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WO2016149417A1 - Compositions de vaccins, adjuvants et méthodes de traitement d'infections urinaires - Google Patents

Compositions de vaccins, adjuvants et méthodes de traitement d'infections urinaires Download PDF

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Publication number
WO2016149417A1
WO2016149417A1 PCT/US2016/022711 US2016022711W WO2016149417A1 WO 2016149417 A1 WO2016149417 A1 WO 2016149417A1 US 2016022711 W US2016022711 W US 2016022711W WO 2016149417 A1 WO2016149417 A1 WO 2016149417A1
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WIPO (PCT)
Prior art keywords
composition
hexaacyl disaccharide
phosphorylated hexaacyl
formulations
adjuvant
Prior art date
Application number
PCT/US2016/022711
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English (en)
Inventor
Gary Eldridge
Steven M. Martin
Original Assignee
Sequoia Sciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US14/660,523 external-priority patent/US9149521B2/en
Priority claimed from US14/800,003 external-priority patent/US9415097B2/en
Application filed by Sequoia Sciences, Inc. filed Critical Sequoia Sciences, Inc.
Priority to CN201680016531.7A priority Critical patent/CN107405395A/zh
Priority to EP16765693.3A priority patent/EP3270962A4/fr
Publication of WO2016149417A1 publication Critical patent/WO2016149417A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0258—Escherichia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention provides novel adjuvant compositions and formulations with excellent stability at refrigerated and room temperatures, and also up to about 37°C, that can be produced at remarkably low costs. These novel adjuvant compositions and formulations are used in vaccines and exhibit superior properties of enhancing immune responses to antigens while causing less severe injection site and systemic reactions.
  • the invention also describes novel vaccine compositions and formulations to treat and prevent urinary tract infections caused by gram-negative bacteria including Escherichia coli and multi-drug resistant E. coli.
  • the invention also provides methods of administration of said novel vaccine formulations and methods of treatment to prevent and treat urinary tract infections caused by gram-negative bacteria including E. coli and multi-drug resistant E. coli.
  • vaccines can have a short shelf life and are prone to expire prior to use.
  • Adjuvants enhance the immune responses to antigens of vaccines.
  • 34 vaccines provided under the Vaccines for Children Program administered by the CDC in the US 20 contain adjuvants.
  • 19 of these vaccines contain alum adjuvants and 1 vaccine contains monophosphoryl lipid A adsorbed to alum (GSK's MPL) as the adjuvant.
  • GlaxoSmithKline's (GSK) Cervarix vaccine containing 3'-0-desacyl-4'- monophosphoryl lipid A adsorbed to alum (GSK's MPL) was licensed in the US for the prevention of cervical cancer caused by human papillomavirus. Because the starting material to produce MPL is isolated from Salmonella minnesota, the final product is a dynamic, complex mixture of hexa-, penta-, and tetraacyl analogues; each of these analogues differ in biological activity. As a result, the mixture of 3'-0-desacyl-4'- monophosphoryl lipid A presents manufacturing, testing, and use challenges that greatly contribute to the expense and supply issues with the vaccine.
  • pandemic vaccine 600 million doses
  • HHS Pandemic Influenza Plan Nov 2005
  • adjuvants to attempt to move toward this seemingly unapproachable vaccination target. Since there is no approved adjuvant for a general flu vaccine in the US, the US national vaccine stockpile was without an alternative and purchased the MF59 adjuvant from Novartis for about $500 million.
  • the MF59 adjuvant was recently discontinued in a clinical study of Fluad Paediatric due to "high vaccine reactogenicity observed in children 9 through 12 years of age, the protocol of study V7P29 was amended to exclude children less than 9 years of age.”
  • the evidence supports that during a national pandemic declaration a significant number of severe reactions will occur due to the use of the MF59 adjuvant and require additional medical care.
  • a synthetic analogue of monophosphoryl lipid A was introduced by Avanti Polar Lipids (Alabaster, Alabama, USA) in around 2004 time period.
