WO2023049747A1 - Streptococcus suis lytic enzyme compositions and methods of use thereof - Google Patents

Streptococcus suis lytic enzyme compositions and methods of use thereof Download PDF

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Publication number
WO2023049747A1
WO2023049747A1 PCT/US2022/076781 US2022076781W WO2023049747A1 WO 2023049747 A1 WO2023049747 A1 WO 2023049747A1 US 2022076781 W US2022076781 W US 2022076781W WO 2023049747 A1 WO2023049747 A1 WO 2023049747A1
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WIPO (PCT)
Prior art keywords
plyss2
clause
veterinary composition
enzyme
combination
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PCT/US2022/076781
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French (fr)
Inventor
Yong-fu LI
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Elanco Us Inc.
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Publication of WO2023049747A1 publication Critical patent/WO2023049747A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
    • 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

Definitions

  • the present disclosure relates to a lytic enzyme of Streptococcus suis as well as formulations and methods utilizing the lytic enzyme for the treatment of animals.
  • Streptococcus suis (S. suis) is a Gram-positive bacterium that is known to be pathogenic in some animals.
  • a lytic enzyme derived from a Streptococcus suis phage is known as “PlySs2.”
  • PlySs2 is an enzyme that hydrolyzes the cell wall of Gram-positive bacteria, including Streptococcus suis.
  • the present disclosure provides veterinary compositions comprising a PlySs2 active compound that are adapted for administration to a non-human animal.
  • the present disclosure is directed to a veterinary composition
  • a veterinary composition comprising a PlySs2 active compound and one or more carriers, wherein the PlySs2 active compound is adapted for administration to a non-human animal.
  • the carrier can be selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof.
  • the veterinary composition can be a single unit dose.
  • the veterinary composition can be a parenteral formulation administered to the non-human animal via a route of administration selected from the group consisting of intravenous, intraarterial, intraperitoneal, intrathecal, intradermal, epidural, intracerebroventricular, intraurethral, intrastemal, intracranial, intratumoral, intramuscular and subcutaneous.
  • a route of administration selected from the group consisting of intravenous, intraarterial, intraperitoneal, intrathecal, intradermal, epidural, intracerebroventricular, intraurethral, intrastemal, intracranial, intratumoral, intramuscular and subcutaneous.
  • the PlySs2 active compound can be a modified PlySs2.
  • the modified PlySs2 comprises an albumin binding domain.
  • the veterinary composition can further comprise a second active compound.
  • the present disclosure is directed to a method of treating a disease in the non-human animal.
  • the method can comprise administering the veterinary composition of the present disclosure to the animal.
  • the disease can be associated with a Gram positive bacterium.
  • the Gram positive bacterium can be a Streptococcus bacterium.
  • the non- human animal can be a swine.
  • the method of treating a disease can comprise administering an amount of PlySs2 between about 1 mg/ kg and about 10 mg/kg to the non-human animal.
  • Administering the veterinary composition to the non-human animal can be performed for about 1 day to about 13 days.
  • the present disclosure is directed to a method of making the veterinary composition comprising a PlySs2 enzyme.
  • the method of making the veterinary composition can include a step of producing a recombinant PlySs2 enzyme from recombinant expression in a bacterium to form the PlySs2 enzyme.
  • the bacterium can y be E. coli.
  • the method of making the veterinary composition can further comprise fermenting the bacterium and isolating the PlySs2 enzyme.
  • the method of making the veterinary composition can also comprise processing the bacterium in a microfluidizer and/or chromatographically purifying the PlySs2 enzyme.
  • the method of making the veterinary composition can further comprise concentrating the PlySs2 enzyme and/or removing one or more endotoxins from the PlySs2 enzyme.
  • the method of making the veterinary composition can also comprise combining the PlySs2 enzyme and the carrier.
  • the present disclosure is directed to a kit comprising a bottle comprising the veterinary composition of the present disclosure.
  • the kit can include the veterinary composition in a parenteral formulation for administration to the non-human animal via a route of administration selected from the group consisting of intravenous, intraarterial, intraperitoneal, intrathecal, intradermal, epidural, intracerebroventricular, intraurethral, intrasternal, intracranial, intratumoral, intramuscular, and subcutaneous.
  • the kit can be used to treat a disease in a non-human animal that is a swine.
  • the bottle included in the kit can comprise an effective amount of the veterinary composition.
  • the effective amount of the veterinary composition can comprise an amount of PlySs2 ranging from about 1 mg/kg to about 10 mg/kg.
  • the veterinary composition in the bottle can be a ready-to-use liquid composition.
  • the veterinary composition in the bottle can be a lyophilized composition.
  • the veterinary composition in the bottle can be a single unit dose.
  • the veterinary composition in the bottle can be a multi-unit dose.
  • the kit can further include further comprising a diluent.
  • the present disclosure is directed to a method of purifying the PlySs2 enzyme.
  • the method of purifying the PlySs2 enzyme can comprise the steps of fermenting the PlySs2 enzyme, flowing the fermented PlySs2 enzyme through the chromatography device, and/or removing one or more endotoxins from the PlySs2 enzyme to form the purified PlySs2 enzyme.
  • the method of purifying the PlySs2 enzyme can also comprise the step of purifying the PlySs2 enzyme to a purity level of about 85% to about 95%.
  • the method of purifying the PlySs2 enzyme can further comprise the step of administering the effective amount of the purified PlySs2 enzyme to the non-human animal.
  • the effective amount of the purified PlySs2 enzyme can range from about 1 mg/kg to about 10 mg/kg.
  • the effective amount of the purified PlySs2 enzyme can also be about 5 mg/kg.
  • the method of purifying the PlySs2 enzyme can further comprise the step of administering the effective amount of the purified PlySs2 enzyme to the non-human animal once.
  • the method of purifying the PlySs2 enzyme can further comprise the step of administering the effective amount of the purified PlySs2 enzyme to the non-human animal once a day for about 13 days.
  • a veterinary composition comprising PlySs2 and one or more carriers.
  • parenteral formulation is selected from the group consisting of intravenous, intraarterial, intraperitoneal, intrathecal, intradermal, epidural, intracerebroventricular, intraurethral, intrastemal, intracranial, intratumoral, intramuscular and subcutaneous.
  • a method of making a veterinary composition comprising a PlySs2 enzyme, wherein the method comprises the step of producing a recombinant PlySs2 enzyme from recombinant expression in a bacterium to form the PlySs2 enzyme.
  • a kit comprising a bottle comprising a veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses
  • a route of administration selected from the group consisting of intravenous, intraarterial, intraperitoneal, intrathecal, intradermal, epidural, intracerebroventricular, intraurethral, intrastemal, intracranial, intratumoral, intramuscular, and subcutaneous.
  • a method of purifying a PlySs2 enzyme comprising a step of fermenting the PlySs2 enzyme.
  • FIG. 1 shows the domains of a PlySs2 protein and a depiction of the PlySs2 amino acid sequence
  • FIG. 2A shows a purification scheme for purifying PlySs2
  • FIG. 2B shows a blot associated with the purification scheme of FIG. 2A and identifies the purified PlySs2;
  • FIG. 3 shows a BSL2 density reduction activity;
  • FIG. 4 shows survival curves in swine after two different dosage regimens comprising PlySs2, TG03 and TG04 treatment groups;
  • FIG. 5 shows the rectal temperature of animals in the study per day for each treatment group
  • FIG. 6 shows the percentage of animals having fever in the study per day for each treatment group
  • FIG. 7A shows exposure and percent survival results for animals in the TG03 and TG04 treatment groups
  • FIG. 7B correlates the day of mortality to the last day of detectable enzyme concentration in animals in the TG03 group of FIG. 7A;
  • FIG. 8A shows the body temperature of the six animals in Pen 6, which received PlySs2 once at a dose of 5 mg/kg (group TG03);
  • FIG. 8B shows the body temperature of the six animals in Pen 10, which received PlySs2 once daily for 13 days at a dose of 5 mg/kg (group TG04);
  • FIG. 9A shows the average exposure of animals in the three different pens which received PlySs2 once at a dose of 5 mg/kg (group TG03);
  • FIG. 9B shows the average exposure of animals in the three different pens which received PlySs2 once daily for 13 days at a dose of 5 mg/kg (group TG04);
  • FIG. 10A shows the plasma concentration of PlySs2 after the animals were administered PlySs2 at a dose of 3 mg/kg.
  • FIG. 10B shows the plasma concentration of PlySs2 after the animals were administered PlySs2 at a dose of 10 mg/kg.
  • the present disclosure provides exemplary veterinary compositions comprising an active pharmaceutical agent or an active compound (e.g., PlySs2).
  • the present disclosure also provides exemplary methods of making or using the veterinary composition.
  • the veterinary composition of the present disclosure exhibits desirable properties and provide benefits for the treatment of disease in an animal (e.g., a non-human animal).
  • the veterinary composition can comprise PlySs2.
  • PlySs2 can be specific in killing harmful bacteria associated with disease in animals (e.g., bacterial disease). Use of PlySs2 for the treatment of bacterial disease can avoid traditional antibiotic treatments of animals and can be utilized as or in an “antibiotic free” treatment course.
  • PlySs2 is a protein, it is biodegradable and advantageously does not accumulate in the body of animals. PlySs2 targets complex cell wall peptidoglycan structures in bacteria and, as a result, PlySs2 can have a lower propensity to develop resistance in animals. Additionally, since PlySs2 hydrolyzes the cell wall of the targeted bacteria, a composition comprising PlySs2 is selective in action and does not inadvertently kill any good or useful bacteria.
  • the veterinary composition can include more than one active compound or active pharmaceutical agent.
  • the veterinary composition can include PlySs2 and one or more antimicrobials, such as an antibiotic.
  • lytic enzymes such as PlySs2
  • administration of the PlySs2 can produce synergy with one or more antibiotics, such as antibiotics co- administered with the PlySs2 or antibiotics that the animal is taking to treat other disease or infectious indications.
  • the veterinary composition can be adapted, utilized, or used to treat an animal.
  • the animal can be a non-human animal.
  • the non-human animal can be any animal that is not a human, such as a ruminant animal, a zoo animal, a domestic animal, a wild animal, a bovine animal, a farm animal, or a companion animal.
  • the non-human animal can be a cattle, a goat, a sheep, a giraffe, an American Bison, an European bison, a yak, a water buffalo, a deer, a camel, an alpaca, a llama, a wildebeest, an antelope, a pronghorn, a nilgai, a swine, a duck, a goose, a chicken, a laying hen, a broiler, a turkey, a quail, an ostrich, a turkey, a pig, a boar, a sow, a gilt, a piglet, a canine, a feline, a rabbit, and/or a ferret.
  • the veterinary composition can also be adapted, utilized, or used to treat a non-human animal that produces food for humans, such as a meat-producing cattle, a milk-producing cattle, a dairy cattle, and/or a chicken capable of
  • the veterinary composition can be adapted, utilized, or used to treat an animal with a disease.
  • the disease can be associated with a microbial infection caused by a fungus, a virus, and/or a bacterium.
  • the disease can be associated with a bacterial infection, such as an infection related to a Gram positive (+) bacterium.
  • the disease can also be associated with a Methicillin Resistant Staphylococcus aureus (MRS A) infection, a Vancomycin Intermediate Staphylococcus aureus (VISA) infection, a Vancomycin Resistant Staphylococcus aureus (VRSA) infection, clinical mastitis, sub-clinical mastitis, septicaemia, inflammation, polyserositis, meningitis, and/or endocarditis.
  • MRS A Methicillin Resistant Staphylococcus aureus
  • VISA Vancomycin Intermediate Staphylococcus aureus
  • VRSA Vancomycin Resistant Staphylococcus aureus
  • the veterinary composition can also be adapted, utilized, or used to treat a disease of an animal infected with Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus agnetis, Staphylococcus simulans, Staphylococcus epide midis, Streptococcus suis, Streptococcus equi, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus sanguinis, Streptococcus pneumonia, Streptococcus pyogenes, group B streptococcus (GBS), group G streptococci (GGS), group E streptococci (GES), Listeria, and Enterococcus faecalis.
