, where the second dose is approximately one-half the first dose unexpectedly provides significant improvement in the efficacy of treatment.
• dalbavancin has been found to form complexes with proteins, e.g. , endogenous proteins, that provide a substantial retention of dalbavancin antibacterial activity and that allow systemic tissue distribution of dalbavancin in a mammal to unexpectedly high levels.
• proteins e.g. , endogenous proteins
• the invention provides protein-dalbavancin complexes that retain physiological antibacterial activity, e.g., the ability to reduce or eliminate a bacterial infection.
• the protein- dalbavancin complexes of the invention further permit systemic tissue distribution of dalbavancin.
• protein-dalbavancin complex refers to a protein molecule bound to at least one dalbavancin molecule. Some embodiments of the invention refer to a plurality of such complexes.
• the binding may be non-covalent or covalent. However, without being limited to any theory, it is believed that the complex constitutes non-covalent binding of one or more molecules of a dalbavancin component to the protein and, as such, the binding may be ionic and/or hydrophobic in nature.
• the complex may be formed in vivo, in vitro, or ex vivo. Often the complex comprises dalbavancin monomer; often, the complex includes a dalbavancin multimer, e.g., a dalbavancin dimer.
• the ratio of protein molecule to dalbavancin molecule is generally 1 : 1 (one dalbavancin molecule per protein molecule) or 0.5:1 (two dalbavancin molecules per protein molecule), although lower ratios are encompassed by the invention.
• the two dalbavancin molecules may bind to the protein as two monomers or as a single dimer.
• the 0.5:1 complex constitutes two molecules of a dalbavancin component in monomeric form bound to different sites on the protein.
• the dalbavancin component(s) of the complex may be any of the components of dalbavancin; if more than one dalbavancin component is bound to the protein, each component may be the same or different from the other component(s).
• Some embodiments provide a plurality of protein-dalbavancin complexes, where the B 0 component of dalbavancin accounts for at least 90% of the dalbavancin molecules (components) in the complexes.
• Some embodiments of the invention provide a plurality of protein-dalbavancin complexes that contain at least about 80, 85, 90, 95, or 98 % B 0 , relative to the total dalbavancin in the complexes, either in an individual in which complexes form in vivo, or in a composition prepared ex vivo or in vitro.
• the protein-dalbavancin complexes contain at least 90% of the B 0 homolog.
• the amount of MAG in the total complexes, relative to overall dalbavancin content, is less than about 4, 3.5, 3, 2.5, 2, 1.5, 1, or 0.5% MAG.
• MAG does not bind substantially to some proteins useful in the protein-dalbavancin complexes of the invention; e.g. , isothermal calorimetry data indicate that MAG does not bind substantially to human serum albumin. Without being limited to theory, this is consistent with a mechanism of binding of dalbavancin to HSA primarily involves the fatty acid side chain group. Percentages in all cases are mole percent.
• the dalbavancin of the protein-dalbavancin complexes of the invention may comprise a pharmaceutically acceptable, non-toxic salt of dalbavancin.
• suitable salts of dalbavancin include salts formed by standard reaction with both organic and inorganic acids such as, for example, hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, trifluoroacetic, trichloroacetic, succinic, citric, ascorbic, lactic, maleic, glutamic, camphoric, glutaric, glycolic, phthalic, tartaric, lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic, and the like acids.
• alkali metal or alkaline earth metal hydroxides such as sodium, potassium, calcium, magnesium, and barium hydroxide
• ammonia and aliphatic, alicyclic, or aromatic organic amines such as methylamine, dimethylamine, diethylamine, ethanolamine, and picoline.
• Dalbavancin is often provided for formation of a protein- dalbancin complex as a hydrochloride salt, which is freely soluble in water.
• the "protein" of the protein-dalbavancin complex may be an endogenous protein.
• endogenous protein refers to a protein (i.e., a type of protein, for example, serum albumin) produced by an individual to whom dalbavancin is administered, or a modified form thereof.
• Modified forms of endogenous proteins include synthetically or recombinantly produced variants.
• Modified forms of endogenous proteins may include functionalities with improved binding to dalbavancin, improved stability in vivo and/or in vitro, and/or enhanced antibacterial activity when complexed with dalbavancin.
• the endogenous protein is an albumin, such as, for example, human serum albumin.
• an "endogenous protein” may be extracted from an individual to whom dalbavancin is to be administered, extracted from another individual, or may be produced recombinantly and/or via chemical synthesis.
• "endogenous protein” refers to a protein type, such as human serum albumin, that is endogenous to the individual to whom dalbavancin is to be administered, whether the source of the protein is, e.g., the individual, another individual, or a recombinant source.
• Recombinant human serum albumin suitable for pharmaceutical administration is described in, e.g., U.S.
• the protein-dalbavancin complexes of the invention may contain any ratio of protein molecule and dalbavancin molecules. In some complexes, the ratio of protein molecules to dalbavancin molecules is 1 :1. In some complexes, the ratio of protein molecule to dalbavancin molecules is about 0.5:1, i.e., two dalbavancin molecules per protein molecule. In the latter complexes, the dalbavancin molecules may bind to the protein as a dimer, or as two monomers.
• complexes with a ratio of protein to dalbavancin of 1 :1 comprise about 90%, 95%, or 99%, of the total protein-dalbavancin complexes in an individual.
• complexes with a ratio of protein to dalbavancin of 1:1 comprise about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of the total protein-dalbavancin complexes.
• the remainder of the complexes contain a ratio of protein molecule to dalbavancin of less than 1:1, e.g. 0.5:1 (two dalbavancin molecules per protein molecule).
• the protein: dalbavancin ratio may be measured by means known in the art.
• an exemplary method is to find the difference between total dalbavancin and free dalabavancin.
• Total dalbavancin may be measured by means described herein.
• Free dalbavancin may be determined by, e.g., microdialysis or ultracentrifugation.
• electrophoresis with, e.g., labeled dalbavancin, may be used to directly measure protein-dalbavancin complexes.
• a protein-dalbavancin complex may be formed in vivo, by administration of dalbavancin to an individual, or ex vivo or in vitro, by contacting dalbavancin and a protein, e.g., an endogenous protein, under suitable conditions.
• the complex may be formed ex vivo or in vitro prior to administration or may be formed in vivo upon administration of dalbavancin.
• the complex is formed upon controlled administration of dalbavancin.
• the complex is formed upon intravenous administration of dalbavancin over a time period of at least about 30 minutes duration.
• Protein-dalbavancin complexes are formed in vivo administering a suitable dose of dalbavancin to an individual, e.g. in a human, about 200 mg, 500 mg, 1000 mg, or more than about 1000 mg of dalbavancin, at a controlled rate, e.g., by controlled intravenous infusion, over a period of about 10, 20, 30, or more than about 30 minutes.
• a suitable dose of dalbavancin to an individual, e.g. in a human, about 200 mg, 500 mg, 1000 mg, or more than about 1000 mg of dalbavancin, at a controlled rate, e.g., by controlled intravenous infusion, over a period of about 10, 20, 30, or more than about 30 minutes.
• protein-dalbavancin complexes in which at least about 90%, or at least about 95%, of the complexes have a ratio of protein molecules to dalbavancin molecules of 1 :1, are formed in vivo by controlled intravenous infusion to a human of a dose of dalbavancin of about 1000 mg over a time period of at least about 30 minutes, so that a serum concentration of dalbavancin (i.e., free dalbavancin and dalbavancin in a complex) of at least about 200 mg/L is reached.