  • Avanti Polar Lipids named this synthetic analogue phosphorylated hexaacyl disaccharide (PHAD), alternatively known as "GLA”.
  • PHAD supplied by Avanti Polar Lipids is provided as a single compound, shown in Figure 1, of approximately 98% purity with a molecular weight of 1763 Daltons.
  • PHAD's purity is in stark contrast to GSK's MPL isolated from Salmonella minnesota that, as described above, exists as a dynamic, complex mixture.
  • PHAD's manufacturing process, supply, use, and stability can be closely monitored and controlled as a pure compound.
  • GSK's Cervarix vaccine contains both monophosphoryl lipid A and alum, because this combination is superior to alum alone (Giannini et al. Vaccine, 2006, 24, p. 5937-5949).
  • a similar increase in efficacy was observed with a vaccine that used a recombinant hepatitis B surface antigen. (Vaccine, 1998, 16(7), p. 708-714).
  • Another example demonstrating the variability of antigen - adjuvant combinations in producing an immune response for a specific antigen is shown in Table 6 of US Patent 6,889,885.
  • IDSA Infectious Disease Society of America
  • Urinary tract infections are one of the most prevalent infectious diseases worldwide and the number one infectious disease suffered by women in the US. Symptoms of UTIs include dysuria (painful urination), urgency to urinate, and suprapubic pain. Acute uncomplicated UTIs occur in an estimated 7 to 11 million women in the US each year. Over half of all adult women will suffer from one or more UTIs in their lifetime with 25-44% of women experiencing a recurrent UTI. In fact, approximately 1,000,000 women and men in the US experience three or more UTI episodes per year. Moreover, recurrence often occurs within 30 to 90 days of infection despite appropriate antibiotic treatment and apparent clearance of the initial infection from the urine.
  • UTIs are most commonly caused by uropathogenic Escherichia coli (UPEC), which can be responsible for up to 85% of community -acquired UTIs.
  • UPEC uropathogenic Escherichia coli
  • a critical pathogenic cascade by which UPEC evade host defenses and rapidly expand in numbers in the urinary tract to cause disease has been uncovered. This work supports the clinical need for a UTI vaccine.
  • FimH plays a significant role in several stages of the pathogenesis cascade, which makes it a prime vaccine target.
  • UPEC strains that lack the FimH adhesin are unable to effectively colonize the bladder.
  • a vaccine against FimH will activate host defenses to recognize and clear UPEC at all stages of infection, even when protected in IBCs or intracellular reservoirs.
  • FimCH vaccine with MF59 as the adjuvant containing squalene was jointly invented by scientists at Medlmmune, Inc. and the laboratory of Professor Scott Hultgren (US Patent 6,500,434; incorporated herein in its entirety).
  • the FimH protein and FimC protein exist as a non-covalent protein complex, FimCH.
  • FimC stabilizes FimH and antibodies are produced against both proteins, however, only antibodies to FimH have been shown to reduce E. coli colonization of bladders in animals.
  • the use of FimCH as an antigen in a vaccine is therefore limited by the requirement of an effective adjuvant.
  • the FimCH vaccine with the MF59 adjuvant containing squalene (an oil- in-water emulsion) elicited an immune response during Phase 1 clinical trials (United States Patent Application 20030138449; incorporated herein in its entirety.).
  • Phase 2 clinical trials were conducted in two distinct populations again with the MF59 adjuvant containing squalene, but women did not produce relevant IgG titers to FimH in either of these trials.
  • the development of Medlmmune's FimCH vaccine with the MF59 adjuvant was discontinued because of these disappointing results.
  • the MF59 adjuvant with squalene has a history of causing severe local injection site and systemic reactions when used with certain antigens. During these Phase 2 clinical trials, women experienced severe injection site reactions and severe systemic reactions. Because of this failure, a vaccine for the treatment or prevention of UTI does not exist in the US.