  • the veterinary composition can result in controlling and/or eliminating about 90% or more of bacteria in the animal. In some embodiments, the veterinary composition can result in controlling or eliminating (e.g., killing) about 90% of disease in animals on a farm. For example, the veterinary composition can result in controlling or eliminating about 90% of Streptococcus suis in animals on a farm with Streptococcus suis related diseases.
  • the veterinary composition can result in controlling or eliminating about 50% to about 100% of bacteria and/or disease in an animal, including any specific or range of percentages comprised therein.
  • the veterinary composition can result in controlling or eliminating about 50% to about 70%, about 70% to about 90%, or about 90% to about 99% of Streptococcus suis in animals on a farm with Streptococcus suis related diseases.
  • the veterinary composition can result in controlling or eliminating about 50% to about 70%, about 70% to about 90%, or about 90% to about 99% of Streptococcus equi, Streptococcus uberis, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus sanguinis, Streptococcus pneumoniae; MRSA, vancomycin-resistant Staphylococcus aureus (VRSA), S. epidermidis, S. pseudintermedius, or Listeria of animals on a farm with the respective disease.
  • Streptococcus equi Streptococcus uberis
  • Streptococcus agalactiae Streptococcus pyogenes
  • Streptococcus sanguinis Streptococcus pneumoniae
  • MRSA vancomycin-resistant Staphylococcus aureus
  • S. epidermidis S. pseudintermedius
  • the veterinary composition can result in about 20% to about 90% improvement in a morbidity rate in the animals with the disease, including any specific or range of percentages comprised therein.
  • the veterinary composition can result in about 40% to about 50%, about 50% to about 60%, or about 60% to about 80% improvement in the morbidity rate in the animals with the disease.
  • the veterinary composition can result in about 20% to about 30%, about 30% to about 50%, or about 50% to about 60% improvement in the mortality rate of the animals with the disease.
  • PlySs2 comprises a catalytic domain.
  • the catalytic domain comprises a catalytic active domain (CAD) and a catalytic binding domain (CBD).
  • the catalytic active domain also referred to as an N-terminal cysteine-histidine aminopeptidase catalytic domain, provides rapid cell wall lysis via hydrolysis.
  • the catalytic binding domain also referred to as a C-terminal SH3b cell-wall binding domain, provides high affinity binding to cell wall peptidoglycans that are essential for bacterial growth. Without being bound by any theory, it is believed that the catalytic binding domain provides targeted pathogen specificity and beneficially allows for the lack of antimicrobial resistance that can be observed with traditional antibiotics.
  • a PlySs2 protein typically comprises about 245 amino acids.
  • FIG. 1 shows the catalytic domains of PlySs2 and a depiction of the PlySs2 amino acid sequence.
  • the PlySs2 is a modified or recombinant PlySs2.
  • the modified and/or recombinant PlySs2 can be synthetically prepared (e.g., manually or automatically in a laboratory) to comprise PlySs2 and an albumin binding domain (ABD).
  • one or more amino acids in PlySs2 are synthetically modified to provide sufficient contact, connection, and/or fusion of the PlySs2 with the ABD to form the modified PlySs2.
  • an ABD can be utilized to increase the half-life of the modified PlySs2 that is present in the veterinary composition.
  • the use of ABD in therapeutic compositions is described, for example, in U.S. Patent No. 10,428,120 as well as published documents US 2014/0170142, US 2020/0040099, US 2017/0114114, JP 2017137320A, JP 2016526014A, JP 6382826B2, and CA 2694139, all of which are incorporated by reference herein in their entireties.
  • PlySs2 can naturally have a half-life of about 4 hours to about 30 hours.
  • the modified PlySs2 comprising the ABD can have an increased half-life ranging from about 5 to about 7 days, including any specific or range of time comprised therein.
  • the veterinary composition can comprise the active compound or the active pharmaceutical agent (e.g., PlySs2) and a carrier.
  • the carrier can be selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof.
  • the carrier can be or can comprise a solid, a liquid, and/or a gaseous compound.
  • the carrier can be or can include a salt, water, and/or saline.
  • the carrier can be or can comprise a solution, a surfactant, a lyophilized composition, a powder, a suspension, a colloid, a crystalline solution, a slurry, a spray, and/or a dispersion.
  • the veterinary composition can be in the form of a gel capsule, a chewable tablet, and/or an injectable solution.
  • the veterinary composition can be administered orally, sublingually, intravenously, intramuscularly, intranasally, intraperitoneally, and/or subcutaneously.
  • the veterinary composition can be administered in a regimen that includes a single unit dose or multi-unit dose.
  • the veterinary composition can be administered a single time using a single unit dose of the veterinary composition.
  • the veterinary composition can be administered using a multi-unit dose by providing the animal more than one injection over a time period, such as for about 1 day to about 13 days.
  • more than one injection (e.g., two injections) of the veterinary composition can be administered for a time period, such as more than 13 days (e.g., 14 days).
  • the veterinary composition can be administered by providing the animal a single treatment dose per episode of the disease.
  • the veterinary composition can comprise an effective amount of the active compound or the active pharmaceutical agent (e.g., PlySs2).
  • the effective amount of the active compound or pharmaceutical component (e.g., PlySs2) in the veterinary composition is the amount of the active compound (e.g., PlySs2) that must be administered to the animal to provide an efficacious reduction or elimination of bacteria and/or disease.
  • the effective amount of the active compound or the active pharmaceutical component (e.g., PlySs2) can range from about 0.001 mg to about 1000 mg, including any amount or range comprised therein.
  • the effective amount of the active compound or the active pharmaceutical agent can depend on the body weight of the animal.
  • the effective amount of the active compound or the active pharmaceutical agent can range from about
  • the effective amount of the active compound can range from about 1 mg/kg to about 5 mg/kg, from about 1 mg/kg to about 10 mg/kg, from about 5 mg/kg to about 10 mg/kg, from about 10 mg/kg to about 20 mg/kg, including any amount or range comprised therein.
  • the animal can be treated by administration of a single dose of the veterinary composition comprising the effective amount of the active compound or the active pharmaceutical agent (e.g., PlySs2).
  • the animal can be treated by administration of multiple doses (e.g. multi-unit dose) of the veterinary composition comprising the effective amount of the active pharmaceutical component (e.g., PlySs2).
  • the multiple doses or multi-unit doses of the veterinary composition comprising the effective amount of the active compound or the active pharmaceutical agent (e.g., PlySs2) can be given on consecutive or intermittent days.
  • the multiple doses of the veterinary composition can be administered to an animal on about 2 to about 25 consecutive days, including any specific or range of days comprised therein, such as for about 2 to about 5, about
  • the veterinary composition can be a controlled release formulation. Preparing the veterinary composition to have a controlled release formulation provides for a slower release of the PlySs2 active compound over time to the animal. Formulating the veterinary composition to have a controlled release comprises using formulations including but not limited to polymer microspheres, polymer nanospheres, hydrogels, and/or in-situ gelling polymers.
  • the controlled release formulation comprises a hydrogel.
  • the hydrogel is a temperature sensitive hydrogel.
  • the controlled release formulation comprising the hydrogel can be a liquid at temperature at or below about 37 degrees to about 42 degrees, including any temperature or range comprised therein.
  • the controlled release formulation comprising the hydrogel can be a gel at temperature greater that about 37 degrees to about 42 degrees, including any temperature or range comprised therein.
  • the hydrogel comprises one or more temperature sensitive polymers.
  • Various temperature sensitive hydrogels and polymers are known in the art. For instance, hydrogels and polymers can be utilized in which the veterinary composition is a liquid at certain temperatures and a gel or a gel-like composition at different temperatures.
  • the controlled release formulation can comprise a nonaqueous carrier.
  • the controlled release formulation can comprise an oil carrier.
  • Formulating the veterinary composition to be associated with the oil provides for a slower release of the PlySs2 over time to the animal. For instance, if the formulation in an oil is administered parenterally to the animal, the oil causes the PlySs2 to be released in a slower manner compared to other, non-oil containing parenteral formulations.
  • the oil of the controlled release formulation can comprise any type of oil, such as a plant oil, an animal oil, an essential oil, and/or any combination thereof.
  • the present disclosure further provides a kit.
  • This kit can comprise a bottle containing the veterinary composition according to the present disclosure adapted for administration to a non-human animal.
  • the veterinary composition in the bottle can be the PlySs2 active compound and one or more carriers.
  • the carrier is selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof.
  • the veterinary composition in the bottle can be a parenteral formulation administered to the non- human animal via a route of administration selected from the group consisting of intravenous, intraarterial, intraperitoneal, intrathecal, intradermal, epidural, intracerebroventricular, intraurethral, intrastemal, intracranial, intratumoral, intramuscular and subcutaneous.
  • a route of administration selected from the group consisting of intravenous, intraarterial, intraperitoneal, intrathecal, intradermal, epidural, intracerebroventricular, intraurethral, intrastemal, intracranial, intratumoral, intramuscular and subcutaneous.
  • the veterinary composition in the bottle can further comprise a second active compound.
  • the second active compound can comprise any antimicrobial or non- antimicrobial compound, such as a fungus, a virus, and/or a bacterium.
  • the second active compound can also be a bacterium that is the same bacterium as the first active compound, such as Streptococcus suis.
  • the second active compound can also be a bacterium that is not the same bacterium as the first active compound, such as Streptococcus suis.
  • the second active compound can be a species of Streptococcus, Staphylococcus, or Listeria.
  • the veterinary composition in the bottle can be in the form of a solution or ready-to-use liquid composition.
  • the veterinary composition in the bottle can be in the form of a lyophilized powder.
  • the veterinary composition in the bottle can be a single unit dose or a multi-unit dose.
  • the bottle can comprise an effective amount of the veterinary composition.
  • the effective amount of the veterinary composition can comprise an effective amount of active compound (e.g., PlySs2) in a single unit dose.
  • the effective amount of the veterinary composition can comprise an effective amount of active compound (e.g., PlySs2) in. in a multi-unit dose.
  • the multi-unit dose of the veterinary composition in the bottle can comprise about 2 to about 5 doses, about 5 to about 10 doses, about 10 to about 30 doses, about 30 to about 50 doses, or any specific number or range of doses comprised therein.
  • the bottle can comprise more than 50 doses.
  • the effective amount of active compound (e.g., PlySs2) can be the amount calculated, experimentally determined, or computationally determined to be able to treat the non-human animal (e.g., swine).
  • the kit can include a diluent.
  • the diluent can be any non-active solute or solution that enables or enhances the administration or activity of the active compound (e.g., PlySs2) to the animal.
  • the diluent of the present disclosure can comprise saline, buffer, and/or water.
  • the kit can further include an instruction manual, which contains information and directions for proper administration and/or dosages of the veterinary composition to one or more types of animals.
  • a method of treating a disease in a non-human animal can include administering the veterinary composition to the non-human animal, the disease can be associated with a Gram positive bacterium (e.g., Streptococcus bacterium).
  • the non-human animal can be any of the animals described previously including, but not limited to a swine.
  • the method of administering the veterinary composition can include administering an amount of PlySs2 from about 1 mg/ kg to about 10 mg/kg including any amount or range comprised therein to the non-human animal.
  • the method of treating a disease in a non-human animal can comprise administering the veterinary composition to the non-human animal once.
  • the method can comprise administering the veterinary composition to the non- human animal once a day for 2 to about 13 consecutive days. In some further embodiments, the method can comprise administering the veterinary composition to the non-human animal for more than 13 days (e.g., 14 days).
  • a method of making or using a veterinary composition comprises a PlySs2 enzyme.
  • This method can include the step of producing a recombinant PlySs2 enzyme from recombinant expression in a bacterium to form the PlySs2 enzyme.
  • the bacterium can be E. coli or any other bacterium that can be utilized for the production of the recombinant PlySs2.
  • the method of producing a recombinant PlySs2 enzyme (e.g., a modified PlySs2 protein) in the bacterium can include fermenting the bacterium and/or isolating the PlySs2 enzyme.
  • the method can include processing the bacterium in a microfluidizer and purifying the PlySs2 enzyme.