• dalbavancin i.e., free dalbavancin and dalbavancin in a complex
• a protein-dalbavancin complex may also be formed in vitro, and either stored in solution, often as a sterile pharmaceutical composition, or lyophilized under conditions in which stability of the complex is retained. If desired, a solution or lyophilized composition including a protein-dalbavancin complex may be sterilized, for example, by e-beam or gamma sterilization methods, or by sterile filtration. Heat sterilization may be used provided there is no denaturation of the protein and/or loss of antibacterial properties.
• In vitro formation may be achieved by contacting the dalbavancin and protein, e.g. , an endogenous protein, under suitable conditions so that complexes with the desired ratio of dalbavancin components, as well as the desired ratio of protein molecules to dalbavancin, are formed.
• the ratio of individual components of dalbavancin may be modulated by adjusting the ratios of individual components in the dalbavancin used to form the complexes.
• dalbavancin that comprises at least about 90%, 95%, or 98% B 0 component, is used in the preparation of the protein-dalbavancin complexes.
• dalbavancin that comprises between about 80% and about 98% Bo is used in the preparation of the protein-dalbavancin complexes.
• the ratio of protein molecules to dalbavancin molecules in the protein-dalbavancin complexes may be modulated by adjusting the molar ratio of protein and dalbavancin in the mixture in which complexes are formed.
• a ratio of protein (e.g., HSA) to dalbavancin of 1 :1 or more results in protein-dalbavancin complexes in which the percentage of complexes where the ratio of protein molecule to dalbavancin is 1:1 is at least 90%, and generally at least 95%.
• the protein used in in vitro formation may be any protein that does not produce an immunological response in the individual to whom it will be delivered sufficient to interfere with the antibacterial effect of dalbavancin.
• the protein may be any endogenous serum protein, e.g., ⁇ -1 glycoprotein or serum albumin (e.g., human serum albumin).
• the protein is an endogenous protein and is human serum albumin, and in some of these embodiments the human serum albumin is recombinant hximan serum albumin, produced by means known in the art. See, e.g. , U.S. Pat. Nos. 5,962,649; 5,986,062; and 6,617,133 which patents are incorporated herein by reference in their entirety.
• a protein-dalbavancin complex typically will retain physiological antibacterial activity for at least 5 days, often at least one week, sometimes at least 15 days, in vitro.
• a plasma level of dalbavancin in a protein-dalbavancin complex is often maintained at a therapeutically effective level of at least about 4, 5, 10, 20, or 30 mg dalbavancin per liter for at least about 5 days.
• a striking feature of the protein-dalbavancin complexes of the invention is their retention of the antibacterial effect of dalbavancin.
• Dalbavancin in serum may be more than 95%.
• protein-bound, but its in vivo activity greatly exceeds what would be expected for a compound with a free fraction of less than 5%.
• the protein- dalbavancin complex retains at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, or more than 50% of the antibacterial activity of free dalbavancin at the same concentration, in vitro or in vivo.
• Another striking feature of the protein-dalbavancin complexes of the invention is that, despite the high percentage of dalbavancin that can exist as a complex with protein, the drug is widely distributed, i.e., its volume of distribution in vivo approximates extracellular water volume.
• Methods are provided for administration of dalbavancin to an individual in need of treatment for a bacterial infection under conditions where a protein-dalbavancin complex forms. Methods are also provided for administration of a protein-dalbavancin complex to an individual in need of treatment for a bacterial infection. Treatment can include prophylaxis, therapy, or cure. Methods include administration of one or more unit doses of dalbavancin in a therapeutically or prophylactically effective amount. Methods also include administration of non-dalbavancin antibiotics in addition to dalbavancin or protein-dalbavancin complex.
• therapeutically effective amount refers to the amount of protein- dalbavancin complex that will render a desired therapeutic outcome (e.g., reduction or elimination of a bacterial infection).
• a therapeutically effective amount may be formed by administration of one or more doses of dalbavancin, or may be administered as one or more doses of protein-dalbavancin complex.
• prophylactically effective amount refers to an amount of protein-dalbavancin complex sufficient to prevent or reduce severity of a futxxre bacterial infection when formed in or administered to an individual who is susceptible to and/or who may contract a bacterial infection by virtue of a medical procedure or stay in the hospital, or exposure to an individual with a bacterial infection.
• Dalbavancin or protein-dalbavancin complex is generally administered in a pharmaceutically acceptable carrier.
• Dalbavancin is often provided as a hydrochloride salt, which is freely soluble in water.
• “individual” refers to a vertebrate, typically a mammal, often a human.
• dalbavancin or protein-dalbavancin complex is administered as a "unit dose" 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 2 days, 5 days, one week, or 10 days, when administered to an individual.
• the novel dosage regimen available for dalbavancin and protein-dalbavancin complexes results in improved efficacy because blood levels of dalbavancin are maintained above minimum bactericidal levels for the entire treatment protocol.
• dalbavancin a novel dosage regimen available for dalbavancin results in improved efficacy because at concentrations required to effect less frequent dosing, dalbavancin exhibits minimal adverse effects in vivo, evidencing a large pharmaceutical window, and further because blood levels of dalbavancin are maintained above minimum bactericidal levels for the entire treatment protocol, evidencing a prolonged serum half-life for dalbavancin.
• the combination of the large pharmaceutical window coupled with prolonged serum half-life permits less frequent dosing of dalbavancin.
• dalbavancin is preferably formulated with a stabilizer which inhibits degradation of one or more of the components of dalbavancin.
• dalbavancin is formulated with a 1 :2 weight ratio of mannitohdalbavancin.
• dalbavancin is formulated with a 1 : 1 :4 weight ratio of mannitol : lactose :dalbavancin.
• dalbavancin is administered under conditions such that a dalbavancin-protein complex forms, and at a dosage that results in therapeutically effective (e.g., bactericidal) plasma levels of the drug for several days, often at least about 5 to about 10 days, often at least about one week.
• protein-dalbavancin complex is administered at a dosage that results in therapeutically effective (e.g., bactericidal) plasma levels of the drug for several days, often at least about 5 to about 10 days, often at least about one week.
• dalbavancin is maintained in plasma at or above the minimum bactericidal concentration of about 4 mg/1 for at least 5 days.
• dalbavancin is maintained at a plasma level of at least about 5 mg/1, often at least about 10 mg/1, often at least about 20 mg/1, often at least about 30 mg/1, often at least about 40 mg/1, for at least 5 days, often at least about one week or longer.
• Plasma levels of dalbavancin may be measured by methods that are well known in the art, such as liquid chromatography, mass spectrometry, or microbiological bioassay.
• An example of a method for quantitating dalbavancin in plasma is provided in Example 5.
• Upper limits for dalbavancin plasma concentration levels are generally dictated by dosages which inhibit unacceptable adverse effects in the patient population treated.
• dalbavancin may be administered, under conditions where protein-dalbavancin complexes form, in a single dose or in multiple doses. In some embodiments, dalbavancin is administered, under conditions where protein-dalbavancin complexes form, weekly for two or more weeks. In one embodiment, dalbavancin is administered, under conditions where protein-dalbavancin complexes form, in at least two doses, often in two doses about 5 to about 10 days apart, more often once a week for two weeks.
• Dalbavancin also may be administered, under conditions where protein-dalbavancin complexes form, in multiple doses two or more days or at least one week apart or in one or more biweekly doses.
• dalbavancin is administered, under conditions where protein- dalbavancin complexes form, weekly, followed by biweekly, or monthly administration.
• dalbavancin is administered, under conditions where protein-dalbavancin complexes form, at weekly intervals for 2, 3, 4, 5, 6, or more weeks.