  • the present invention addresses many problems of prior art adjuvants, vaccines and pharmaceutical compositions described herein.
  • the present invention provides novel liquid adjuvant compositions and formulations which provides many unexpected and advantageous properties unknown in the art of adjuvant and
  • liquid adjuvant compositions and formulations that exhibit room temperature stability for about more than 6 months and up to about 37°C for about 60 or more days is provided.
  • the novel liquid adjuvant compositions and formulations can be stored at refrigerated or room temperature conditions facilitating its shelf life during shipping and storage and lowering its delivery costs.
  • the adjuvant formulations of the invention described herein address many current obstacles in vaccine administration by enabling a low cost and unexpectedly and remarkably stable adjuvant formulation which enhances an immune response to E. coli antigen with less severe injection site and systemic reactions.
  • the invention described herein contributes to reducing this problem by treating urinary tract infections caused by gram-negative bacteria including E. coli.
  • a composition comprising one synthetically produced adjuvant phosphorylated hexaacyl disaccharide or its derivative, 3-deacyl- phosphorylated hexaacyl disaccharide, and a buffer selected from the group consisting of citrate, succinate, and phosphate at about 25 mM to about 50 mM, preferably 28 mM to about 50 mM, and most preferably 30 mM to about 50 mM.
  • These novel phosphorylated hexaacyl disaccharide compositions are preferably aqueous buffered suspensions.
  • the composition can be used in a variety of ways in the vaccine and pharmaceutical context.
  • composition significantly improves the stability of phosphorylated hexaacyl disaccharide or its derivative, 3-deacyl- phosphorylated hexaacyl disaccharide, in suspension and achieves exceptional stability at room temperature and up to and at about 37°C.
  • the compositions also exhibit excellent stability at refrigerated temperature as well. This represents a significant advancement in adjuvant and pharmaceutical technology by providing an efficient and economical phosphorylated hexaacyl disaccharide composition that does not require refrigeration for long term stability.
  • novel adjuvant formulations as an aqueous buffered suspension include one synthetically produced adjuvant phosphorylated hexaacyl disaccharide or its derivative, 3-deacyl-phosphorylated hexaacyl disaccharide, a buffer selected from the group consisting of citrate, succinate, and phosphate at about 10 mM to about 50 mM, preferably about 25 mM to about 50 mM, more preferably 28 mM to about 50 mM, and most preferably 30 mM to about 50 mM, and preferably one synthetically produced phosphatidylcholine.
  • novel adjuvant formulations are preferably aqueous buffered suspensions.
  • the adjuvant formulations have excellent long-term stability when stored at refrigerated and room temperatures and excellent stability up to and at about 37°C. These formulations can be produced at remarkably low costs.
  • the novel adjuvant formulations described herein do not require lyophilization, or equivalent process for room temperature stability or stability up to and at about 37°C.
  • the adjuvant formulations include a specific buffer and optionally and preferably one or more synthetically produced phosphatidylcholines selected from the group consisting of DMPC, DPPC, DSPC, DOPC, and POPC, preferably DPPC, and one synthetically produced adjuvant, phosphorylated hexaacyl disaccharide or its derivative, 3-deacyl-phosphorylated hexaacyl disaccharide, in a molar ratio of about 1 : 1 to 40: 1 (phosphatidylcholine: phosphorylated hexaacyl disaccharide), preferably about 1 : 1 to 20: 1 (phosphatidylcholine: phosphorylated hexaacyl disaccharide), more preferably about 2: 1 to 5: 1 (phosphatidylcholine: phosphorylated hexaacyl
  • the adjuvant formulations include only a single adjuvant phosphorylated hexaacyl disaccharide or its derivative, 3-deacyl-phosphorylated hexaacyl disaccharide, in citrate, succinate or phosphate buffers at specified concentrations as described herein, and preferably a single phosphatidylcholine. No other ingredients are required to produce the unexpected long- term stability at room temperature. Further the adjuvant formulation can be produced at low cost. In this regard, the long-term stability of these adjuvant formulations at room temperature is remarkable and is achieved without the use of cholesterol,
  • Another aspect of this invention is adjuvant formulations that do not need two or more phosphatidylcholines or the addition of a phosphatidylglycerol. As shown in the examples, two or more phosphatidylcholines or one or more phosphatidylglycerols can be added to these formulations, but preferably it is not needed to achieve the remarkable long-term stability demonstrated herein.