  • the purifying step can comprise chromatographically purifying the PlySs2 enzyme in a chromatography device.
  • the method can include concentrating the PlySs2 enzyme and/or removing one or more endotoxins from the PlySs2 enzyme.
  • the method can also include combining the purified PlySs2 enzyme with a carrier in order to make or use the veterinary composition.
  • a method of purifying a PlySs2 enzyme comprises the steps of fermenting the PlySs2 enzyme, flowing the fermented PlySs2 enzyme through the chromatography device, and/or removing one or more endotoxins from the PlySs2 enzyme to form the purified PlySs2 enzyme.
  • the method of purifying PlySs2 can comprise purifying the PlySs2 enzyme to a purity level of about 70% to about 99%, including any percentage or range comprised therein. More specifically, the method of purifying PlySs2 can comprise purifying the PlySs2 enzyme to a purity level of about 70% to about 80, about 80% to 90%, about 90% to about 95%, or about 95% to about 99%.
  • the method of purifying PlySs2 can also include combining the purified PlySs2 enzyme with a carrier in order to make or use the veterinary composition that can be administered to any non-human animal.
  • the method can also include administering the effective amount of the purified PlySs2 enzyme to the non-human animal.
  • the method can further include administering to the non-human animal about 0.5 mg/kg to about 15 mg/kg, including any specific amount or range of enzyme comprised therein.
  • the method can include administering to the non-human animal about 5 mg/kg to about 10 mg/kg, about 10 mg/kg to about 15 mg/kg, including any amount or range of enzyme comprised therein.
  • the method can include administering about 5 mg/kg of the purified PlySs2 enzyme to the non-human animal.
  • the method can further include administering the effective amount of the purified PlySs2 enzyme to the non-human animal once.
  • the method can further include administering the effective amount of the purified PlySs2 enzyme to the non-human animal once a day for a period of consecutive days.
  • the period of consecutive days can range from 1 day to 13 days. In some embodiments, the period of consecutive days can be more than 13 days (e.g., 14 days).
  • the instant example describes an exemplary process for preparing PlySs2.
  • the expression platform for preparing PlySs2 was E. coli TOPIC, pBAD24, inducible with arabinose. Fermentation was performed on a pilot scale in 30-L fermenters. Cell processing was performed using a microfluidizer and purification was done via flow through IEX chromatography. Finally, the PlySs2 was concentrated and underwent diafiltration and endotoxin removal. A purification scheme for purifying PlySs2 is shown in FIG. 2A. A blot associated with the purification scheme is illustrated in FIG. 2B.
  • FIG. 2B illustrates 4 lanes showing total lysate, a soluble fraction of the lysate, total insoluble lysate, and a purified PlySs2.
  • FIG. 2B identifies the purified PlySs2 as a 26 kDa molecule.
  • the yield of the PlySs2 production process was 10 to 30 g (purified) PlySs2 per 30 L fermenter, with 85-95% purity by sodium dodecyl sulfate (SDS).
  • the formulation of PlySs2 was prepared to be 13 mg/ml, in 10 mM Tris-HCl (pH 7.4, 20% glycerol). Preliminary analysis indicated that activity/stability was retained at 4 °C for at least 1 month (via activity assay and visual inspection) and that activity was retained after 3 year frozen storage (via activity assay).
  • a BSL2 density reduction activity assay was performed and results are shown in FIG. 3. Briefly, S. suis was washed with buffer and diluted to ODeoo ⁇ 1.5 as substrate. PlySs2 was serially diluted to seven different targeted concentrations (40, 20, 10, 5, 2.5, 1.25, and 0.625 pg/ml). A density reduction assay (DRA) assay was performed by mixing 25 pl cells with 25 pl PlySs2 in a 96-well half area flat bottom clear plate. Then, ODeoo was read at room temperature for 1 hr with shaking between each read. The ODeoo for each of the concentrations is shown in FIG. 3.
  • DPA density reduction assay
  • the instant example evaluates the effectiveness of PlySs2 at two different dosage regimens in swine.
  • Veterinary compositions comprising PlySs2 were administered individually to swine intramuscularly (IM) in order to reduce the incidence of S. suis in a challenge model.
  • IM intramuscularly
  • the example utilized an established intranasal challenge model.
  • 2 ml of 1 % acetic acid was given to animals by intranasal (IN) application.
  • IN intranasal
  • animals were given 4 mL of a challenge of S. suis via IN application.
  • control or PlySs2 treatments were performed.
  • the live phase was 21 days long.
  • the primary variable of the instant example was mortality of the animals and secondary variables included body temperature, clinical signs, blood septicemia, lung lesions, bacteria isolation (necropsy), average daily gain (ADG), and body weight.
  • In vivo exposure of PlySs2 was evaluated via blood collection on: Day 0 (prior challenge), Day 1 (prior first treatment), Day 2, Day 3, Day 4, Day 7, Day 9, Day 11, Day 14, Day 16, and Day 21.
  • FIG. 5 shows the rectal temperature of animals in the study per day for each treatment group.
  • FIG. 6 shows the percentage of animals having fever in the study per day for each treatment group.
  • FIG. 5 shows that the animals in the groups treated with the active compound had lower temperature than the animals treated with either baytril or sterile buffer.
  • FIG. 6 shows that the percentage of animals with fever in the groups treated with the active compound was lower than the percentage of animals with fever in the groups treated with either baytril or a sterile buffer.
  • FIG. 7 A shows exposure and percent survival results for animals in the TG03 and TG04 treatment groups. About 83.3% of the treated animals were shown to survive on day 21 post treatment if they were administered the active compound for 13 consecutive days. Alternatively, if the animals were administered the active compound only once, about 11.8% of treated animals were shown to survive on day 21 post treatment.
  • FIG. 7B correlates the day of mortality to the last day of detectable enzyme concentration in animals in the TG03 group. The majority of animals did not show any detectable level of enzyme concentration more than 2 days after being administered the active compound.
  • FIGS. 8A and 8B there was no observed correlation between exposure and body temperature. Without aligning with any theory, FIG. 8A suggests that there can be influence of individual innate immunity in Subject 64.
  • FIG. 8A shows results of the 6 animals in Pen 6, which received PlySs2 once at a dose of 5 mg/kg (group TG03).
  • FIG. 8B shows results of the 6 animals in Pen 10, which received PlySs2 once daily for 13 days at a dose of 5 mg/kg (group TG04).
  • FIGS. 9A and 9B shows individual exposures to PlySs2 for various animals in the PlySs2 treatment groups. As illustrated in FIG.
  • mice in Pens 1, 6, and 9 received PlySs2 once at a dose of 5 mg/kg (group TG03). Most of these animals exhibited a declining plasma concentration of the enzyme after day 1, the only day that the animals were administered the active compound.
  • animals in Pens 3, 4, and 10 received PlySs2 once daily for 13 days at a dose of 5 mg/kg (group TG04). Most of these animals exhibited a declining plasma concentration of the enzyme after day 13, the last day that these animals were administered the active compound.
  • the instant example shows that a daily dosing schedule (i.e., once daily dosing of PlySs2) correlated with efficacy and improved survival of animals. A reduction in clinical signs was observed and animals did not have any injection site reactions.
  • the instant example evaluates pharmacokinetics of PlySs2 at different dosages.
  • Animals were administered PlySs2 at a dose of 3 mg/kg and at a dose of 10 mg/kg. There were four animals in each group.
  • the results of the pharmacokinetic study for a dose of 3 mg/kg for each animal is shown in FIG. 10A.
  • the results of the pharmacokinetic study for a dose of 10 mg/kg for each animal is shown in FIG. 10B.
  • FIGS. 10A and 10B illustrate the plasma concentration of PlySs2 over a period of time after the animals were dosed.
  • the maximum concentration of PlySs2 (Cmax) was dose responsive, but not dose proportional. Furthermore, the area under the curve of PlySs2 (AUC) did not clearly increase with increase in dose. The half-life of PlySs2 was generally around 4 hours in duration, with two subjects observed to have a half-life of greater than 30 hours.
  • embodiments “comprising,” “including,” or “having” an element or a plurality of elements having a particular property can include additional such elements not having that property.
  • the term “comprising” or “comprises” refers to a composition, compound, formulation, or method that is inclusive and does not exclude additional elements, components, and/or method steps.
  • the term “comprising” also refers to a composition, compound, formulation, or method embodiment of the present disclosure that is inclusive and does not exclude additional elements, components, or method steps.
  • the phrase “consisting of’ or “consists of’ refers to a compound, composition, formulation, or method that excludes the presence of any additional elements, components, or method steps.
  • the term “consisting of’ also refers to a compound, composition, formulation, or method of the present disclosure that excludes the presence of any additional elements, components, or method steps.
  • the phrase “consisting essentially of’ or “consists essentially of’ refers to a composition, compound, formulation, or method that is inclusive of additional elements, components, or method steps that do not materially affect the characteristic(s) of the composition, compound, formulation, or method.
  • the phrase “consisting essentially of’ also refers to a composition, compound, formulation, or method of the present disclosure that is inclusive of additional elements, components, or method steps that do not materially affect the characteristic(s) of the composition, compound, formulation, or method steps.
  • Approximating language can be applied to modify any quantitative representation that could permissibly vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as “about,” and “substantially” is not to be limited to the precise value specified. In some instances, the approximating language can correspond to the precision of an instrument for measuring the value.
  • range limitations can be combined and/or interchanged. Such ranges are identified and include all the sub-ranges contained therein unless context or language indicates otherwise.
  • the terms “can” and “can be” indicate a possibility of an occurrence within a set of circumstances; a possession of a specified property, characteristic or function; and/or qualify another verb by expressing one or more of an ability, capability, or possibility associated with the qualified verb. Accordingly, usage of “can” and “can be” indicates that a modified term is apparently appropriate, capable, or suitable for an indicated capacity, function, or usage, while taking into account that in some circumstances, the modified term can sometimes not be appropriate, capable, or suitable. [0092] It is to be understood that the above description is intended to be illustrative, and not restrictive. For example, the above-described embodiments (and/or aspects thereof) can be used individually, together, or in combination with each other.

Abstract

The present disclosure provides veterinary compositions comprising PlySs2, along with methods of using, making, or producing the PlySs2-containing veterinary composition, which exhibits desirable properties and provide benefits for the treatment of disease in animals.

Description

STREPTOCOCCUS SUIS LYTIC ENZYME COMPOSITIONS AND METHODS OF USE THEREOF
TECHNICAL FIELD
[0001] The present disclosure relates to a lytic enzyme of Streptococcus suis as well as formulations and methods utilizing the lytic enzyme for the treatment of animals.
BACKGROUND
[0002] Streptococcus suis (S. suis) is a Gram-positive bacterium that is known to be pathogenic in some animals. A lytic enzyme derived from a Streptococcus suis phage is known as “PlySs2.” PlySs2 is an enzyme that hydrolyzes the cell wall of Gram-positive bacteria, including Streptococcus suis. There is a need for new antibiotics to non-human animals. Accordingly, the present disclosure provides veterinary compositions comprising a PlySs2 active compound that are adapted for administration to a non-human animal.
SUMMARY
[0003] In an aspect, the present disclosure is directed to a veterinary composition comprising a PlySs2 active compound and one or more carriers, wherein the PlySs2 active compound is adapted for administration to a non-human animal. The carrier can be selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof. The veterinary composition can be a single unit dose.
[0004] The veterinary composition can be a parenteral formulation administered to the non-human animal via a route of administration selected from the group consisting of intravenous, intraarterial, intraperitoneal, intrathecal, intradermal, epidural, intracerebroventricular, intraurethral, intrastemal, intracranial, intratumoral, intramuscular and subcutaneous.
[0005] The PlySs2 active compound can be a modified PlySs2. The modified PlySs2 comprises an albumin binding domain. The veterinary composition can further comprise a second active compound.
[0006] In a second aspect, the present disclosure is directed to a method of treating a disease in the non-human animal. The method can comprise administering the veterinary composition of the present disclosure to the animal. The disease can be associated with a Gram positive bacterium. The Gram positive bacterium can be a Streptococcus bacterium. The non- human animal can be a swine. [0007] The method of treating a disease can comprise administering an amount of PlySs2 between about 1 mg/ kg and about 10 mg/kg to the non-human animal. Administering the veterinary composition to the non-human animal can be performed for about 1 day to about 13 days.
