News Column

Patent Issued for Disk Diffusion Assay for Oritavancin

September 8, 2014



By a News Reporter-Staff News Editor at Clinical Trials Week -- A patent by the inventor Belley, Adam (Beaconsfield, CA), filed on July 26, 2012, was published online on August 26, 2014, according to news reporting originating from Alexandria, Virginia, by NewsRx correspondents (see also Biotechnology Companies).

Patent number 8815535 is assigned to The Medicines Company (Parsippany, NJ).

The following quote was obtained by the news editors from the background information supplied by the inventors: "Antimicrobial susceptibility testing (ASTs) devices provide critical information that aids in selection of effective and appropriate antimicrobial therapy. Several AST devices are available from device manufacturers and they include disk diffusion assays, E-test strips (AB bioMerieux), and automated systems such as VITEK.TM. (bioMerieux), Phoenix (Becton Dickinson) and Microscan WALKAWAY.TM. (Siemens). Because of its simplicity, the disk diffusion assay is a widely used AST device.

"The investigational lipoglycopeptide oritavancin exhibits an inherent capacity to bind to plastic and glass surfaces which has consequently confounded measurement of its true potency in susceptibility tests (Arhin, F F, et al. 2008. Antimicrob Agents Chemother 52:1597-603). To date, only the broth microdilution (BMD) minimal inhibitory concentration (MIC) test is a reference method accepted by the Clinical Laboratory Standards Institute (CLSI) for use in determining the susceptibility of a bacterial isolate to oritavancin (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, CLSI document M7-A8, 8th ed. Clinical and Laboratory Standards Institute, Wayne, Pa. 2009; Performance Standards for Antimicrobial Susceptibility Testing; Eighteenth Informational Supplement, CLSI document M100-S18, 7th ed. Clinical and Laboratory Standards Institute, Wayne, Pa. 2008). Recent developments have demonstrated that inclusion of 0.002% polysorbate-80 (P80) during BMD MIC testing minimizes loss of oritavancin due to non-specific binding and allows for accurate determination of the MIC of a clinical isolate (Arhin, F F, et al. 2008. Antimicrob Agents Chemother 52:1597-603).

"Because BMD MIC testing is technically cumbersome, other methods that simplify susceptibility testing for oritavancin would be advantageous to clinical microbiologists. Previous studies that evaluated the feasibility of an agar-based disk diffusion assay for oritavancin concluded that slow diffusion of oritavancin in agar results in zones of clearance (zone diameters) that do not meet the minimum requirements recommended by the CLSI (.gtoreq.15 mm) under standard testing conditions (Sahm, D F, et al. 2003. Feasibility study to assess disk diffusion as a method for testing the susceptibility of bacterial pathogens to oritavancin. Unpublished internal report; Turnbull, B. 2007. Disk diffusion feasibility study for oritavancin susceptibility testing. Unpublished internal report).

"Development of a disk diffusion assay that permits accurate determination of the susceptibility of a bacterial isolate to oritavancin, under the minimum requirements recommended by the CLSI, would greatly aid in rapid and accurate selection of therapy using oritavancin."

In addition to the background information obtained for this patent, NewsRx journalists also obtained the inventor's summary information for this patent: "The present invention is directed to disk diffusion assays for determining susceptibility of bacteria to a glycopeptide antibiotic.

"In a first embodiment, the present invention is directed to a disk diffusion assay for determining susceptibility of bacteria to a glycopeptide antibiotic that comprises: a) impregnating a paper disk with a solution comprising a glycopeptide antibiotic and polysorbate 80, b) placing the impregnated disk on the surface of a bacteria-coated media plate, c) incubating the plate of b) under conditions promoting bacterial growth, and d) measuring a zone of inhibition around the disk.

"In certain aspects of this embodiment, polysorbate 80 present is present in the solution at a concentration of between about 0.002% and about 10% v/v, or at a concentration of between about 1% and about 8% v/v. In other aspects, polysorbate 80 present is present in the solution at a concentration of about 2.5% or about 5% v/v.

"In certain aspects of this embodiment, the glycopeptide antibiotic is present in the solution at a concentration of between about 0.5 and about 50 mg/ml, or at a concentration of between about 1 and about 20 mg/ml. In related aspects, the paper disk is impregnated with about 180 ug of the glycopeptide antibiotic, or with about 360 ug of the glycopeptide antibiotic.