  • concentrations of citrate, succinate, or phosphate buffer to about 10 mM to about 50 mM to achieve the remarkable stability of the invention described herein is believed to be due to addition of preferred excipients, preferably phosphatidylcholine, at the defined molar ratio of the preferred aspect of the invention of phosphatidylcholine to phosphorylated hexaacyl disaccharide or its derivative, 3-deacyl-phosphorylated hexaacyl disaccharide, described herein.
  • the preferred buffer concentrations are about 25 mM to about 50 mM, even more preferably 28 mM to about 50 mM, and most preferably 30 mM to about 50 mM.
  • the pH is in a range of about 4.0 to about 7.5, preferably about 4.5 to about 6.5, more preferably about 5.0 to 6.0.
  • this exceptional stability at room temperature is not present when formulated in water, acetate buffer, PBS, or citrate or phosphate buffers at or greater than 100 mM. Instead the stability is produced by citrate, succinate, or phosphate at concentrations of about 10 mM to about 50 mM, but preferably about 25 mM to about 50 mM, more preferably 28 mM to about 50 mM, and most preferably 30 mM to about 50 mM.
  • Another embodiment of the invention includes a non-ionic surfactant, preferably polysorbate 80, to reduce the aggregation of particles of the invention.
  • a non-ionic surfactant preferably polysorbate 80
  • Removing the need for lyophilization is a significant advantage and unexpected breakthrough because many costly steps and risks have been eliminated.
  • Another aspect of this invention is that these adjuvant formulations are superior at enhancing an immune response to an antigen while causing significantly less severe injection site and systemic reactions during administration compared to the prior art.
  • Another aspect of this invention is adjuvant formulations substantially free of
  • metabolizable oils including squalene, and substantially free of cholesterol.
  • cholesterol is necessary for adjuvant formulations or liposomes to function.
  • the current invention produces all its benefits as described herein without requiring cholesterol.
  • the advantages of the adjuvant formulations and pharmaceutical compositions described herein include: room temperature stability as an aqueous buffered suspension for at least 6 months, and / or stability up to and at about 37°C for about 60 or more days; less severe injection site and systemic reactions per administration while enhancing immune responses to antigens; and, lower cost of production or manufacturing with fewer materials or components and lower concentration of the materials or components.
  • the inventive adjuvant formulations provide these three combined major benefits not previously achieved by adjuvants that are synthetic analogues of MLA or MPL as alternatives to alum-based adjuvants.
  • novel vaccine compositions containing the novel adjuvant formulations for use to treat and prevent urinary tract infections caused by gram-negative bacteria including Escherichia coli and multi-drug resistant E. coli are provided.
  • Methods of administration of said novel vaccine compositions and methods of treatment to prevent and treat urinary tract infections caused by gram-negative bacteria including E. coli and multi-drug resistant E. coli are also provided.
  • Another aspect of the invention is vaccine compositions that induce the production of antibodies against FimH in a human with recurrent urinary tract infections.
  • a vaccine kit comprising the phosphorylated hexaacyl disaccharide compositions or formulations or vaccine compositions, along with administration directions and instructions for storage are provided.
  • the instructions provide for the exposure of the phosphorylated hexaacyl disaccharide compositions or formulations at room temperature and up to and about 37°C.
  • These instructions describing storage, shipping, and exposure temperatures may be approved by a government regulatory authority including the US FDA or European Medicines Agency.