[0008] In a third aspect, the present disclosure is directed to a method of making the veterinary composition comprising a PlySs2 enzyme. The method of making the veterinary composition can include a step of producing a recombinant PlySs2 enzyme from recombinant expression in a bacterium to form the PlySs2 enzyme. The bacterium can y be E. coli.
[0009] The method of making the veterinary composition can further comprise fermenting the bacterium and isolating the PlySs2 enzyme. The method of making the veterinary composition can also comprise processing the bacterium in a microfluidizer and/or chromatographically purifying the PlySs2 enzyme. The method of making the veterinary composition can further comprise concentrating the PlySs2 enzyme and/or removing one or more endotoxins from the PlySs2 enzyme. The method of making the veterinary composition can also comprise combining the PlySs2 enzyme and the carrier.
[0010] In a fourth aspect, the present disclosure is directed to a kit comprising a bottle comprising the veterinary composition of the present disclosure. The kit can include the veterinary composition in a parenteral formulation for administration to the non-human animal via a route of administration selected from the group consisting of intravenous, intraarterial, intraperitoneal, intrathecal, intradermal, epidural, intracerebroventricular, intraurethral, intrasternal, intracranial, intratumoral, intramuscular, and subcutaneous. The kit can be used to treat a disease in a non-human animal that is a swine.
[0011] The bottle included in the kit can comprise an effective amount of the veterinary composition. The effective amount of the veterinary composition can comprise an amount of PlySs2 ranging from about 1 mg/kg to about 10 mg/kg. The veterinary composition in the bottle can be a ready-to-use liquid composition. The veterinary composition in the bottle can be a lyophilized composition. The veterinary composition in the bottle can be a single unit dose. The veterinary composition in the bottle can be a multi-unit dose. The kit can further include further comprising a diluent.
[0012] In a fifth aspect, the present disclosure is directed to a method of purifying the PlySs2 enzyme. The method of purifying the PlySs2 enzyme can comprise the steps of fermenting the PlySs2 enzyme, flowing the fermented PlySs2 enzyme through the chromatography device, and/or removing one or more endotoxins from the PlySs2 enzyme to form the purified PlySs2 enzyme. The method of purifying the PlySs2 enzyme can also comprise the step of purifying the PlySs2 enzyme to a purity level of about 85% to about 95%. The method of purifying the PlySs2 enzyme can further comprise the step of administering the effective amount of the purified PlySs2 enzyme to the non-human animal. The effective amount of the purified PlySs2 enzyme can range from about 1 mg/kg to about 10 mg/kg. The effective amount of the purified PlySs2 enzyme can also be about 5 mg/kg.
[0013] The method of purifying the PlySs2 enzyme can further comprise the step of administering the effective amount of the purified PlySs2 enzyme to the non-human animal once. The method of purifying the PlySs2 enzyme can further comprise the step of administering the effective amount of the purified PlySs2 enzyme to the non-human animal once a day for about 13 days.
[0014] The following numbered embodiments are contemplated and are non-limiting:
1. A veterinary composition comprising PlySs2 and one or more carriers.
2. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the PlySs2 is a recombinant PlySs2.
3. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the PlySs2 is a recombinant PlySs2 produced from recombinant expression in E. coli.
4. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the PlySs2 is a purified PlySs2.
5. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the PlySs2 is a chromatographic ally purified PlySs2.
6. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the PlySs2 is a purified PlySs2 purified from E. coli.
7. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof.
8. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof.
9. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier comprises one or more salts.
10. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier does not comprise salt.
11. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier comprises water. 12. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is water.
13. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier comprises liquid.
14. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is a liquid.
15. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier comprises a solution.
16. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is a solution.
17. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier comprises saline.
18. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is saline.
19. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier comprises a soft gel capsule.
20. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is a soft gel capsule.
21. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier comprises powder.
22. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is a powder.
23. The veterinary composition of clause 22, any other suitable clause, or any combination of suitable clauses, wherein the powder is contained in a capsule.
24. The veterinary composition of clause 22, any other suitable clause, or any combination of suitable clauses, wherein the powder is contained in a gel cap.
25. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier comprises a solid.
26. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is a solid.
27. The veterinary composition of clause 26, any other suitable clause, or any combination of suitable clauses, wherein the solid is a chewable tablet.
28. The veterinary composition of clause 27, any other suitable clause, or any combination of suitable clauses, wherein the chewable tablet is flavored. 29. The veterinary composition of clause 27, any other suitable clause, or any combination of suitable clauses, wherein the chewable tablet is not flavored.
30. The veterinary composition of clause 26, any other suitable clause, or any combination of suitable clauses, wherein the solid is a chewy treat.
31. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier comprises a mixture of a solid and a liquid.
32. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is a mixture of a solid and a liquid.
33. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier comprises gas.
34. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is a gas.
35. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier comprises a dispersion.
36. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is a dispersion.
37. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier comprises a suspension.
38. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is a suspension.
39. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier comprises a solution suspension.
40. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is a solution suspension.
41. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier comprises a crystalline suspension.
42. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is a crystalline suspension.
43. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier comprises a slurry.
44. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is a slurry.
45. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier comprises a spray. 46. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is a spray.
47. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is wet.
48. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier is dry.
49. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the carrier comprises a surfactant.
50. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition comprises a further active pharmaceutical ingredient (API).
51. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition comprises a second therapeutic agent.
52. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is lyophilized.
53. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is a unit dose.
54. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is a single unit dose.
55. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is a multi-unit dose.
56. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is a parenteral formulation.
57. The veterinary composition of clause 56, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is selected from the group consisting of intravenous, intraarterial, intraperitoneal, intrathecal, intradermal, epidural, intracerebroventricular, intraurethral, intrastemal, intracranial, intratumoral, intramuscular and subcutaneous.
58. The veterinary composition of clause 56, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intravenous.
59. The veterinary composition of clause 56, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intraarterial.
60. The veterinary composition of clause 56, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intraperitoneal. 61. The veterinary composition of clause 56, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intrathecal.
62. The veterinary composition of clause 56, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intradermal.
63. The veterinary composition of clause 56, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is epidural.
64. The veterinary composition of clause 56, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intracerebroventricular.
65. The veterinary composition of clause 56, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intraurethral.
66. The veterinary composition of clause 56, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intrasternal.
67. The veterinary composition of clause 56, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intracranial.
68. The veterinary composition of clause 56, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intratumoral.
69. The veterinary composition of clause 56, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intramuscular.
70. The veterinary composition of clause 56, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is subcutaneous.
71. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the PlySs2 is a modified PlySs2.
72. The veterinary composition of clause 71, any other suitable clause, or any combination of suitable clauses, wherein the modified PlySs2 comprises PlySs2 and an albumin binding domain (ABD).
73. The veterinary composition of clause 72, any other suitable clause, or any combination of suitable clauses, wherein the PlySs2 contacts the ABD.
74. The veterinary composition of clause 72, any other suitable clause, or any combination of suitable clauses, wherein the PlySs2 is connected with the ABD.
75. The veterinary composition of clause 72, any other suitable clause, or any combination of suitable clauses, wherein the PlySs2 is fused with the ABD.
76. The veterinary composition of clause 72, any other suitable clause, or any combination of suitable clauses, wherein the PlySs2 comprises an amino acid sequence comprising one or more amino acid modifications. 77. The veterinary composition of clause 76, any other suitable clause, or any combination of suitable clauses, wherein the amino acid sequence comprising one or more amino acid modifications is modified for contacting the ABD.
78. The veterinary composition of clause 76, any other suitable clause, or any combination of suitable clauses, wherein the amino acid sequence comprising one or more amino acid modifications is modified for connection with the ABD.
79. The veterinary composition of clause 76, any other suitable clause, or any combination of suitable clauses, wherein the amino acid sequence comprising one or more amino acid modifications is modified for fusing with the ABD.
80. The veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is a controlled release formulation.
81. The veterinary composition of clause 80, any other suitable clause, or any combination of suitable clauses, wherein the controlled release formulation comprises a hydrogel.
82. The veterinary composition of clause 81, any other suitable clause, or any combination of suitable clauses, wherein the hydrogel is a temperature sensitive hydrogel.
83. The veterinary composition of clause 81, any other suitable clause, or any combination of suitable clauses, wherein the hydrogel comprises one or more temperature sensitive polymers.
84. The veterinary composition of clause 80, any other suitable clause, or any combination of suitable clauses, wherein the controlled release formulation is a formulation in an oil.
85. The veterinary composition of clause 84, any other suitable clause, or any combination of suitable clauses, wherein the formulation in the oil comprises a veterinary acceptable oil.
86. The veterinary composition of clause 84, any other suitable clause, or any combination of suitable clauses, wherein the formulation in the oil comprises a PlySs2 powder.
87. The veterinary composition of clause 84, any other suitable clause, or any combination of suitable clauses, wherein the formulation in the oil comprises a lyophilized PlySs2 powder.
88. The veterinary composition of clause 84, any other suitable clause, or any combination of suitable clauses, wherein the formulation in the oil is a suspension.
89. The veterinary composition of clause 88, any other suitable clause, or any combination of suitable clauses, wherein the suspension comprises the PlySs2 suspended in the oil.
90. The veterinary composition of clause 88, any other suitable clause, or any combination of suitable clauses, wherein the suspension comprises a lyophilized PlySs2 powder suspended in the oil. 91. A method of treating a disease in an animal, said method comprising the step of administering the veterinary composition to the animal, wherein the disease is associated with a
Gram positive bacterium.
92. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the disease is an infection associated with the Gram positive bacterium.
93. The method of clause 92, any other suitable clause, or any combination of suitable clauses, wherein the Gram positive bacterium is a Staphylococcus bacterium.
94. The method of clause 93, any other suitable clause, or any combination of suitable clauses, wherein the Staphylococcus bacterium is Staphylococcus aureus.
95. The method of clause 93, any other suitable clause, or any combination of suitable clauses, wherein the Staphylococcus bacterium is Staphylococcus pseudintermedius.
96. The method of clause 93, any other suitable clause, or any combination of suitable clauses, wherein the Staphylococcus bacterium is Staphylococcus agnetis.
97. The method of clause 93, any other suitable clause, or any combination of suitable clauses, wherein the Staphylococcus bacterium is Staphylococcus simulans.
98. The method of clause 93, any other suitable clause, or any combination of suitable clauses, wherein the Staphylococcus bacterium is Staphylococcus epidermidis.
99. The method of clause 92, any other suitable clause, or any combination of suitable clauses, wherein the Gram positive bacterium is a Streptococcus bacterium.
100. The method of clause 99, any other suitable clause, or any combination of suitable clauses, wherein the Streptococcus bacterium is Streptococcus suis.
101. The method of clause 99, any other suitable clause, or any combination of suitable clauses, wherein the Streptococcus bacterium is Streptococcus equi.
102. The method of clause 99, any other suitable clause, or any combination of suitable clauses, wherein the Streptococcus bacterium is Streptococcus agalactiae.
103. The method of clause 99, any other suitable clause, or any combination of suitable clauses, wherein the Streptococcus bacterium is Streptococcus dysgalactiae.
104. The method of clause 99, any other suitable clause, or any combination of suitable clauses, wherein the Streptococcus bacterium is Streptococcus uberis.
105. The method of clause 99, any other suitable clause, or any combination of suitable clauses, wherein the Streptococcus bacterium is Streptococcus sanguinis.
106. The method of clause 99, any other suitable clause, or any combination of suitable clauses, wherein the Streptococcus bacterium is Streptococcus pneumonia.
107. The method of clause 99, any other suitable clause, or any combination of suitable clauses, wherein the Streptococcus bacterium is Streptococcus pyogenes. 108. The method of clause 99, any other suitable clause, or any combination of suitable clauses, wherein the Streptococcus bacterium is group B streptococcus (GBS).
109. The method of clause 99, any other suitable clause, or any combination of suitable clauses, wherein the Streptococcus bacterium is group G streptococci (GGS).