"In certain aspects of this embodiment, the impregnated disk is maintained at room temperature for at least about 12 hours, for at least about 4 days, for at least about 8 days, or for at least about 30 days, before it is placed on the media plate.

"In certain aspects of this embodiment, the plate of b) on which the disk has been placed is maintained at room temperature for at least about 1 hour, at least about 3 hours or at least about 6 hours, before the incubating step c). Alternatively, the plate of b) is maintained at temperature of about 4.degree. C. for at least about 1 hour, at least about 3 hours or at least about 6 hours, before the incubating step c). In certain assays, the incubating step c) is conducted at about 37.degree. C. As an example, in one aspect the plate of b) is maintained at room temperature for at least about 3 hours before the incubating step c), and the incubating step c) is conducted at about 37.degree. C., or the plate of b) is maintained at about 4.degree. C. for at least about 3 hours before the incubating step c), and the incubating step c) is conducted at about 37.degree. C.

"In certain aspects of this embodiment, the glycopeptide antibiotic is oritavancin.

"In certain aspects of this embodiment, the zone of inhibition measured in d) is at least about 15 mm in diameter.

"In a second embodiment, the present invention is directed to a disk diffusion assay for determining susceptibility of bacteria to a glycopeptide antibiotic that comprises: a) impregnating a paper disk with a solution comprising a glycopeptide antibiotic and polysorbate 80, wherein polysorbate 80 is present in the solution at a concentration of between about 0.002% and about 10% v/v, b) maintaining the impregnated disk at room temperature for at least about 8 days, c) placing the impregnated disk on the surface of a bacteria-coated media plate, and maintaining the plate at 4.degree. C. for at least about 1 hour, d) incubating the plate of c) under conditions promoting bacterial growth, and e) measuring a zone of inhibition around the disk.

"In certain aspects of this embodiment, polysorbate 80 is present in the solution at a concentration of between about 1% and about 8% v/v. In other aspects, polysorbate 80 is present in the solution at a concentration of about 2.5% or about 5% v/v.

"In certain aspects of this embodiment, the glycopeptide antibiotic is present in the solution at a concentration of between about 0.5 and about 50 mg/ml, or at a concentration of between about 1 and about 20 mg/ml. In related aspects, the paper disk is impregnated with about 180 ug of the glycopeptide antibiotic, or about 360 ug of the glycopeptide antibiotic.

"In certain aspects of this embodiment, the impregnated disk is maintained at room temperature for at least about 4 days, at least about 8 days or for at least about 30 days.

"In certain aspects of this embodiment, the plate on which the disk has been placed is maintained at about 4.degree. C. for at least about 3 hour, or the plate is maintained at 4.degree. C. for at least about 6 hours. In other aspects, the plate of c) is maintained at room temperature for at least about 3 hours before the incubating step d), and the incubating step d) is conducted at about 37.degree. C., or the plate of c) is maintained at about 4.degree. C. for at least about 3 hours before the incubating step d), and the incubating step d) is conducted at about 37.degree. C. As an example, in one aspect the plate of c) is maintained at room temperature for at least about 3 hours before the incubating step d), and the incubating step d) is conducted at about 37.degree. C., or the plate of c) is maintained at about 4.degree. C. for at least about 3 hours before the incubating step d), and the incubating step d) is conducted at about 37.degree. C.

"In certain aspects of this embodiment, the glycopeptide antibiotic is oritavancin.

"In certain aspects of this embodiment, the zone of inhibition measured in e) is at least about 15 mm in diameter.

"In a third embodiment, the present invention is directed to a disk diffusion assay for determining susceptibility of bacteria to oritavancin that comprises: a) impregnating a paper disk with a solution comprising oritavancin and polysorbate 80, wherein polysorbate 80 is present in the solution at a concentration of about 5% v/v, b) maintaining the impregnated disk at room temperature for at least about 8 days, c) placing the impregnated disk on the surface of a bacteria-coated media plate, and maintaining the plate at 4.degree. C. for at least about 3 hours, d) incubating the plate of c) under conditions promoting bacterial growth, and e) measuring a zone of inhibition around the disk.

"In certain aspects of this embodiment, oritavancin is present in the solution at a concentration of between about 0.5 and about 50 mg/ml, or at a concentration of between about 1 and about 20 mg/ml. In related aspects, the paper disk is impregnated with about 180 ug of oritavancin, or about 360 ug of oritavancin.