• protein-dalbavancin complex may be administered in a single dose or in multiple doses. In some embodiments, protein-dalbavancin complex is administered weekly for two or more weeks. In one embodiment, protein-dalbavancin complex is administered in at least two doses, often in two doses about 5 to about 10 days apart, more often once a week for two weeks. Protein-dalbavancin complex also may be administered in multiple doses two or more days or at least one week apart or in one or more biweekly doses. In some embodiments, protein-dalbavancin complex is administered weekly, followed by biweekly, or monthly administration. In some embodiments, protein-dalbavancin complex is administered at weekly intervals for 2, 3, 4, 5, 6, or more weeks.
• dalbavancin is administered under conditions where protein- dalbavancin complexes form, or where protein-dalbavancin complex is administered
• most advantageously daily dosing is not required because higher, less frequent doses are used.
• Single or multiple doses may range in dalbavancin content, for example, from about 0.1 to about 5 grams.
• a single dose with a dalbavancin content of about 0.1 to about 4 grams, e.g., about 3 grams, may be administered for various infection treatments.
• each dose may range in dalbavancin content, for example, from about 0.25 to about 5.0 grams.
• dialbavancin content of a dose refers to the amount of dalbavancin present whether administered as free dalbavancin or as part of a protein-dalbavancin complex, or as a mixture of the two.
• the dalbavancin content of the dose may be, for example, 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 e.g., about 3 grams.
• a single dose containing about 1, 1.5, 2, 2.5, or 3 grams of dalbavancin is administered for treatment of a bacterial infection.
• the dalbavancin content of the dose may be, for example, about 0.1 to about 3 grams, or about 0.1 to about 1 gram, e.g., about 0.5 or about 0.25 gram.
• the individual dosages may be the same or different.
• Each dose may be given in the form of free dalbavancin (under conditions where a protein-dalbavancin complex forms), or in the form of protein-dalbavancin complex, or a mixture of the two.
• a first, higher dose is administered, that is, for example, about 1.5 to 3 times higher in dalbavancin content, than one or more subsequent doses.
• the first dose may contain 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 contain about 0.8 to about 2 g and the second dose about 0.4 to about 1 gram, or the first dose may contain about 0.4 to about 3 g and the second dose about 0.2 to 1.5 g.
• At least two dosages are administered wherein the first dosage includes about twice as much dalbavancin as subsequent dosages.
• a first dosage contains about 1 gram of dalbavancin and a subsequent dosage contains about 0.5 gram.
• a first dosage contains about 0.5 gram of dalbavancin and a subsequent dosage contains about 0.25 gram.
• dalbavancin or protein-dalbavancin complex is administered in two doses of equal or different amount two or more days or at least about one week apart. Often, two doses containing about 0.2 to about 1.5 grams of dalbavancin are administered about 5 to about 10 days apart, more often about 1 week apart. In one embodiment, a first dosage containing about 1 gram of dalbavancin and a second dosage containing about 0.5 gram of dalbavancin are administered about 1 week apart.
• the time between doses may range, for example, from about 5 to about 10 days, often about one week.
• Dose frequency 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 or more days.
• the number of doses given can be, for example, one, two, three, four, five, six or more doses, each dose after the initial dose being given after the selected dosage interval.
• the trough level or the level of dalbavancin in plasma after a first dose of dalbavancin or protein-dalbavancin complex and just prior to administration of a second dose, is at least about 4 mg/1.
• the trough level at the end of a dosing interval such as about one week is at least about 10 mg/1, more preferably at least about 20 mg/1, more preferably at least about 30 mg/1, and even more preferably at least about 40 mg/1.
• Dalbavancin or protein-dalbavancin complex can be administered parenterally, e.g. , intramuscularly (i.m.), intravenously (i.v.), subcutaneously (s.c), intraperitoneally (i.p.), or intrathecally (i.t).
• the dosing schedule and actual dosage administered may vary depending on such factors as the nature and severity of the infection, the age, weight, and general health of the patient and the tolerance of a particular patient to dalbavancin or protein-dalbavancin complex, but will be ascertainable to health professionals.
• an intravenous dose containing one gram of dalbavancin is followed by an intravenous dose containing 0.5 gram dalbavancin one week later.
• Administration and delivery of dalbavancin or protein-dalbavancin complex to the patient, e.g., intravenously, can be done at a controlled rate, so that the concentration in the blood does not increase too quickly or cause precipitation to occur.
• the infusion duration can be, for example, about 1 minute to about 2 hours.
• an infusion duration of about 30 minutes may be used where the dose contains about 0.5 to about 1 gram dalbavancin.
• the infusion duration for a dose containing about 0.5 to about 1 gram of dalbavancin may be shorter, e.g., about 1, 2, 5, 10, 15, 20, or 25 minutes.
• Intravenous administration of dalbavancin or protein-dalbavancin complex under controlled rate conditions can generate concentrations of dalbavancin in the body that are in great excess of what can be achieved at physiological pH in vitro.
• the content of dalbavancin administered may be any of the dosages disclosed herein.
• the dalbavancin dose is generally chosen such that the drag will remain at a therapeutically or prophylactically effective (i.e., bactericidal) plasma level for an extended period of time, often at least 5 days, more often about one week or longer.
• Administration of a dose of dalbavancin or protein-dalbavancin complex which produces and maintains bactericidal concentrations for at least about one week (or about 5 to about 10 days) is preferred.
• a bactericidal concentration is defined as the concentration of dalbavancin required to kill at least 99% of the bacteria present at the initiation of an in vitro experiment over a 24 hour period.
• a minimum bactericidal concentration of dalbavancin in plasma is typically about 4 mg/1.
• indications that can be treated include both complicated and uncomplicated skin and soft tissue infections (SSTI), blood stream infections (BSI), catheter- related blood stream infections (CRBSI), osteomyelitis, prosthetic joint infections, surgical prophylaxis, endocarditis, hospital or community acquired pneumonia, pneumococcal pneumonia, empiric treatment of febrile neutropenia, joint space infections, and device infections (e.g., pace makers and internal cardiac defibrillators).
• Gram-positive or antibiotic- resistant bacterial infections may be treated, such as a Staphylococcus, Streptococcus, Neisseria, or Clostridium genus infection, in particular Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Streptococcus pyogenes, Groups A andC Streptococcus, Neisseria gonorrhoeae, or Clostridium difficile.
• Staphylococcus, Streptococcus, Neisseria, or Clostridium genus infection in particular Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Streptococcus pyogenes, Groups A andC Streptococcus, Neisseria gonorrhoeae, or Clostridium difficile.
• the invention provides methods for treatment of skin and soft tissue infections (SSTIs). Patients who may benefit from this treatment may have either deep or superficial infections. SSTI may involve deeper soft tissue and/or require significant surgical intervention, such as for example a major abscess, infected ulcer, major burn, or deep and extensive cellulitis. Infected surgical wounds may also be treated.
• SSTI skin and soft tissue infections
• the clinical presentation of skin and skin structure infection may vary from mild folliculitis to severe necrotizing fasciitis.
• the mode of acquisition may also vary with community-acquired skin and skin structure infections, which are often preceded by injuries resulting from occupational exposure or recreational activities, and are usually associated with a greater diversity of pathogens.
• Hospital-acquired skin and skin structure infections are generally associated with surgical procedures, the development of pressure sores, and catheterization.
• Post-surgical infections are the third most frequent nosocomial infection and account for 17% of all nosocomial infections reported to the National Nosocomial Infection Surveillance System (NNIS).
• NIS National Nosocomial Infection Surveillance System
• the most frequent source of infection is the patient's endogenous flora.