110. The method of clause 99, any other suitable clause, or any combination of suitable clauses, wherein the Streptococcus bacterium is group E streptococci (GES).
111. The method of clause 91 , any other suitable clause, or any combination of suitable clauses, wherein the Gram positive bacterium is a Listeria bacterium.
112. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the Gram positive bacterium is an Enterococcus bacterium.
113. The method of clause 112, any other suitable clause, or any combination of suitable clauses, wherein the Enterococcus bacterium is Enterococcus faecalis.
114. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the disease is a Methicillin Resistant Staphylococcus aureus (MRSA) infection.
115. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the disease is a Vancomycin Intermediate Staphylococcus aureus (VISA) infection.
116. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the disease is a Vancomycin Resistant Staphylococcus aureus (VRSA) infection.
117. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the disease is mastitis.
118. The method of clause 117, any other suitable clause, or any combination of suitable clauses, wherein the mastitis is clinical mastitis.
119. The method of clause 117, any other suitable clause, or any combination of suitable clauses, wherein the mastitis is sub-clinical mastitis.
120. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the disease is septicaemia.
121. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the disease is inflammation.
122. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the disease is polyarthritis.
123. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the disease is polyserositis. 124. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the disease is meningitis.
125. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the disease is endocarditis.
126. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition reduces one or more symptoms associated with the disease in the animal.
127. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the animal is a ruminant.
128. The method of clause 127, any other suitable clause, or any combination of suitable clauses, wherein the ruminant is selected from the group consisting of cattle, goats, sheep, giraffes, American Bison, European bison, yaks, water buffalo, deer, camels, alpacas, llamas, wildebeest, antelope, pronghorn, and nilgai.
129. The method of clause 127, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a zoo animal.
130. The method of clause 127, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a domestic animal.
131. The method of clause 127, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a wild animal.
132. The method of clause 127, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a farm animal.
133. The method of clause 127, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a livestock animal.
134. The method of clause 127, any other suitable clause, or any combination of suitable clauses, wherein the ruminant animal is a bovine animal.
135. The method of clause 134, any other suitable clause, or any combination of suitable clauses, wherein the bovine animal is a cattle.
136. The method of clause 134, any other suitable clause, or any combination of suitable clauses, wherein the cattle is a meat-producing cattle.
137. The method of clause 134, any other suitable clause, or any combination of suitable clauses, wherein the cattle is a milk-producing cattle.
138. The method of clause 134, any other suitable clause, or any combination of suitable clauses, wherein the milk-producing cattle is a dairy cattle.
139. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the animal is a poultry. 140. The method of clause 139, any other suitable clause, or any combination of suitable clauses, wherein the poultry is selected from the group of ducks, geese, chickens, chickens, laying hens, broilers, turkeys, quails, ostriches, and turkeys.
141. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the animal is a swine.
142. The method of clause 141, any other suitable clause, or any combination of suitable clauses, wherein the swine is selected from the group consisting of pigs, boars, sows, gilts, and piglets.
143. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the animal is a companion animal.
144. The method of clause 143, any other suitable clause, or any combination of suitable clauses, wherein the companion animal is selected from the group consisting of canine, feline, rabbit, and ferret.
145. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is administered at an amount of between 0.001 to 1000 mg of PlySs2.
146. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is administered at an amount of between 0.1 to 10 mg of PlySs2.
147. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is administered at an amount of between 1 to 50 mg of the PlySs2.
148. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is administered at an amount of between 10 to 40 mg of the PlySs2.
149. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is administered at an amount of between 20 to 40 mg of the PlySs2.
150. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is administered at an amount of 30 mg of the PlySs2.
151. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is administered at a dose of 0.001 to 1000 mg of PlySs2 per kg of animal body weight. 152. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is administered at a dose of 0.1 to 100 mg of PlySs2 per kg of animal body weight.
153. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is administered at a dose of 1 to 10 mg of PlySs2 per kg of animal body weight.
154. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is administered at a dose of 1 to 5 mg of PlySs2 per kg of animal body weight.
155. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is administered at a dose of 5 mg of PlySs2 per kg of animal body weight.
156. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is administered at a dose of 10 mg of PlySs2 per kg of animal body weight.
157. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is administered at a dose of 15 mg of PlySs2 per kg of animal body weight.
158. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is administered at a dose of 20 mg of PlySs2 per kg of animal body weight.
159. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is a parenteral formulation.
160. The method of clause 159, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is selected from the group consisting of intravenous, intraarterial, intraperitoneal, intrathecal, intradermal, epidural, intracerebroventricular, intraurethral, intrastemal, intracranial, intratumoral, intramuscular and subcutaneous.
161. The method of clause 159, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intravenous.
162. The method of clause 159, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intraarterial.
163. The method of clause 159, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intraperitoneal.
164. The method of clause 159, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intrathecal. 165. The method of clause 159, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intradermal.
166. The method of clause 159, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is epidural.
167. The method of clause 159, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intracerebroventricular.
168. The method of clause 159, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intraurethral.
169. The method of clause 159, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intrasternal.
170. The method of clause 159, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intracranial.
171. The method of clause 159, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intratumoral.
172. The method of clause 159, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is intramuscular.
173. The method of clause 159, any other suitable clause, or any combination of suitable clauses, wherein the parenteral formulation is subcutaneous.
174. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed in conjunction with feeding the animal.
175. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed while feeding the animal.
176. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed concurrently with feeding the animal.
177. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed simultaneously with feeding the animal.
178. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed independently of feeding the animal. 179. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed after feeding the animal.
180. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed daily.
181. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed once per day.
182. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed twice per day.
183. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed up to three times per day.
184. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed up to four times per day.
185. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed up to five times per day.
186. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed up to six times per day.
187. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed once per week.
188. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed twice per week.
189. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed once every two weeks. 190. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed twice every two weeks.
191. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed for 7 days.
192. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed for 10 days.
193. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed for 1 to about 13 days.
194. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the step of administering the veterinary formulation to the animal is performed for 21 days.
195. A method of making a veterinary composition comprising a PlySs2 enzyme, wherein the method comprises the step of producing a recombinant PlySs2 enzyme from recombinant expression in a bacterium to form the PlySs2 enzyme.
196. The method of clause 195, any other suitable clause, or any combination of suitable clauses, wherein the bacterium is E. coli.
197. The method of clause 195, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises the step of fermenting the bacterium.
198. The method of clause 195, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises the step of isolating the PlySs2 enzyme.
199. The method of clause 195, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises the step of processing the bacterium in a microfluidizer.
200. The method of clause 195, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises the step of chromatographically purifying the PlySs2 enzyme.
201. The method of clause 195, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises the step of concentrating the PlySs2 enzyme.
202. The method of clause 195, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises the step of removing one or more endotoxins from the PlySs2 enzyme. 203. The method of clause 195, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises the step of combining the PlySs2 enzyme and a carrier.
204. The method of clause 195, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is used to treat a disease.
205. The method of clause 204, any other suitable clause, or any combination of suitable clauses, wherein the disease is an infection associated with a Gram positive bacterium.
206. The method of clause 195, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition reduces one or more symptoms associated with the disease in a non-human animal.
207. A kit comprising a bottle comprising a veterinary composition of clause 1, any other suitable clause, or any combination of suitable clauses
208. The kit of clause 207, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition is a parenteral formulation for administration to a non- human animal via a route of administration selected from the group consisting of intravenous, intraarterial, intraperitoneal, intrathecal, intradermal, epidural, intracerebroventricular, intraurethral, intrastemal, intracranial, intratumoral, intramuscular, and subcutaneous.
209. The kit of clause 207, any other suitable clause, or any combination of suitable clauses, wherein the animal disease is associated with a swine.
210. The kit of clause 207, any other suitable clause, or any combination of suitable clauses, wherein the bottle comprises an effective amount of the veterinary composition.
211. The kit of clause 207, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the veterinary composition comprises an amount of PlySs2 ranging from about 1 mg/kg to about 10 mg/kg.
212. The kit of clause 207, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition in the bottle is a ready-to-use liquid composition.
213. The kit of clause 207, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition in the bottle is a lyophilized composition.
214. The kit of clause 207, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition in the bottle is a single unit dose.
215. The kit of clause 207, any other suitable clause, or any combination of suitable clauses, wherein the veterinary composition in the bottle is a multi-unit dose.
216. The kit of clause 207, any other suitable clause, or any combination of suitable clauses, further comprising a diluent. 217. The kit of clause 207, any other suitable clause, or any combination of suitable clauses, further comprising an instruction manual which contains the information for administration of the veterinary composition.
218. A method of purifying a PlySs2 enzyme, wherein the method comprises a step of fermenting the PlySs2 enzyme.
219. The method of clause 218, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises the step of flowing the fermented PlySs2 enzyme through a chromatography device.
220. The method of clause 218, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises the step of removing one or more endotoxins from the PlySs2 enzyme to form a purified PlySs2 enzyme.
221. The method of clause 218, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises the step of purifying the PlySs2 enzyme to a purity level of about 85% to about 95%.
222. The method of clause 218, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises the step of administering an effective amount of the purified PlySs2 enzyme to a non-human animal.
223. The method of clause 218, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the purified PlySs2 enzyme ranges from about 1 mg/kg to about 10 mg/kg.
224. The method of clause 218, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the purified PlySs2 enzyme is about 5 mg/kg.
225. The method of clause 218, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises the step of administering the effective amount of the purified PlySs2 enzyme to the non-human animal once.
226. The method of clause 218, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises the step of administering the effective amount of the purified PlySs2 enzyme to the non-human animal once a day for about 13 days.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows the domains of a PlySs2 protein and a depiction of the PlySs2 amino acid sequence;
[0016] FIG. 2A shows a purification scheme for purifying PlySs2;
[0017] FIG. 2B shows a blot associated with the purification scheme of FIG. 2A and identifies the purified PlySs2; [0018] FIG. 3 shows a BSL2 density reduction activity;
[0019] FIG. 4 shows survival curves in swine after two different dosage regimens comprising PlySs2, TG03 and TG04 treatment groups;
[0020] FIG. 5 shows the rectal temperature of animals in the study per day for each treatment group;
[0021] FIG. 6 shows the percentage of animals having fever in the study per day for each treatment group;
[0022] FIG. 7A shows exposure and percent survival results for animals in the TG03 and TG04 treatment groups;
[0023] FIG. 7B correlates the day of mortality to the last day of detectable enzyme concentration in animals in the TG03 group of FIG. 7A;
[0024] FIG. 8A shows the body temperature of the six animals in Pen 6, which received PlySs2 once at a dose of 5 mg/kg (group TG03);
[0025] FIG. 8B shows the body temperature of the six animals in Pen 10, which received PlySs2 once daily for 13 days at a dose of 5 mg/kg (group TG04);
[0026] FIG. 9A shows the average exposure of animals in the three different pens which received PlySs2 once at a dose of 5 mg/kg (group TG03);
[0027] FIG. 9B shows the average exposure of animals in the three different pens which received PlySs2 once daily for 13 days at a dose of 5 mg/kg (group TG04);
[0028] FIG. 10A shows the plasma concentration of PlySs2 after the animals were administered PlySs2 at a dose of 3 mg/kg; and
[0029] FIG. 10B shows the plasma concentration of PlySs2 after the animals were administered PlySs2 at a dose of 10 mg/kg.
DETAILED DESCRIPTION
[0030] The present disclosure provides exemplary veterinary compositions comprising an active pharmaceutical agent or an active compound (e.g., PlySs2). The present disclosure also provides exemplary methods of making or using the veterinary composition. The veterinary composition of the present disclosure exhibits desirable properties and provide benefits for the treatment of disease in an animal (e.g., a non-human animal).