"In certain aspects of this embodiment, the impregnated disk is maintained at room temperature for at least about 30 days.

"In certain aspects of this embodiment, the plate is maintained in step c) at about 4.degree. C. for at least about 6 hours, or the incubating step d) is conducted at about 37.degree. C., or both.

"In certain aspects of this embodiment, the zone of inhibition measured in e) is at least about 15 mm in diameter.

"In a fourth embodiment, the present invention is directed to a disk diffusion assay for determining susceptibility of bacteria to a glycopeptide antibiotic that comprises: a) impregnating a paper disk with a solution comprising a glycopeptide antibiotic, polysorbate 80 and Span 80, b) placing the impregnated disk on the surface of a bacteria-coated media plate, c) incubating the plate of b) under conditions promoting bacterial growth, and d) measuring a zone of inhibition around the disk.

"In certain aspects of this embodiment, the glycopeptide antibiotic is present in the solution at a concentration of between about 0.5 and about 5 mg/ml. In a specific aspect, the glycopeptide antibiotic is present in the solution at a concentration of about 1.25 mg/ml. In related aspects, the paper disk is impregnated with about 25 ug of the glycopeptide antibiotic.

"In certain aspects of this embodiment, polysorbate 80 and Span 80 are present in the solution at a ratio of between about 75:25 and 25:75 v/v, between about 60:40 and 40:60 v/v, or between about 55:45 and 45:55 v/v, or polysorbate 80 and Span 80 are present in the solution at a ratio of 50:50 v/v. In related aspects, the combined concentration of polysorbate 80 and Span 80 present in the solution is between about 2.5% and about 5.5% v/v, or the combined concentration of polysorbate 80 and Span 80 present in the solution is about 4%. In a specific aspect, polysorbate 80 and Span 80 are present in the solution at a ratio of between about 55:45 and about 45:55 v/v, and at a concentration of between about 3% and about 5% v/v. In a further specific aspect, polysorbate 80 and Span 80 are present in the solution at a ratio of about 50:50 v/v and at a concentration of about 4% v/v.

"In certain aspects of this embodiment, the impregnated disk is dried at room temperature before placement on the media plate. The impregnated disk is placed on the media plate within a few hours of drying, such as within about 1, 2, 3, 4, 5 or 6 hours of drying.

"In certain aspects of this embodiment, the incubating step c) is conducted at about 37.degree. C.

"In certain aspects of this embodiment, the glycopeptide antibiotic is oritavancin.

"In certain aspects of this embodiment, the zone of inhibition measured in d) is at least about 15 mm in diameter.

"In a fifth embodiment, the present invention is directed to a disk diffusion assay for determining susceptibility of bacteria to a glycopeptide antibiotic that comprises: a) impregnating a paper disk with a solution comprising a glycopeptide antibiotic, polysorbate 80 and Span 80, wherein the glycopeptide antibiotic is present at a concentration of about 1.25 mg/ml, and wherein the polysorbate 80 and Span 80 are present in the solution at a concentration of about 4% v/v and in a ratio of about 50:50 v/v, b) placing the impregnated disk on the surface of a bacteria-coated media plate, c) incubating the plate of b) under conditions promoting bacterial growth, and d) measuring a zone of inhibition around the disk.

"In certain aspects of this embodiment, the paper disk is impregnated with about 25 ug of the glycopeptide antibiotic.

"In certain aspects of this embodiment, the impregnated disk is dried at room temperature before placement on the media plate.

"In certain aspects of this embodiment, the incubating step c) is conducted at about 37.degree. C.

"In certain aspects of this embodiment, the glycopeptide antibiotic is oritavancin.

"In certain aspects of this embodiment, the zone of inhibition measured in d) is at least about 15 mm in diameter."

URL and more information on this patent, see: Belley, Adam. Disk Diffusion Assay for Oritavancin. U.S. Patent Number 8815535, filed July 26, 2012, and published online on August 26, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=8815535.PN.&OS=PN/8815535RS=PN/8815535

Keywords for this news article include: Alcohols, Antibacterial, Antibiotics, Antimicrobials, Bacterial Infections and Mycoses, Biotechnology Companies, Biotechnology Company, Clinical Trials and Studies, Glycopeptides, Organic Chemicals, Polyethylene Glycols, Polysorbates, The Medicines Company, Therapy, bioMerieux.

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Source: Clinical Trials Week


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