• Staphylococcus aureus, coagulase-negative staphylococci, and Enterococcus spp. are the pathogens most frequently isolated from SSTIs.
• Symptoms of SSTI infections may include erythema, tenderness or pain, heat or localized warmth, drainage or discharge, swelling or induration, redness, or fluctuance.
• Patients that may benefit from treatment with the methods of the invention include those with deep or complicated infections or infections that require surgical intervention, or patients with underlying diabetes mellitus or peripheral vascular disease. These infections are often caused by Gram-positive bacteria such as Staphylococcus or Streptococcus species, such as Staphylococcus aureus or Streptococcus pyogenes.
• Methods for treatment of a skin or soft tissue bacterial infection include administering a therapeutically effective amount of dalbavancin to an individual in need of treatment, in a form (i.e., dalbavancin or protein-dalbavancin complex), amount, and according to a dosing regime as discussed above.
• dalbavancin or protein-dalbavancin complex is administered intravenously in two doses, often about 5 to about 10 days apart, more often about 1 week apart.
• the first dosage contains, at least twice as much dalbavancin as the second dosage.
• the first dosage contains about 1000 mg of dalbavancin and the second dosage contains about 500 mg of dalbavancin.
• the invention also provides methods for prophylactic prevention of the onset of a bacterial infection, for example an infection caused by Staphylococcus aureus, or by a Neisseria or Clostridium genus bacterium.
• a prophylactically effective amoxmt of dalbavancin or protein-dalbavancin complex is administered to an individual who may be susceptible to contracting a bacterial infection, for example through a medical procedure.
• dalbavancin or protein-dalbavancin complex is administered in an amount sufficient to provide a prophylactically effective plasma level of dalbavancin for at least about 3 days, at least about 5 days, or at least about one week or longer.
• Dalbavancin or protein- dalbavancin complex may be administered, for example, parenterally, e.g., via intramuscular (i.m), intravenous (i.v.), intraperitoneal (i.p.), subcutaneous (s.c), or intrathecal (i.t.) injection, prior or subsequent to surgery as a preventative step against infection.
• Dalbavancin or protein- dalbavancin complex may be administered immediately prior or subsequently to, 1 or more days or about one week prior or subsequently to, or during an invasive medical procedure such as surgery or a stay in a medical care facility such as a hospital to prevent infection.
• a prophylactic method may be used in any situation in which it is possible or likely that an individual may contract a bacterial infection, including situations in which an individual has been exposed to or is likely to be exposed to a bacterially infected individual.
• dalbavancin or protein-dalbavancin complex may be administered as either a single dose or as two or more doses of equal or different amount that are administered several days to about one week apart.
• dalbavancin or protein-dalbavancin complex may be administered prior to or simultaneously with insertion of an intravenous catheter in order to prevent a bloodstream related infection.
• dalbavancin or protein-dalbavancin complex may be administered in a single dose or in multiple doses, according to any of the dosing schemes described above.
• dalbavancin or protein-dalbavancin complex is administered as a single dose containing about 0.1 to about 3 grams of dalbavancin, or about 0.1 to about 1 gram, e.g., about 0.25 gram or about 0.5 gram.
• a single dose containing about 0.25 gram is administered intravenously over a time frame of about 2 minutes to about 1 hour, e.g., about 30 minutes.
• dalbavancin or protein-dalbavancin complex is administered intravenously simultaneously with administration of another pharmaceutical (e.g., antibiotic) treatment.
• another pharmaceutical e.g., antibiotic
• dalbavancin or protein-dalbavancin complex may be administered either simultaneously or sequentially with at least one other antibiotic.
• at least one other antibiotic that is bactericidally effective against one or more Gram-negative bacterial species and/or a Gram- positive bacterial strain against which dalbavancin is not effective is administered in addition to dalbavancin or protein-dalbavancin complex.
• dalbavancin or protein- dalbavancin complex and at least one antibiotic that is bactericidally effective against at least one Gram-negative bacterial species is administered as a mixture.
• compositions of the invention may be in the form of a unit dose of dalbavancin or protein-dalbavancin complex that contains 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 longer when the composition is administered to an individual, and a pharmaceutically acceptable carrier.
• a therapeutically or prophylactically effective plasma level of dalbavancin is at least about 4 mg per liter of plasma.
• Plasma levels of dalbavancin may be measured by well known methods in the art, such as those described above.
• Dalbavancin or the dalbavancin component of a protein-dalbavancin complex may optionally be in a pharmaceutically acceptable form for administration to an individual, optionally as a pharmaceutically acceptable, non-toxic salt.
• Suitable salts of dalbavancin include salts formed by standard reaction with both organic and inorganic acids such as, for example, hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, trifluoroacetic, trichloroacetic, succinic, citric, ascorbic, lactic, maleic, glutamic, camphoric, glutaric, glycolic, phthalic, tartaric, laxiric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic, and the like acids.
• organic and inorganic acids such as, for example, hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, trifluoroacetic, trichloroacetic, succinic, citric, ascorbic, lactic, maleic, glutamic, camphoric, glutaric, glycolic, phthalic, tartaric, l
• a pharmaceutically acceptable aqueous formulation of dalbavancin or protein-dalbavancin complex is provided that is suitable for parenteral administration, such as, for example, intravenous injection.
• parenteral administration such as, for example, intravenous injection.
• methods well known in the art may be used, and any pharmaceutically acceptable carriers, diluents, excipients, or other additives normally used in the art may be used.
• a pharmaceutical composition for parenteral administration includes dalbavancin or protein-dalbavancin complex and a physiologically acceptable diluent such as deionized water, physiological saline, 5% dextrose, water miscible solvent (e.g., ethyl alcohol, polyethylene glycol, propylene glycol, etc.), non-aqueous vehicle (e.g., oil such as corn oil, cottonseed oil, peanut oil, and sesame oil), or other commonly used diluent.
• a physiologically acceptable diluent such as deionized water, physiological saline, 5% dextrose, water miscible solvent (e.g., ethyl alcohol, polyethylene glycol, propylene glycol, etc.), non-aqueous vehicle (e.g., oil such as corn oil, cottonseed oil, peanut oil, and sesame oil), or other commonly used diluent.
• a physiologically acceptable diluent such
• the formulation may additionally include a solubilizing agent such as polyethylene glycol, polypropylene glycol, or other known solubilizing agent, buffers for stabilizing the solution (e.g., citrates, acetates, and phosphates) and/or antioxidants (e.g., ascorbic acid or sodium bisulfite).
• a solubilizing agent such as polyethylene glycol, polypropylene glycol, or other known solubilizing agent
• buffers for stabilizing the solution e.g., citrates, acetates, and phosphates
• antioxidants e.g., ascorbic acid or sodium bisulfite
• suitable pharmaceutical carriers and their formulations are described in "Remington's Pharmaceutical Sciences” by E. W. Martin.
• pharmaceutical preparations of the invention may also be prepared to contain acceptable levels of particulates (e.g., particle-free) and to be non-pyrogenic. (e.g., meeting the requirements of an injectable in the U.S. Pharmacopei
• a pharmaceutical composition is provided by dissolving a dried (e.g., lyophilized) dose of dalbavancin, often containing a stabilizer or mixture of stabilizers, in an amount of water and preferably deionized water in a volume sufficient for solubilization.
• a dried (e.g., lyophilized) dose of dalbavancin often containing a stabilizer or mixture of stabilizers, in an amount of water and preferably deionized water in a volume sufficient for solubilization.
• the amoxmt of water sufficient for solubilization is approximately 10 mL and the resulting pH of the dalbavancin solution is above 3.0, and about 3.5 to 4.5.