[0031] Specifically, in one aspect, the veterinary composition can comprise PlySs2. PlySs2 can be specific in killing harmful bacteria associated with disease in animals (e.g., bacterial disease). Use of PlySs2 for the treatment of bacterial disease can avoid traditional antibiotic treatments of animals and can be utilized as or in an “antibiotic free” treatment course. [0032] Since PlySs2 is a protein, it is biodegradable and advantageously does not accumulate in the body of animals. PlySs2 targets complex cell wall peptidoglycan structures in bacteria and, as a result, PlySs2 can have a lower propensity to develop resistance in animals. Additionally, since PlySs2 hydrolyzes the cell wall of the targeted bacteria, a composition comprising PlySs2 is selective in action and does not inadvertently kill any good or useful bacteria.
[0033] Alternatively, or additionally, the veterinary composition can include more than one active compound or active pharmaceutical agent. For example, the veterinary composition can include PlySs2 and one or more antimicrobials, such as an antibiotic. Moreover, given their mechanism of action, lytic enzymes, such as PlySs2, administered with other antimicrobials can provide a synergistic effect in treating bacterial diseases in animals. In some embodiments, administration of the PlySs2 can produce synergy with one or more antibiotics, such as antibiotics co- administered with the PlySs2 or antibiotics that the animal is taking to treat other disease or infectious indications.
[0034] The veterinary composition can be adapted, utilized, or used to treat an animal. The animal can be a non-human animal. For example, the non-human animal can be any animal that is not a human, such as a ruminant animal, a zoo animal, a domestic animal, a wild animal, a bovine animal, a farm animal, or a companion animal. Specifically, the non-human animal can be a cattle, a goat, a sheep, a giraffe, an American Bison, an European bison, a yak, a water buffalo, a deer, a camel, an alpaca, a llama, a wildebeest, an antelope, a pronghorn, a nilgai, a swine, a duck, a goose, a chicken, a laying hen, a broiler, a turkey, a quail, an ostrich, a turkey, a pig, a boar, a sow, a gilt, a piglet, a canine, a feline, a rabbit, and/or a ferret. The veterinary composition can also be adapted, utilized, or used to treat a non-human animal that produces food for humans, such as a meat-producing cattle, a milk-producing cattle, a dairy cattle, and/or a chicken capable of laying eggs.
[0035] The veterinary composition can be adapted, utilized, or used to treat an animal with a disease. The disease can be associated with a microbial infection caused by a fungus, a virus, and/or a bacterium. In an exemplary embodiment, the disease can be associated with a bacterial infection, such as an infection related to a Gram positive (+) bacterium.
[0036] The disease can also be associated with a Methicillin Resistant Staphylococcus aureus (MRS A) infection, a Vancomycin Intermediate Staphylococcus aureus (VISA) infection, a Vancomycin Resistant Staphylococcus aureus (VRSA) infection, clinical mastitis, sub-clinical mastitis, septicaemia, inflammation, polyserositis, meningitis, and/or endocarditis. The veterinary composition can also be adapted, utilized, or used to treat a disease of an animal infected with Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus agnetis, Staphylococcus simulans, Staphylococcus epide midis, Streptococcus suis, Streptococcus equi, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus sanguinis, Streptococcus pneumonia, Streptococcus pyogenes, group B streptococcus (GBS), group G streptococci (GGS), group E streptococci (GES), Listeria, and Enterococcus faecalis.
[0037] In some embodiments, the veterinary composition can result in controlling and/or eliminating about 90% or more of bacteria in the animal. In some embodiments, the veterinary composition can result in controlling or eliminating (e.g., killing) about 90% of disease in animals on a farm. For example, the veterinary composition can result in controlling or eliminating about 90% of Streptococcus suis in animals on a farm with Streptococcus suis related diseases.
[0038] In other embodiments, the veterinary composition can result in controlling or eliminating about 50% to about 100% of bacteria and/or disease in an animal, including any specific or range of percentages comprised therein. For example, the veterinary composition can result in controlling or eliminating about 50% to about 70%, about 70% to about 90%, or about 90% to about 99% of Streptococcus suis in animals on a farm with Streptococcus suis related diseases. In some embodiments, the veterinary composition can result in controlling or eliminating about 50% to about 70%, about 70% to about 90%, or about 90% to about 99% of Streptococcus equi, Streptococcus uberis, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus sanguinis, Streptococcus pneumoniae; MRSA, vancomycin-resistant Staphylococcus aureus (VRSA), S. epidermidis, S. pseudintermedius, or Listeria of animals on a farm with the respective disease.
[0039] The veterinary composition can result in about 20% to about 90% improvement in a morbidity rate in the animals with the disease, including any specific or range of percentages comprised therein. The veterinary composition can result in about 40% to about 50%, about 50% to about 60%, or about 60% to about 80% improvement in the morbidity rate in the animals with the disease. The veterinary composition can result in about 20% to about 30%, about 30% to about 50%, or about 50% to about 60% improvement in the mortality rate of the animals with the disease.
[0040] Generally, PlySs2 comprises a catalytic domain. The catalytic domain comprises a catalytic active domain (CAD) and a catalytic binding domain (CBD). The catalytic active domain, also referred to as an N-terminal cysteine-histidine aminopeptidase catalytic domain, provides rapid cell wall lysis via hydrolysis. The catalytic binding domain, also referred to as a C-terminal SH3b cell-wall binding domain, provides high affinity binding to cell wall peptidoglycans that are essential for bacterial growth. Without being bound by any theory, it is believed that the catalytic binding domain provides targeted pathogen specificity and beneficially allows for the lack of antimicrobial resistance that can be observed with traditional antibiotics.
[0041] A PlySs2 protein typically comprises about 245 amino acids. The PlySs2 protein includes the CAD and the CBD (26 kDa, pl = 9.7). FIG. 1 shows the catalytic domains of PlySs2 and a depiction of the PlySs2 amino acid sequence.
[0042] In certain embodiments, the PlySs2 is a modified or recombinant PlySs2. For instance, the modified and/or recombinant PlySs2 can be synthetically prepared (e.g., manually or automatically in a laboratory) to comprise PlySs2 and an albumin binding domain (ABD). In some embodiments, one or more amino acids in PlySs2 are synthetically modified to provide sufficient contact, connection, and/or fusion of the PlySs2 with the ABD to form the modified PlySs2.
[0043] Generally, an ABD can be utilized to increase the half-life of the modified PlySs2 that is present in the veterinary composition. The use of ABD in therapeutic compositions is described, for example, in U.S. Patent No. 10,428,120 as well as published documents US 2014/0170142, US 2020/0040099, US 2017/0114114, JP 2017137320A, JP 2016526014A, JP 6382826B2, and CA 2694139, all of which are incorporated by reference herein in their entireties. For example, PlySs2 can naturally have a half-life of about 4 hours to about 30 hours. However, albumin has a circulatory half-life of about 19 days and undergoes the neonatal Fc receptor (FcRn) mediated recycling pathway in a body of a subject (e.g., a nonhuman animal). Therefore, the modified PlySs2 comprising the ABD can have an increased half-life ranging from about 5 to about 7 days, including any specific or range of time comprised therein.
[0044] In some embodiments, the veterinary composition can comprise the active compound or the active pharmaceutical agent (e.g., PlySs2) and a carrier. The carrier can be selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof. The carrier can be or can comprise a solid, a liquid, and/or a gaseous compound. The carrier can be or can include a salt, water, and/or saline. The carrier can be or can comprise a solution, a surfactant, a lyophilized composition, a powder, a suspension, a colloid, a crystalline solution, a slurry, a spray, and/or a dispersion. The veterinary composition can be in the form of a gel capsule, a chewable tablet, and/or an injectable solution.
[0045] The veterinary composition can be administered orally, sublingually, intravenously, intramuscularly, intranasally, intraperitoneally, and/or subcutaneously. The veterinary composition can be administered in a regimen that includes a single unit dose or multi-unit dose. For example, the veterinary composition can be administered a single time using a single unit dose of the veterinary composition. Alternatively, the veterinary composition can be administered using a multi-unit dose by providing the animal more than one injection over a time period, such as for about 1 day to about 13 days. In some embodiments, more than one injection (e.g., two injections) of the veterinary composition can be administered for a time period, such as more than 13 days (e.g., 14 days). Alternatively, the veterinary composition can be administered by providing the animal a single treatment dose per episode of the disease.
[0046] The veterinary composition can comprise an effective amount of the active compound or the active pharmaceutical agent (e.g., PlySs2). The effective amount of the active compound or pharmaceutical component (e.g., PlySs2) in the veterinary composition is the amount of the active compound (e.g., PlySs2) that must be administered to the animal to provide an efficacious reduction or elimination of bacteria and/or disease. The effective amount of the active compound or the active pharmaceutical component (e.g., PlySs2) can range from about 0.001 mg to about 1000 mg, including any amount or range comprised therein.
[0047] The effective amount of the active compound or the active pharmaceutical agent (e.g., PlySs2) can depend on the body weight of the animal. For example, the effective amount of the active compound or the active pharmaceutical agent (e.g., PlySs2) can range from about
1 mg/kg to about 20 mg/kg, including any amount or range comprised therein. Specifically, the effective amount of the active compound (e.g., PlySs2) can range from about 1 mg/kg to about 5 mg/kg, from about 1 mg/kg to about 10 mg/kg, from about 5 mg/kg to about 10 mg/kg, from about 10 mg/kg to about 20 mg/kg, including any amount or range comprised therein.
[0048] In some embodiments, the animal can be treated by administration of a single dose of the veterinary composition comprising the effective amount of the active compound or the active pharmaceutical agent (e.g., PlySs2). In other embodiments, the animal can be treated by administration of multiple doses (e.g. multi-unit dose) of the veterinary composition comprising the effective amount of the active pharmaceutical component (e.g., PlySs2). The multiple doses or multi-unit doses of the veterinary composition comprising the effective amount of the active compound or the active pharmaceutical agent (e.g., PlySs2) can be given on consecutive or intermittent days. For example, the multiple doses of the veterinary composition can be administered to an animal on about 2 to about 25 consecutive days, including any specific or range of days comprised therein, such as for about 2 to about 5, about
2 to about 10, or about 2 to about 15 consecutive days, including any specific or range of consecutive days comprised therein. [0049] In certain embodiments, the veterinary composition can be a controlled release formulation. Preparing the veterinary composition to have a controlled release formulation provides for a slower release of the PlySs2 active compound over time to the animal. Formulating the veterinary composition to have a controlled release comprises using formulations including but not limited to polymer microspheres, polymer nanospheres, hydrogels, and/or in-situ gelling polymers.
[0050] In some embodiments, the controlled release formulation comprises a hydrogel. In an embodiment, the hydrogel is a temperature sensitive hydrogel. In some embodiments, the controlled release formulation comprising the hydrogel can be a liquid at temperature at or below about 37 degrees to about 42 degrees, including any temperature or range comprised therein. The controlled release formulation comprising the hydrogel can be a gel at temperature greater that about 37 degrees to about 42 degrees, including any temperature or range comprised therein. In an embodiment, the hydrogel comprises one or more temperature sensitive polymers. Various temperature sensitive hydrogels and polymers are known in the art. For instance, hydrogels and polymers can be utilized in which the veterinary composition is a liquid at certain temperatures and a gel or a gel-like composition at different temperatures.
[0051] In certain embodiments, the controlled release formulation can comprise a nonaqueous carrier. For example, the controlled release formulation can comprise an oil carrier. Formulating the veterinary composition to be associated with the oil provides for a slower release of the PlySs2 over time to the animal. For instance, if the formulation in an oil is administered parenterally to the animal, the oil causes the PlySs2 to be released in a slower manner compared to other, non-oil containing parenteral formulations. The oil of the controlled release formulation can comprise any type of oil, such as a plant oil, an animal oil, an essential oil, and/or any combination thereof.
[0052] The present disclosure further provides a kit. This kit can comprise a bottle containing the veterinary composition according to the present disclosure adapted for administration to a non-human animal. The veterinary composition in the bottle can be the PlySs2 active compound and one or more carriers. The carrier is selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof. The veterinary composition in the bottle can be a parenteral formulation administered to the non- human animal via a route of administration selected from the group consisting of intravenous, intraarterial, intraperitoneal, intrathecal, intradermal, epidural, intracerebroventricular, intraurethral, intrastemal, intracranial, intratumoral, intramuscular and subcutaneous.