  • one or more of the kit components is the phosphorylated hexaacyl disaccharide compositions or formulations in a syringe.
  • the phosphorylated hexaacyl disaccharide composition and formulations and vaccine compositions are sterile compositions and sterile pharmaceutical compositions, more preferably the sterile phosphorylated hexaacyl disaccharide compositions and formulations are contained in a sterile syringe.
  • Figure 1 shows the chemical structure of one salt of phosphorylated hexaacyl disaccharide.
  • Figure la shows a preferred 3-deacyl-phosphorylated hexaacyl disaccharide salt.
  • Figure 2 shows the chemical structure of DPPC.
  • Figure 3 is a graph illustrating the indirect ELISA of FimCH and Q133K using IgG anti-FimH.
  • Figure 4 is a graph illustrating the potency assay analyzing FimCH and
  • Figure 5 is a graph illustrating the evaluation of small molecule inhibitors in the potency assay.
  • Two small molecules, 4-methylumbelliferyl-a-D-mannopyranoside (UFMP) and methyl-a-D-mannopyranoside (MDMP) inhibit mannose binding to FimH.
  • Figure 6 is a representative chromatogram of the FimCH drug substance sample by CEX-HPLC.
  • Figure 7 is a graph illustrating the protection from E. coli Infection following FimCH/PHAD immunization of mice.
  • Figure 8 is an example chromatogram of DPPC and PHAD by HPLC. DETAILED DESCRIPTION OF THE INVENTION
  • “About 50 mM” refers to the buffer concentrations described herein means between 50 mM and less than 100 mM. As shown in the examples, 100 mM or greater of the specified buffers is not as effective in enabling long-term room temperature stability. The upper end of buffer concentrations is typically evaluated at two-fold increments. With reference to about 50 mM, the specified buffer concentrations of the invention are preferably less than 90 mM, preferably less than 80 mM, more preferably less than 70 mM, and most preferably less than 60 mM.
  • Acceptable carrier refers to a carrier that is not deleterious to the other ingredients of the composition and is not deleterious to material to which it is to be applied.
  • adjuvant refers to an agent that, when present in an effective amount, increases the antigenic response; a substance enhancing the immune response to an antigen; or an agent that stimulates antibody production to an antigen. Numerous naming conventions or terminologies exist in the art. Without reference to a specific naming convention, the adjuvant compositions as described herein may simply be referred to as adjuvant formulations or adjuvant preparations.
  • administering refers to any means of providing a compound or composition to a subject.
  • Colloid refers to one or more chemicals, compounds, or substances microscopically dispersed throughout an aqueous buffered solution or another substance.
  • the adjuvant formulations described herein can also be described as colloid.
  • a colloidal dispersion is Fungizone, which consists of Amphotericin B-sodium desoxycholate for parenteral administration.
  • Critical micelle concentration refers to the concentration of surfactant(s) above which micelles form and all additional surfactants added to the system go to micelles.
  • DLPC refers to l,2-dilauroyl-sn-glycero-3-phosphocholine.
  • DMPC refers to l,2-dimyristoyl-sn-glycero-3-phosphocholine.
  • DOPC refers to l,2-dioleoyl-sn-glycero-3-phosphocholine.
  • DPPG refers to l,2-dipalmitoyl-sn-glycero-3-phospho-(l'-rac-glycerol).
  • DSPC refers to l,2-distearoyl-sn-glycero-3-phosphocholine.
  • Effective amount refers to a sufficient amount of FimCH or truncated FimH or other antigen in a vaccine composition that is administered to a human to elicit an immune response against FimH or other antigen, or sufficient amount of an adjuvant, preferably phosphorylated hexaacyl disaccharide or its derivative, 3-deacyl- phosphorylated hexaacyl disaccharide, to elicit an increased immune response to antigen.