• the pH of the dalbavancin solution in a drip bag is about 4.5.
• the second amoxmt of aqueous solution may be deionized or sterile, or both deionized and sterile.
• compositions for parenteral administration may be made up in sterile vials containing one or more unit doses of dalbavancin or protein-dalbavancin complex in a therapeutically or prophylactically effective amoxmt as described above, optionally including an excipient, under conditions in which bactericidal effectiveness of dalbavancin is retained.
• the composition may be in the form of a dry (e.g., lyophilized) powder.
• a physiologically acceptable diluent may be added and the solution withdrawn via syringe for administration to a patient.
• a pharmaceutical formulation as described above may be sterilized by any acceptable means including, for example, by e-beam or gamma sterilization methods, or by sterile filtration.
• a typical formulation for parenteral administration may contain dalbavancin, alone or as part of a protein-dalbavancin complex, at a concentration such as about 0.1 to about 100 mg, about 0.5 to about 50 mg, about 1 to about 10 mg, or about 2 to about 4 mg of dalbavancin per ml of final preparation.
• a pharmaceutical composition in accordance with the invention includes a mixture of dalbavancin or protein-dalbavancin complex and one or more additional antibiotics.
• at least one non-dalbavancin antibiotic in the mixture is bactericidally effective against one or more species of Gram-negative bacteria and/or one or more Gram-positive bacterial strains against which dalbavancin is not effective.
• the mixture may also include a pharmaceutically acceptable carrier as described above.
• pharmaceutical compositions of the invention include one or more stabilizing substances which inhibit degradation of one or more of the components of dalbavancin to less active or inactive materials.
• stabilizing substance refers to a substance that stabilizes the level of one or more of the constituent components of dalbavancin, for example, B 0 , in the composition.
• a “stabilizing effective amount” refers to an amount of a stabilizer sufficient to enhance long-term stability of one or more components of a dalbavancin composition.
• a stabilizing effective amoxmt may be provided by a mixture of two or more stabilizing substances, each of which alone is not present in an amount sufficient to provide a stabilizing effect.
• stabilizers include, for example, nonionic substances such as sugars, e.g., mono-, di-, or polysaccharides, or derivatives thereof, sugar alcohols, or polyols.
• stabilizing substances include, for example, mannitol, lactose, sucrose, sorbitol, glycerol, cellulose, trehalose, maltose, raffinose, or mixtures thereof.
• the pharmaceutical composition includes a weight ratio of 1 :2 mannitol: dalbavancin. In another embodiment, the pharmaceutical composition includes a weight ratio of 1 : 1 :4 mannitol:lactose:dalbavancin. Often, the pH of a pharmaceutical composition of the invention is, for example, about 3 to about 5, for example about 3.5 or about
• one or more procedures may be employed to reduce formation of MAG.
• freeze drying of dalbavancin in the presence of a stabilizing substance, such as mannitol may be employed to reduce the amount of MAG formed.
• dalbavancin compositions are often at lower than ambient temperature, such as at about 5° C, to enhance stability.
• kits for use in methods of treatment or prophylaxis of bacterial infections include a pharmaceutical composition of the invention, for example including at least one unit dose of dalbavancin or protein-dalbavancin complex, and instructions providing information to a health care provider regarding usage for treating or preventing a bacterial infection. Instructions may be provided in printed form or in the form of an electronic medium such as a floppy disc, CD, or DVD, or in the form of a website address where such instructions may be obtained. Instructions may include information on administration conditions for dalbavancin that lead to the formation of a protein-dalbavancin complex.
• a unit dose of dalbavancin or protein-dalbavancin complex includes a dosage such that when administered to an individual, a therapeutically or prophylactically effective plasma level of dalbavancin is maintained in the individual for at least 5 days.
• a kit includes two unit dosages to be administered at least 5 days apart, often about one week apart, often including a first dosage of dalbavancin or protein-dalbavancin complex that is about 1.5 to about 3 times higher than the second dosage.
• Dalbavancin or protein-dalbavancin complex is often included as a sterile aqueous pharmaceutical composition or lyophilized composition.
• Kits of the invention may include, in addition to dalbavancin or protein-dalbavancin complex, a non-dalbavancin antibiotic or mixture of non-dalbavancin antibiotics, for use with dalbavancin or protein-dalbavancin complex as described in the methods above.
• kits of the invention may include information on preparation and administration of the kit contents (e.g., dalbavancin or protein-dalbavancin complex, and optionally non-dalbavancin antibiotics), possible adverse reactions, recommended dosages, dosing schedules, and the like.
• instructions may describe, for example, administration of dalbavancin under conditions so that a protein-dalbavancin complex forms, in order to treat or to prevent a bacterial infection.
• instructions may describe, for example, administration of protein-dalbavancin complex in order to treat or prevent a bacterial infection.
• instructions may describe, for example, administration of dalbavancin under conditions sush that a protein-dalbavancin complex forms, and simultaneous or sequential administration of non-dalbavancin antibiotics, in order to treat or to prevent a bacterial infection.
• instructions may describe, for example, administration of protein-dalbavancin complex, and simultaneous or sequential administration of non-dalbavancin antibiotics, in order to treat or to prevent a bacterial infection.
• Suitable packaging refers to a solid matrix or material customarily used in a system and capable of holding within fixed limits a dalbavancin or protein-dalbavancin complex composition suitable for administration to an individual.
• materials include glass and plastic (e.g., polyethylene, polypropylene, and polycarbonate) bottles, vials, paper, plastic, and plastic-foil laminated envelopes and the like. If e-beam sterilization techniques are employed, the packaging should have sufficiently low density to permit sterilization of the contents.
• Kits may also optionally include equipment for administration of dalbavancin or protein-dalbavancin complex, such as, for example, syringes or equipment for intravenous administration, and/or a sterile buffered solution for preparing a lyophilized composition for administration.
• equipment for administration of dalbavancin or protein-dalbavancin complex such as, for example, syringes or equipment for intravenous administration, and/or a sterile buffered solution for preparing a lyophilized composition for administration.
• DMEPA 3-(dimethylamino)-propylamine
• NMP N-methyl-2-pyrrolidone
• the intent-to-treat (ITT) population included all patients who received at least one dose of study drug (all randomized study subjects).
• the microbiological intent-to-treat (MITT) population were all ITT patients who had a culture-confirmed Gram-positive pathogen at baseline.
• the clinically-evaluable population were defined as those who 1) fulfilled all study entry criteria, 2) had no change in antimicrobial therapy for Gram-positive infection following Day 4, except for oral step-down therapy (only applied to standard of care group), 3) returned for the follow-up (FU) assessment visit (unless a treatment failure), and 4) did not receive a non- protocol approved concomitant antimicrobial (unless a treatment failxxre).
• the microbiologically-evaluable population was the subset of clinically-evaluable patients who had a culture-confirmed Gram-positive pathogen at baseline. [0125] The study populations are shown in Table 2.
• the effectiveness of the three treatment regimens was determined by assessing the patients' clinical response and the documented or presumed microbiological responses.
• the primary efficacy endpoint was clinical response at the follow-up visit for the clinically evaluable population.
• Clinical response, for both EOT and FU visits, was categorized as success (cure or improvement) or failure (including indeterminate results). Patients classified as successes must not have received additional systemic antibacterial treatment for their infection. Failure was defined as persistence of one or more local or systemic signs and symptoms of SSTI such that treatment with new or additional systemic antibacterial agents was required for the SSTI.
• Microbiological outcome a secondary efficacy variable, was assessed in the subpopulation of patients who had microbiologically documented SSTI (i.e., at least one identified baseline pathogen).