[0053] In some embodiments, the veterinary composition in the bottle can further comprise a second active compound. The second active compound can comprise any antimicrobial or non- antimicrobial compound, such as a fungus, a virus, and/or a bacterium. The second active compound can also be a bacterium that is the same bacterium as the first active compound, such as Streptococcus suis. The second active compound can also be a bacterium that is not the same bacterium as the first active compound, such as Streptococcus suis. For example, the second active compound can be a species of Streptococcus, Staphylococcus, or Listeria.
[0054] In one embodiment of the kit, the veterinary composition in the bottle can be in the form of a solution or ready-to-use liquid composition. Alternatively, the veterinary composition in the bottle can be in the form of a lyophilized powder. The veterinary composition in the bottle can be a single unit dose or a multi-unit dose.
[0055] In one embodiment, the bottle can comprise an effective amount of the veterinary composition. In some embodiments, the effective amount of the veterinary composition can comprise an effective amount of active compound (e.g., PlySs2) in a single unit dose. Alternatively, the effective amount of the veterinary composition can comprise an effective amount of active compound (e.g., PlySs2) in. in a multi-unit dose. For example, the multi-unit dose of the veterinary composition in the bottle can comprise about 2 to about 5 doses, about 5 to about 10 doses, about 10 to about 30 doses, about 30 to about 50 doses, or any specific number or range of doses comprised therein. In some embodiments of the kit, the bottle can comprise more than 50 doses. The effective amount of active compound (e.g., PlySs2) can be the amount calculated, experimentally determined, or computationally determined to be able to treat the non-human animal (e.g., swine).
[0056] In a further embodiment, the kit can include a diluent. The diluent can be any non-active solute or solution that enables or enhances the administration or activity of the active compound (e.g., PlySs2) to the animal. In an exemplary embodiment, the diluent of the present disclosure can comprise saline, buffer, and/or water. In yet another embodiment, the kit can further include an instruction manual, which contains information and directions for proper administration and/or dosages of the veterinary composition to one or more types of animals.
[0057] In some embodiments, a method of treating a disease in a non-human animal can include administering the veterinary composition to the non-human animal, the disease can be associated with a Gram positive bacterium (e.g., Streptococcus bacterium). The non-human animal can be any of the animals described previously including, but not limited to a swine. The method of administering the veterinary composition can include administering an amount of PlySs2 from about 1 mg/ kg to about 10 mg/kg including any amount or range comprised therein to the non-human animal. [0058] In one embodiment, the method of treating a disease in a non-human animal can comprise administering the veterinary composition to the non-human animal once. In other embodiments, the method can comprise administering the veterinary composition to the non- human animal once a day for 2 to about 13 consecutive days. In some further embodiments, the method can comprise administering the veterinary composition to the non-human animal for more than 13 days (e.g., 14 days).
[0059] In some embodiments, a method of making or using a veterinary composition comprises a PlySs2 enzyme. This method can include the step of producing a recombinant PlySs2 enzyme from recombinant expression in a bacterium to form the PlySs2 enzyme. The bacterium can be E. coli or any other bacterium that can be utilized for the production of the recombinant PlySs2.
[0060] The method of producing a recombinant PlySs2 enzyme (e.g., a modified PlySs2 protein) in the bacterium can include fermenting the bacterium and/or isolating the PlySs2 enzyme. In some embodiments, the method can include processing the bacterium in a microfluidizer and purifying the PlySs2 enzyme. In an exemplary embodiment, the purifying step can comprise chromatographically purifying the PlySs2 enzyme in a chromatography device. Additionally, the method can include concentrating the PlySs2 enzyme and/or removing one or more endotoxins from the PlySs2 enzyme. The method can also include combining the purified PlySs2 enzyme with a carrier in order to make or use the veterinary composition.
[0061] A method of purifying a PlySs2 enzyme is also described herein. In one embodiment, the method of purifying the PlySs2 enzyme comprises the steps of fermenting the PlySs2 enzyme, flowing the fermented PlySs2 enzyme through the chromatography device, and/or removing one or more endotoxins from the PlySs2 enzyme to form the purified PlySs2 enzyme. The method of purifying PlySs2 can comprise purifying the PlySs2 enzyme to a purity level of about 70% to about 99%, including any percentage or range comprised therein. More specifically, the method of purifying PlySs2 can comprise purifying the PlySs2 enzyme to a purity level of about 70% to about 80, about 80% to 90%, about 90% to about 95%, or about 95% to about 99%.
[0062] The method of purifying PlySs2 can also include combining the purified PlySs2 enzyme with a carrier in order to make or use the veterinary composition that can be administered to any non-human animal. The method can also include administering the effective amount of the purified PlySs2 enzyme to the non-human animal. The method can further include administering to the non-human animal about 0.5 mg/kg to about 15 mg/kg, including any specific amount or range of enzyme comprised therein. For example, the method can include administering to the non-human animal about 5 mg/kg to about 10 mg/kg, about 10 mg/kg to about 15 mg/kg, including any amount or range of enzyme comprised therein. In some embodiments, the method can include administering about 5 mg/kg of the purified PlySs2 enzyme to the non-human animal.
[0063] The method can further include administering the effective amount of the purified PlySs2 enzyme to the non-human animal once. Alternatively, the method can further include administering the effective amount of the purified PlySs2 enzyme to the non-human animal once a day for a period of consecutive days. The period of consecutive days can range from 1 day to 13 days. In some embodiments, the period of consecutive days can be more than 13 days (e.g., 14 days).
[0064] Various figures are included in the present disclosure and are identified in the Examples described herein.
EXAMPLES
EXAMPLE 1: Process for Preparing PlySs2
[0065] The instant example describes an exemplary process for preparing PlySs2. The expression platform for preparing PlySs2 was E. coli TOPIC, pBAD24, inducible with arabinose. Fermentation was performed on a pilot scale in 30-L fermenters. Cell processing was performed using a microfluidizer and purification was done via flow through IEX chromatography. Finally, the PlySs2 was concentrated and underwent diafiltration and endotoxin removal. A purification scheme for purifying PlySs2 is shown in FIG. 2A. A blot associated with the purification scheme is illustrated in FIG. 2B.
[0066] FIG. 2B illustrates 4 lanes showing total lysate, a soluble fraction of the lysate, total insoluble lysate, and a purified PlySs2. FIG. 2B identifies the purified PlySs2 as a 26 kDa molecule. The yield of the PlySs2 production process was 10 to 30 g (purified) PlySs2 per 30 L fermenter, with 85-95% purity by sodium dodecyl sulfate (SDS).
[0067] For the instant example, the formulation of PlySs2 was prepared to be 13 mg/ml, in 10 mM Tris-HCl (pH 7.4, 20% glycerol). Preliminary analysis indicated that activity/stability was retained at 4 °C for at least 1 month (via activity assay and visual inspection) and that activity was retained after 3 year frozen storage (via activity assay).
[0068] Furthermore, a BSL2 density reduction activity assay was performed and results are shown in FIG. 3. Briefly, S. suis was washed with buffer and diluted to ODeoo ~ 1.5 as substrate. PlySs2 was serially diluted to seven different targeted concentrations (40, 20, 10, 5, 2.5, 1.25, and 0.625 pg/ml). A density reduction assay (DRA) assay was performed by mixing 25 pl cells with 25 pl PlySs2 in a 96-well half area flat bottom clear plate. Then, ODeoo was read at room temperature for 1 hr with shaking between each read. The ODeoo for each of the concentrations is shown in FIG. 3.
EXAMPLE 2: Efficacy Study of PlySs2 in Swine
[0069] In the instant example, an efficacy study in swine was evaluated. Parameters of the study are shown in Table 1:
Table 1 - Study Parameters
Figure imgf000030_0001
[0070] Mortality of the various treatment groups is shown in Table 2.
Table 2 - Mortality
Figure imgf000030_0002
[0071] By day 7, treatment with PlySs2 appears to reduce mortality. All mortalities occur by approximately day 14.
EXAMPLE 3: Efficacy Study of PlySs2 via Streptococcus suis Challenge
[0072] The instant example evaluates the effectiveness of PlySs2 at two different dosage regimens in swine. Veterinary compositions comprising PlySs2 were administered individually to swine intramuscularly (IM) in order to reduce the incidence of S. suis in a challenge model.
[0073] Briefly, the example utilized an established intranasal challenge model. On Day 0 of the challenge, 2 ml of 1 % acetic acid was given to animals by intranasal (IN) application. About one to two hours later, animals were given 4 mL of a challenge of S. suis via IN application. On Day 1, control or PlySs2 treatments were performed. The live phase was 21 days long.
[0074] The primary variable of the instant example was mortality of the animals and secondary variables included body temperature, clinical signs, blood septicemia, lung lesions, bacteria isolation (necropsy), average daily gain (ADG), and body weight. In vivo exposure of PlySs2 was evaluated via blood collection on: Day 0 (prior challenge), Day 1 (prior first treatment), Day 2, Day 3, Day 4, Day 7, Day 9, Day 11, Day 14, Day 16, and Day 21.
Inspection of injection sites were performed daily and animals were also observed approximately 10 to 15 minutes after each injection. Treatment groups for the instant example are shown in Table 3. All animals were challenged with S. suis and all treatment groups included 18 animals (6 animals per pen; 3 total pens):
Table 3 - Treatment Groups
Figure imgf000031_0001
[0075] The survival curve results for each treatment group are shown in FIG. 4. Further, mortality statistics and reasons for removal are shown in Tables 4 and 5, respectively. All animals found dead or euthanized were necropsied and documented. In the event that an animal was severely moribund as determined by the attending veterinarian, the animal was euthanized prior to the study end date (Day 21). Table 4 - Mortality Percentage
Figure imgf000032_0001
Table 5 - Reasons for Removal
Figure imgf000032_0002
[0076] FIG. 5 shows the rectal temperature of animals in the study per day for each treatment group. FIG. 6 shows the percentage of animals having fever in the study per day for each treatment group. FIG. 5 shows that the animals in the groups treated with the active compound had lower temperature than the animals treated with either baytril or sterile buffer. FIG. 6 shows that the percentage of animals with fever in the groups treated with the active compound was lower than the percentage of animals with fever in the groups treated with either baytril or a sterile buffer.
[0077] Identification of S. suis by location for each treatment group is shown in Table 6. The percentage of animals affected by S. suis at each location is shown for each treatment group. Total lung lesions observed were not statistically significant between groups and ADG was greater in the PlySs2 (13X) group compared to other groups. Table 6 - Percent affected by S. suis
Figure imgf000033_0001
[0078] The percentage of abnormal clinical scores (i.e., greater than zero) for various clinical evaluations for each treatment group is shown in Table 7. There were few events identified for cough and for abdominal distention.
Table 7 - Clinical Scores Greater Than Zero (>0)
Figure imgf000033_0002
Figure imgf000034_0001
0079] The instant example demonstrated variable exposure to PlySs2. Further, exposure was not directly related to morbidity. However, multiple dosing was correlated to improved survival of animals as animals that got daily doses (i.e., animals in the TG04 group) exhibited better survival data. FIG. 7 A shows exposure and percent survival results for animals in the TG03 and TG04 treatment groups. About 83.3% of the treated animals were shown to survive on day 21 post treatment if they were administered the active compound for 13 consecutive days. Alternatively, if the animals were administered the active compound only once, about 11.8% of treated animals were shown to survive on day 21 post treatment. FIG. 7B correlates the day of mortality to the last day of detectable enzyme concentration in animals in the TG03 group. The majority of animals did not show any detectable level of enzyme concentration more than 2 days after being administered the active compound.