• a microbiologic response was assessed for each Gram-positive pathogen identified at baseline (i.e., eradication, presumed eradication, persistence, presumed persistence). For patients for whom follow-up cultures were not performed, the microbiologic responses for baseline pathogens were presumed on the basis of the clinical response.
• Microbiologic response by patient at the EOT and FU visits was graded as success (i.e., all Gram-positive organisms eradicated or presumed eradicated) or failure (i.e., at least one Gram- positive organism persisted or presumed to have persisted, multiple pathogens with partial eradication).
• success i.e., all Gram-positive organisms eradicated or presumed eradicated
• failure i.e., at least one Gram- positive organism persisted or presumed to have persisted, multiple pathogens with partial eradication.
• colonization and superinfection were assessed.
• a patient's bacteriological response could also include recurrence.
• aureus eradication rates were higher for the two-dose dalbavancin group (90%) compared with single-dose dalbavancin (50%) and standard of care (60%) treatments. Similar findings were observed for the MITT population; two-dose dalbavancin eradicated 80% of MRSA isolates (Table 5).
• microbiological success rates at EOT and FU are summarized in Table 6. Comparable microbiologic success rates were reported at both visits for patients treated with two-dose dalbavancin and standard of care regimens (approximately 64% to 77%), whereas those given a single dose of dalbavancin had lower rates of success ( ⁇ 40%).
• microbiologic success rates at EOT/FU in the microbiologically-evaluable population paralleled clinical response findings: 38.5%/ 27.3% for single-dose dalbavancin, 72.7%/72.7% for two-dose dalbavancin, and 71.4%/64.3% for standard of care therapy. Similar findings were observed for the MITT population (data not shown).
• Dalbavancin plasma concentrations were determined using validated liquid chromatography and mass spectrophotometer methods. The lower limit of quantitation was 500 ng/ml for plasma.
• AE adverse events
• S. aureus was the most frequently isolated organism at baseline. In this trial, approximately 83% of patients were infected with S. aureus and 38% of all S. aureus strains were MRSA. Most infections (80%) were caused by a single pathogen. The MICs for dalbavancin against Gram-positive isolates, including MRSA, ranged from 0.016 to 0.25 mg/L. [0144] Microbiological success rates paralleled those of clinical response for the clinically- evaluable population.
• Example 2 Pharmacokinetics and Renal Excretion of Dalbavancin in Healthy Subjects
• the primary objectives of this study were to characterize the pharmacokinetics of dalbavancin and to calculate the extent of renal excretion in healthy subjects receiving a therapeutic dose of the drag. This was an open label, non-comparative, study.
• Plasma and urine samples were collected on study days 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, and 42. Blood samples were drawn into heparinized tubes and centrifuged. Plasma was separated and stored frozen at -20°C until time of assay. Plasma and urine samples were assayed for dalbavancin using validated LC/MS/MS methods. The lower limit of quantitation of the assay was 500 ng/mL for urine and plasma.
• Plasma concentrations of dalbavancin versus time are shown for all subjects in Figure 1. Pharmacokinetic parameters are presented in Table 9. Concentrations were similar across all subjects. Peak plasma concentrations were approximately 300 mg/L and were achieved immediately following the end of infusion. Dalbavancin shows an apparent volume of distribution of more than 10 L and is assumed to be well distributed in the extracellular fluid. [0154] Dalbavancin was slowly eliminated with a t 2 of 9-12 days. The total drug clearance was 0.0431 ⁇ 0.0074 L/hr. The estimated fraction of drag excreted unchanged into urine was 42% of the administered dose, and renal clearance was estimated as 0.018 L/h. The variability observed across subjects was low with a coefficient of variation of less than 22% across all pharmacokinetic parameters.
• Adverse events were recorded and assessed for severity and relationship to study drag.
• Laboratory data chemistry panel, CBC with differential, urinalysis
• ECG, physical examination, and vital signs were obtained, and changes from baseline were assessed.
• Dalbavancin was well-tolerated in this study. No subject deaths or serious adverse events were reported during this study and no subject was prematurely withdrawn from study due to an AE.
• ALT value 46 IU/L, upper limit of normal 40 IU/L
• eosinophilia value 0.5 x 10 3 / ⁇ L, upper limit of normal 0.4 x 10 3 / ⁇ L
• elevated LDH value 303 IU/L, upper limit of normal 90 IU/L
• ALT value 54 IU/L, upper limit of normally 40 IU/L
• elevated AST value 42 IU/L, upper limit of normal 40 IU/L
• tinnitus in one subject.
• a single 1000 mg IV dose of dalbavancin was well-tolerated. Following a single intravenous infusion of 1000 mg, plasma concentrations of dalbavancin above 45 mg/1 are maintained for at least seven days. This is above concentrations known to be bactericidal (4-32 mg/1). This supports the use of dalbavancin as a once- weekly regimen.
• the urinary elimination profile indicates that renal excretion is an important elimination pathway, with approximately 40% excreted in urine. This finding is consistent with observations in animals. Since the kidneys are not the exclusive elimination route, a dosing adjustment for dalbavancin may not be necessary in renally impaired patients.
• Example 3 Protein Binding of Dalbavancin using Isothermal Titration Microcalorimetry [0160] Binding of dalbavancin to proteins was measured by isothermal titration microcalorimetry (ITC) in 20 mM phosphate, 150 mM NaCl, pH 7.4 at 25 and 37 °C using a Microcal VP-ITC instrument. In atypical experiment, 25 x 10 ⁇ l of protein ( ⁇ 150 ⁇ M) was injected into a calorimeter cell containing dalbavancin solution ( ⁇ 5 ⁇ M). Actual protein and dalbavancin concentrations were determined by measuring absorbence at 280 nm.
• ITC isothermal titration microcalorimetry
• Control experiments included injections of protein into buffer (in the absence of dalbavancin) to account for the heats of dilution of protein under identical conditions. For comparison, similar experiments with some necessary modifications were performed using teicoplanin. [0161] Experiments with dalbavancin were conducted with each of the following proteins: human albumin; dog albumin; rat albumin; bovine albumin; and human -glycoprotein. Teicoplanin was studied with human albumin and ⁇ -glycoprotein. A comparison of binding affinities at two different temperatures is shown in Table 10. Table 10. Comparison of apparent binding affinities (Ka, X 10 5 M "1 )
• Dog albumin 0.62 ( ⁇ 0.09) 0.50 ( ⁇ 0.13)
• Bovine albumin 1.38 ( ⁇ 0.14) ⁇ -glycoprotein 1.84 ( ⁇ 0.36) 4.8 ( ⁇ 2.3) Teicoplanin
• ⁇ H the enthalpy of binding (the size of signal related to binding)
• N number of binding sites (assuming the binding model is correct) [0163] Assuming non-competitive binding, N is the (relative) number of moles of injectant required to saturate all the available binding sites in the sample.
• dalbavancin is the "sample” and the protein (HSA, etc.) is the “injectant.”
• each protein apparently binds two dalbavancin molecules. It is possible that dalbavancin forms a dimer that binds 1:1 with a protein. Results of binding stoichiometry modeling suggests that two dalbavancin molecules are bound to one molecule of protein, unlike teicoplanin, which exhibits 1 : 1 binding.
• Table 11 presents the calculated percent bound for antibiotic concentrations in the range 1-500 ⁇ M, assuming physiological concentrations of human serum albumin (6 x 10 "4 M) and ⁇ -glycopeptide (1.5 x 10 "5 M). To relate this to the clinical situation, the peak concentration of dalbavancin in man is approximately 300 mg/L, or 165 ⁇ M.
• the binding and formation of a 2:1 dalbavancimprotein complex also explains the prolonged half-life, and the apparent volume of distribution, which approximates extracellular water volume.
• the low affinity helps explain the observed in vivo activity, which greatly exceeds what would be expected for a compoxmd with a free fraction close to 1%.
• the high capacity for plasma proteins helps to explain the relatively high plasma concentrations achieved in spite of poor solubility of the compound at physiological pH.
• Concentration-time profiles were determined for more than 40 tissues, including kidney, liver, spleen, blood, plasma, lung, and skin. Concentrations and t 2 values of drug- derived radioactivity in tissues, including skin, were comparable to those observed in plasma. Dalbavancin was found to be rapidly and extensively distributed with all tissues having quantifiable concentrations of drug-derived radioactivity within 12 hours after post-infusion. Most tissues reached maximum concentration (C max ) within 24 h after the dose. Recovered radioactivity after 5 days was ⁇ 5% of the dose in any single tissue. By 70 days after the dose, only the carcass retained > 1% (2.34%) of the administered radioactivity. Thus, dalbavancin did not accumulate in any single tissue, organ, or blood cellular component.
• bile samples were collected from bile duct cannulated rats (4 animals) through 384 h (16 days) post-dose. Almost 11% of the dose was recovered in the bile over 384 h after the dose. This represents the majority of the drug-derived radioactivity found in feces.
• dalbavancin Stock solutions of dalbavancin were prepared by dissolving dalbavancin in deionized water to prepare a 1000 ⁇ g/ml solution, followed by serial dilutions in deionized water to prepare 500, 50 and 10 ⁇ g/ml solutions.
• Calibration standards of 100, 60, and 40 ⁇ g/ml dalbavancin concentration were prepared by spiking human plasma with appropriate volumes of a 1000 ⁇ g/ml dalbavancin stock solution prepared as described above.
• Calibration standards of 20 and 10 ⁇ g/ml concentration were prepared by spiking human plasma with appropriate volumes of a 500 ⁇ g/ml dalbavancin stock solution, and a calibration standard of 0.5 ⁇ g/ml was prepared by spiking human plasma with an appropriate volume of a 10 ⁇ g/ml stock solution.
• Quality control standards of 90 and 30 ⁇ g/ml dalbavancin were prepared by spiking human plasma with an appropriate volume of a 1000 ⁇ g/ml dalbavancin stock solution prepared as described above.
• a quality control standard of 1.5 ⁇ g/ml was prepared by spiking human plasma with an appropriate volume of a 50 ⁇ g/ml solution.
• a 30 ⁇ g/ml working solution of internal standard BI-K0098 which is the diethyl- amino-propyl-amino derivative of A-40926, was prepared as follows. Approximately lOmg of BI-K0098 was dissolved in approximately 10 ml of mobile phase A (80% of lOmM Ammonium Formiate/Formic Acid, pH 3 (v/v), 10% of Acetonitrile (v/v), and 10% 2-Propanol (v/v)) to make a 1000 ⁇ g/ml internal standard stock solution. The stock solution (300 ⁇ l) was then diluted to a volume of 10 ml with mobile phase A to make a 30 ⁇ g/ml internal standard solution.
• mobile phase A 80% of lOmM Ammonium Formiate/Formic Acid, pH 3 (v/v), 10% of Acetonitrile (v/v), and 10% 2-Propanol (v/v)
• Samples were prepared as follows for quantitative determination of dalbavancin concentration in plasma. To 50 ⁇ l of calibration or quality control standards prepared as described above, lOO ⁇ l of internal standard working standard solution was added and mixed. The mixture was permitted to equilibrate for five minutes at room temperature, followed by addition of 250 ⁇ l of acetonitrile. The mixture was then vortexed for 10 seconds, followed by centrifugation for 1 minute at about 10,000 rpm on an ALC micro-centrifugette 4214. Supernatants were transferred to clean tubes and evaporated to dryness in a Savant Speed- Vac System at about 40 °C. Samples were then resuspended in 150 ⁇ l of mobile phase A.
• the HPLC system was coupled to a PE SCIEX API-2000 triple quadrapole mass spectrometer, with turbo ion spray operating in a positive ionization mode. Air was used to generate a spray in the ion source. Probe temperature was set at 500° C with nitrogen as curtain gas. Multiple reactions monitoring (MRM) was employed using nitrogen as collision gas. The analytes were detected by monitoring the following ion transitions: 909.3 Da ⁇ 1429.3 Da for dalbavancin, and 923.3 Da ⁇ 1457.3 Da for the internal standard (BI-K0098). To avoid mass spectrometer contamination, a post-column flow diversion in the first minute and 2.5 minutes after the beginning of the chromatographic run was performed.
• MRM Multiple reactions monitoring
• dalbavancin The stability of dalbavancin in plasma samples was tested by analyzing three replicates quality control standards of human plasma samples, prepared as described above, at two different concentrations, 1.5 and 90 ⁇ g/ml. Detectable dalbavancin concentration was stable after three cycles of freeze-thaw treatment. Dalbavancin concentration in processed samples was stable after 24 hours at room temperature. No reduction in dalbavancin concentration with respect to time zero samples was observed. Example 6. Dalbavancin Mass Spectroscopy Analysis.
• the data indicate that the population ratio of dalbavancin multimer to dalbavancin monomer increases with increasing concentration. This may help to explain the high drag loading capacities that may be administered to an individual.
• the role of multimer as a depot of monomer may decrease the tendency of higher concentration samples to form precipitates and enhance the concentrations which may be administered to an individual.
• the presence of multimers may also allow rapid administration of a dose of dalbavancin to an individual.
• a non-limiting example of a method of determining the population ratio of dalbavancin multimer to monomer is provided, for example, by determining the ratio between peak intensities of ions A and B as shown in Figure 3. Dividing the intensity of peak A by the intensity of peak B provides one measure of the population ratio of dalbavancin multimer to monomer.
• ionic groups such as a carboxylate group on a first dalbavancin monomer, aid in the stabilization of dalbavancin multimers by forming ionic interactions with oppositely charged ions, such as tertiary nitrogen groups, on a second dalbavancin monomer.
• ionic interactions can be influenced by pH. It is believed that the increasing tendency of dalbavancin to be present as a multimer at higher pH is indication that ionic interactions are important in multimer stabilization. In particular, it is believed that dalbavancin multimers are destabilized at lower pH, presumably due to interruption of the ionic interactions contributing to multimer stability as certain functional groups, such as carboxylate groups, may be protonated at lower pH values.
• a similar glycopeptide antibiotic does not show multimeric complexes in solution at various concentrations. This supports the indication that structurally similar compoxmds fail to form multimeric species in solution, and that this phenomenon may play an important role in the activity of the dalbavancin.
• Example 7 Matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry of protein-dalbavancin complexes
• 10 ul HSA, 0.150 mM was mixed with 10 ul dalbavancin solution (from 0.075 mM, 0.15 mM, 0.3 mM and 1.5 mM) and incubated for 60 min at 37°C.
• the samples were prepared for analysis using the dried droplet technique. Spectra were obtained on a BRUKER FLEX III, tof mass spectrometer previously tuned and calibrated using standard bovine serum albumin, acquiring and averaging spectra generated by 200 laser shots.