[0080] As shown in FIGS. 8A and 8B, there was no observed correlation between exposure and body temperature. Without aligning with any theory, FIG. 8A suggests that there can be influence of individual innate immunity in Subject 64. FIG. 8A shows results of the 6 animals in Pen 6, which received PlySs2 once at a dose of 5 mg/kg (group TG03). FIG. 8B shows results of the 6 animals in Pen 10, which received PlySs2 once daily for 13 days at a dose of 5 mg/kg (group TG04). FIGS. 9A and 9B shows individual exposures to PlySs2 for various animals in the PlySs2 treatment groups. As illustrated in FIG. 9A, animals in Pens 1, 6, and 9 received PlySs2 once at a dose of 5 mg/kg (group TG03). Most of these animals exhibited a declining plasma concentration of the enzyme after day 1, the only day that the animals were administered the active compound. As illustrated in FIG. 9B, animals in Pens 3, 4, and 10 received PlySs2 once daily for 13 days at a dose of 5 mg/kg (group TG04). Most of these animals exhibited a declining plasma concentration of the enzyme after day 13, the last day that these animals were administered the active compound.
[0081] The instant example shows that a daily dosing schedule (i.e., once daily dosing of PlySs2) correlated with efficacy and improved survival of animals. A reduction in clinical signs was observed and animals did not have any injection site reactions.
EXAMPLE 4: Pharmacokinetic Analysis of PlySs2 Compositions
[0082] The instant example evaluates pharmacokinetics of PlySs2 at different dosages. Animals were administered PlySs2 at a dose of 3 mg/kg and at a dose of 10 mg/kg. There were four animals in each group. The results of the pharmacokinetic study for a dose of 3 mg/kg for each animal is shown in FIG. 10A. The results of the pharmacokinetic study for a dose of 10 mg/kg for each animal is shown in FIG. 10B. FIGS. 10A and 10B illustrate the plasma concentration of PlySs2 over a period of time after the animals were dosed.
[0083] For the instant example, the maximum concentration of PlySs2 (Cmax) was dose responsive, but not dose proportional. Furthermore, the area under the curve of PlySs2 (AUC) did not clearly increase with increase in dose. The half-life of PlySs2 was generally around 4 hours in duration, with two subjects observed to have a half-life of greater than 30 hours.
[0084] The features illustrated or described in connection with one exemplary embodiment or aspect can be combined with any other feature or element of any other embodiment or aspect described herein. Such modifications and variations are intended to be included within the scope of the present disclosure. Further, a person skilled in the art will recognize that terms commonly known to those skilled in the art can be used interchangeably herein.
[0085] The above embodiments and aspects are described in sufficient detail to enable those skilled in the art to practice what is claimed and it is to be understood that other embodiments can be utilized and that logical, mechanical, and electrical changes can be made without departing from the spirit and scope of the claims. The detailed description is, therefore, not to be taken in a limiting sense.
[0086] As used herein, an element or step recited in the singular and proceeded with the word “a” or “an” should be understood as not excluding plural of said elements or steps, unless such exclusion is explicitly stated. Furthermore, references to “one embodiment” of the presently described subject matter are not intended to be interpreted as excluding the existence of additional embodiments that also incorporate the recited features. Specified numerical ranges of units, measurements, and/or values include, consist essentially or, or consist of all the numerical values, units, measurements, and/or ranges including or within those ranges and/or endpoints, whether those numerical values, units, measurements, and/or ranges are explicitly specified in the present disclosure or not.
[0087] Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terms “first,” “second,” “third,” and the like, as used herein do not denote any order or importance, but rather are used to distinguish one element from another. The term “or” and “and/or” is meant to be inclusive and mean either or all of the listed items. In addition, the terms “connected” and “coupled” are not restricted to physical or mechanical connections or couplings, and can include electrical connections or couplings, whether direct or indirect. [0088] Moreover, unless explicitly stated to the contrary, embodiments “comprising,” “including,” or “having” an element or a plurality of elements having a particular property can include additional such elements not having that property. The term “comprising” or “comprises” refers to a composition, compound, formulation, or method that is inclusive and does not exclude additional elements, components, and/or method steps. The term “comprising” also refers to a composition, compound, formulation, or method embodiment of the present disclosure that is inclusive and does not exclude additional elements, components, or method steps. The phrase “consisting of’ or “consists of’ refers to a compound, composition, formulation, or method that excludes the presence of any additional elements, components, or method steps.
[0089] The term “consisting of’ also refers to a compound, composition, formulation, or method of the present disclosure that excludes the presence of any additional elements, components, or method steps. The phrase “consisting essentially of’ or “consists essentially of’ refers to a composition, compound, formulation, or method that is inclusive of additional elements, components, or method steps that do not materially affect the characteristic(s) of the composition, compound, formulation, or method. The phrase “consisting essentially of’ also refers to a composition, compound, formulation, or method of the present disclosure that is inclusive of additional elements, components, or method steps that do not materially affect the characteristic(s) of the composition, compound, formulation, or method steps.
[0090] Approximating language, as used herein throughout the specification and claims, can be applied to modify any quantitative representation that could permissibly vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as “about,” and “substantially” is not to be limited to the precise value specified. In some instances, the approximating language can correspond to the precision of an instrument for measuring the value. Here and throughout the specification and claims, range limitations can be combined and/or interchanged. Such ranges are identified and include all the sub-ranges contained therein unless context or language indicates otherwise.
[0091] As used herein, the terms “can” and “can be” indicate a possibility of an occurrence within a set of circumstances; a possession of a specified property, characteristic or function; and/or qualify another verb by expressing one or more of an ability, capability, or possibility associated with the qualified verb. Accordingly, usage of “can” and “can be” indicates that a modified term is apparently appropriate, capable, or suitable for an indicated capacity, function, or usage, while taking into account that in some circumstances, the modified term can sometimes not be appropriate, capable, or suitable. [0092] It is to be understood that the above description is intended to be illustrative, and not restrictive. For example, the above-described embodiments (and/or aspects thereof) can be used individually, together, or in combination with each other. In addition, many modifications can be made to adapt a particular situation or material to the teachings of the subject matter set forth herein without departing from its scope. While the dimensions and types of materials described herein are intended to define the parameters of the disclosed subject matter, they are by no means limiting and are exemplary embodiments. Many other embodiments will be apparent to those of skill in the art upon reviewing the above description. The scope of the subject matter described herein should, therefore, be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.
[0093] This written description uses examples to disclose several embodiments of the subject matter set forth herein, including the best mode, and also to enable a person of ordinary skill in the art to practice the embodiments of disclosed subject matter, including making and using the devices or systems and performing the methods. The patentable scope of the subject matter described herein is defined by the claims, and can include other examples that occur to those of ordinary skill in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.
[0094] While only certain features of the invention have been illustrated and described herein, many modifications and changes will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

WHAT IS CLAIMED IS:
1. A veterinary composition comprising a PlySs2 active compound and one or more carriers, wherein the PlySs2 active compound is adapted for administration to a nonhuman animal.
2. The veterinary composition of claim 1, wherein the carrier is selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof.
3. The veterinary composition of claim 1, wherein the veterinary composition is a single unit dose.
4. The veterinary composition of claim 1, wherein the veterinary composition is a parenteral formulation administered to the non-human animal via a route of administration selected from the group consisting of intravenous, intraarterial, intraperitoneal, intrathecal, intradermal, epidural, intracerebroventricular, intraurethral, intrasternal, intracranial, intratumoral, intramuscular and subcutaneous.
5. The veterinary composition of claim 1, wherein the PlySs2 active compound is a modified PlySs2, and the modified PlySs2 comprises an albumin binding domain.
6. The veterinary composition of claim 1, wherein the veterinary composition further comprises a second active compound.
7. A method of treating a disease in a non-human animal, said method comprising administering the veterinary composition of any of claims 1 to 6 to the non-human animal, wherein the disease is associated with a Gram positive bacterium.
8. The method of claim 7, wherein the Gram positive bacterium is a Streptococcus bacterium.
9. The method of claim 7, wherein the non-human animal is a swine.
10. The method of claim 7, wherein administering the veterinary composition comprises administering an amount of PlySs2 between about 1 mg/ kg and about 10 mg/kg to the non-human animal.
11. The method of claim 7, wherein administering the veterinary composition to the animal is performed for about 1 day to about 13 days, wherein after 13 days the animal survival rate ranges from about 83.3% to about 100%.
12. A method of making a veterinary composition comprising a PlySs2 enzyme, the method comprising the step of producing a recombinant PlySs2 enzyme from recombinant expression in a bacterium to form the PlySs2 enzyme, wherein the bacterium is E. coli.
- 36 -
13. The method of claim 12, further comprising fermenting the bacterium and isolating the PlySs2 enzyme.
14. The method of claim 12, further comprising processing the bacterium in a microfluidizer and chromatographically purifying the PlySs2 enzyme.
15. The method of claim 12, further comprising concentrating the PlySs2 enzyme and removing one or more endotoxins from the PlySs2 enzyme.
16. The method of claim 12, further comprising combining the PlySs2 enzyme and a carrier.
17. A kit comprising a bottle comprising the veterinary composition of any of claims 1 to 6.
18. The kit of claim 17, wherein the veterinary composition is a parenteral formulation for administration to a non-human animal via a route of administration selected from the group consisting of intravenous, intraarterial, intraperitoneal, intrathecal, intradermal, epidural, intracerebroventricular, intraurethral, intrasternal, intracranial, intratumoral, intramuscular, and subcutaneous.
19. The kit of claim 17, wherein the animal disease is associated with a swine.
20. The kit of claim 17, wherein the bottle comprises an effective amount of the veterinary composition.
21. The kit of claim 17, wherein the effective amount of the veterinary composition comprises an amount of PlySs2 ranging from about 1 mg/kg to about 10 mg/kg.
22. The kit of claim 17, wherein the veterinary composition in the bottle is a ready-to-use liquid composition.
23. The kit of claim 17, wherein the veterinary composition in the bottle is a lyophilized composition.
24. The kit of claim 17, wherein the veterinary composition in the bottle is a single unit dose.
25. The kit of claim 17, wherein the veterinary composition in the bottle is a multi-unit dose.
26. The kit of any of claims 18 to 25, further comprising a diluent.
27. A method of purifying a PlySs2 enzyme, the method comprising the steps of fermenting the PlySs2 enzyme, flowing the fermented PlySs2 enzyme through a chromatography device, and removing one or more endotoxins from the PlySs2 enzyme to form a purified PlySs2 enzyme.
- 37 -
28. The method of claim 27, further comprising the step of purifying the PlySs2 enzyme to a purity level of about 85% to about 95%.
29. The method of claim 27, further comprising the step of administering an effective amount of the purified PlySs2 enzyme to a non-human animal.
30. The method of claim 29, wherein the effective amount of the purified PlySs2 enzyme ranges from about 1 mg/kg to about 10 mg/kg.
31. The method of claim 30, wherein the effective amount of the purified PlySs2 enzyme is about 5 mg/kg.
32. The method of claim 27, further comprising the step of administering the effective amount of the purified PlySs2 enzyme to the non-human animal once.
33. The method of claim 27, further comprising the step of administering the effective amount of the purified PlySs2 enzyme to the non-human animal once a day for about 13 days.
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Citations (5)

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Publication number Priority date Publication date Assignee Title
US20090264830A1 (en) * 2005-09-29 2009-10-22 Infa S.A. Kit for Parenteral Administration of Medicaments
US20130337528A1 (en) * 2010-12-10 2013-12-19 Tracy Thompson Compositions for separation methods
US20150225464A1 (en) * 2012-09-25 2015-08-13 Affibody Ab Albumin binding polypeptide
US20200149025A1 (en) * 2011-04-21 2020-05-14 The Rockefeller University Streptococcus bacteriophage lysins for detection and treatment of gram positive bacteria
US20210032294A1 (en) * 2018-02-26 2021-02-04 Contrafect Corporation MODIFIED PlySs2 LYSINS AND USES THEREOF

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090264830A1 (en) * 2005-09-29 2009-10-22 Infa S.A. Kit for Parenteral Administration of Medicaments
US20130337528A1 (en) * 2010-12-10 2013-12-19 Tracy Thompson Compositions for separation methods
US20200149025A1 (en) * 2011-04-21 2020-05-14 The Rockefeller University Streptococcus bacteriophage lysins for detection and treatment of gram positive bacteria
US20150225464A1 (en) * 2012-09-25 2015-08-13 Affibody Ab Albumin binding polypeptide
US20210032294A1 (en) * 2018-02-26 2021-02-04 Contrafect Corporation MODIFIED PlySs2 LYSINS AND USES THEREOF

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