• Matrix 9 parts of DHB-9 (2,5- dihydroxy-benzoic acid) saturated in acetonitrile/H 2 O (50:50), 1 part of sinapinic acid saturated in acetonitrile/H 2 O (50:50). 0.5 ul of sample solution and 0.5 ul of matrix solution were mixed and placed on the laser target. [00179]
• Dalbavancin binds to the protein as the monomer (1 HSA + 1 dalbavancin). At very high dalbavancin:protein ratios (1:2, 1:10), the presence of complexes containing 2 molecules of dalbavancin per protein molecule can be observed.
• Isothermal titration calorimetry was performed using a Microcal VP-ITC instrument at 25 °C and 37 °C using standard operating procedures. See, e.g., Wisemanet al., Anal. Biochem. (1989) 179, 131-137; Cooper, et al, Philos. Trans. R. Soc. Lond. Ser. A-Math. Phys. Eng. Sci. (1993) 345, 23-35; Cooper, A, Isothermal Titration Microcalorimetry in C. Jones, B. Mulloy and A. H. Thomas (Eds.), Microscopy, Optical Spectroscopy, and Macroscopic Techniques.
• Vancomycin 10 1.1 6.90E+05 1.45 -9.49 -6.80 0
• K app ,L KLDHSA (for large [HSA])
• Figure 13B depicts ligand binding to the dalbavancin dimer in solution, and (more weakly) to the dalbavancin monomers attached to HSA. This is consistent with the non-competitive binding of dalbavancin to both tri-peptide and to HSA, with variable apparent stoichiometries.
• this Example shows that HSA reduces the binding affinity of dalbavancin for tri-peptide ligand in a manner consistent with a non-competitive mechanism and that dalbavancin bound to HSA retains its ability to bind tri-peptide ligands, albeit with reduced affinity.
• the pH of the permeate solution was adjusted at pH 7 with 30 % sulfuric acid (stored at 20 °C).
• 25 m of filtered broth containing 6.62 Kg of A- 40926 (268 mg/L) was obtained.
• the deacetylation yield was 66.2 %. If the microfiltration process was carried out for longer time and higher extraction volume than those employed in this process, the yield can be increased up to 90 %.
• A-40926 contained in filtered broth was purified on a polyamide column, as described below.
• the amount reported in this description is about 1/10 of the amount usually worked in an industrial preparation and is representative of the current production method.
• the A-40926 filtered broth (9000 L; assay 0.275 mg/L; A-40926 2475 g; pH 6 ⁇ 0.2; temperature 10 ⁇ 3 °C) was loaded into the column at about 5 g of activity per liter of resin (activity/resin ratio of 5-8 g/L was usually used).
• the column was washed with the following solutions: 3 BV (1500 L) of a solution at pH 6 prepared by dissolving 7.5 Kg of sodium carbonate in 1500 L of deminerahzed water and adjusting the pH with acetic acid; 4 BV (2000 L) of a solution at pH 8 prepared by dissolving 10 Kg of sodium carbonate in 2000 L of deminerahzed water and adjusting the pH with acetic acid; 1.5 BV (750 L ) of a solution at pH 9 prepared by dissolving 4 Kg of sodium carbonate in 750 L of deminerahzed water and adjusting the pH with acetic acid.
• A-40926 was recovered from the column by eluting with 4 BV (2000 L) of a buffer solution at pH 10 prepared by dissolving 10 Kg of sodium carbonate in 2000 L of deminerahzed water and adjusting the pH with acetic acid. The fractions containing purified A-40926
• the resin used for the purification was regenerated with 1.5 BV of 1 : 1 mixture of isopropanol / 5 % NaOH followed by a washing with 5 BV of deminerahzed water.
• the solution coming from the column was subject to several rounds of dilution/concentration steps to eliminate most of the inorganic salts in the solution.
• the solution was concentrated to 80 L by nanofiltration using a membrane with a cut-off of 250 D, diluted with 80 L of deminerahzed water, and re-concentrated at the starting volume (80 L) by nanofiltration. This operation was repeated at least 5 times.
• the pH of the final solution (80 L , pH 7.5) was adjusted at pH 6.3 with 23 % HC1.
• the solution was then diluted with 80 L of acetone, and its pH was adjusted again at pH 2.6 with 23 % HC1.
• Solid A-40926 was dried under reduced pressure at 30-35 °C in a static drier until the residual acetone was below 2 % and the water was less than 10 %. The product was then sieved through a 50 mesh sieve obtaining 2.08 Kg of purified A-40926 (HPLC assay 81.4 %; water 6.2 %; sulphated ashes 4.8 %). The yield, starting from the activity loaded on the column, was 68.4 %.
• Dalbavncin (BI-397) was prepared from the natural glycopeptide A-40926 through a three-step synthesis as described in Malabarba and Donadio (1999), Drags of the Future, 24(8):839-846. Specifically, A-40926 was first subject to an esterification step to make MA, which was then subject to an amidation step to make MA-A-1. A final hydrolysis step then converted MA-A-1 into dalbavancin.
• Washing with water was done primarily to remove sulphates from MA.
• DMSO 1.6 L was placed into a 10 L round bottomed flask, equipped with a mechanical stirrer and a thermometer, and cooled with an ice bath below 10 °C. HCl 37% (1 L) was then slowly added under stirring maintaining the temperature of the mixture below 25 °C.
• DCC dicyclohexylcarbodiammide
• the second portion of the starting solution coming from the hydrolysis step (76.5 L) was diluted with H O (56 L) to lower the DMSO content under 5% (v/v) and acidified to pH 2.87 with 3.0 L of 1 N HCl.
• the portion was then purified as previously described in the purification of the first portion.
• XAD-7 HP resin (8L) was suspended in a 1 :1 water/methanol solution, filtered, and loaded into a proper glass-column (internal diameter 12 cm) with a peristaltic pump.
• A-40926 was finally eluted with 8 B V (64 L) of a 1 : 1 water/acetone mixture acidified with 5 mL of acetic acid / L of water. 16 fractions of 4 L each were collected. The rich fractions (from 5 to 15) in which A-40926 concentration was greater than 0.5 g/L were gathered together obtaining a solution containing 163.4 g of A-40926 (43 L, 3.8 g/L ). The column yield was 81.3 %. The other fractions (200 L) containing 0.23 g/L (45.3g; 22.2 %) of less pure A-
• the retentate was concentrated to 1/10 of the starting volume (4 IS), the same volume of water was added and the solution obtained was concentrated again. This concentration/dilution step was repeated three times in order to reduce the residual acetone to 0.25 %.
• a 300 mL portion of the A-40926 solution (19.9 g of A-40926) was further concentrated to 100 mL by using a laboratory scale ultrafilter and then heated at 60-65 °C.
• the pH of this solution was adjusted at 7 (30% NaOH), and 1.2 mL of 5:1 acetone/isopropanol mixture per mL of concentrated solution was added drop wise at this temperature.
• the resulted mixture was left to cool at 20 °C. After 1.5 hours, the solid obtained was filtered, washed on the filter with acetone, and dried at 40 °C for 15 hours. 20.6 g of product (HPLC assay 82.0 %; A- 40926 16.9 g) was obtained.
• the precipitation yield was 84.9 %.
• the overall yield, starting from the filtered broth, was about 64 %.
• the resin was washed with the following three solutions: 1050 mL (3 BV) of aqueous solution of sodium carbonate (5 g/L) adjusted at pH 6 with acetic acid; 1750 mL (5 BV) of aqueous solution of sodium carbonate (5 g/L) adjusted at pH 8 with acetic acid; 3150 mL (9 BV) of aqueous solution of sodium carbonate (5 g/L) adjusted at pH 9 with acetic acid. [0269] The activity was then eluted with 10 BV of deminerahzed water. 20 fractions of 500 mL each were collected.

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