  • an adjuvant preferably phosphorylated hexaacyl disaccharide or its derivative, 3-deacyl- phosphorylated hexaacyl disaccharide
  • injection site reaction refers to pain, tenderness, redness, and / or swelling at the site of administration or injection site.
  • invention means at least some embodiments of the present invention; references to various feature(s) of the "invention” or “present invention” throughout this document do not mean that all claimed embodiments or methods include the referenced feature(s).
  • Label refers to all labels and other written, printed, or graphic matter upon any article or any of its containers or wrappers, or accompanying such article and, therefore, includes any package inserts or information sheets that accompany vaccine or adjuvant compositions or formulations of the invention.
  • Less severe injection site and systemic reactions refers to less severe or Grade 3 injection site reactions and / or systemic reactions as compared to commercial vaccine Cervarix as detailed herein and in its product information documents and investigational vaccine adjuvant GLA-SE as described herein and in Treanor et al.
  • Liposome refers to generally vesicles that consist of a lipid bilayer membrane surrounding a hydrophilic core.
  • Low cost or “low costs” refers to a composition with the components or materials at the lowest concentrations sufficient to achieve the novel characteristics of the invention.
  • “Lyoprotectants” refers to materials, chemicals, or excipients primarily used to protect materials from freezing damage or other impairment during manufacturing, storage and use or improving reconstitution including enabling appropriate solvation prior to use, and also includes these materials, chemicals, or excipients used to modify osmolality or adjust tonicity, and include but not limited to sorbitol, mannitol, mannose, erythritol, xylitol, glycerol, sucrose, dextrose, trehalose, maltose, lactose, and cellobiose.
  • Methodabolizable oil refers primarily to squalene, or closely related analogues of squalene as used as an adjuvant in vaccine formulations or adjuvant formulations, but also refers to medium-chain triglycerides including Miglyol 810 and oils from vegetables, animals, or fish when used in vaccine or adjuvant formulations as excipients, or to create an adjuvant effect, or to produce emulsions. Examples include grapeseed oil, soybean oil, coconut oil, olive oil, sunflower oil, com oil, and shark liver oil.
  • Micelle refers to an aggregate of surfactant molecules dispersed in an aqueous buffered solution with the hydrophilic head regions in contact with surrounding aqueous buffered solution, sequestering the hydrophobic single-tail regions in the center of the micelle.
  • MCA monophosphoryl lipid A
  • MPL refers to 3 '-O-desacyl-4' -monophosphoryl lipid A.
  • “Pharmaceutically acceptable carrier” refers to a carrier that is not deleterious to the other ingredients of the composition and is not deleterious to the human or other animal recipient thereof. In the context of the other ingredients of the
  • not deleterious means that the carrier will not react with or degrade the other ingredients or otherwise interfere with their efficacy. Interference with the efficacy of an ingredient does not, however, refer to mere dilution of the ingredient.
  • Phosphorylated hexaacyl disaccharide is a Toll-like receptor 4 agonist and refers to phosphorylated hexaacyl disaccharide, its acid or other pharmaceutically acceptable salts of phosphorylated hexaacyl disaccharide.
  • the structure of a preferred phosphorylated hexaacyl disaccharide salt is shown in Figure 1, which is available from Avanti Polar Lipids (PHAD).
  • Phosphorylated hexaacyl disaccharide, as used herein may be fully or partially synthetic or non-synthetic, although fully synthetic is preferred.
  • 3-deacyl-phosphorylated hexaacyl disaccharide is a Toll-like receptor 4 agonist and refers to 3-deacyl-phosphorylated hexaacyl disaccharide, its acid or other pharmaceutically acceptable salts of 3-deacyl-phosphorylated hexaacyl disaccharide.
  • the structure of a preferred 3-deacyl-phosphorylated hexaacyl disaccharide salt is shown in Figure 1A, which is available from Avanti Polar Lipids.
  • 3-deacyl-phosphorylated hexaacyl disaccharide, as used herein may be fully or partially synthetic or non-synthetic, although fully synthetic is preferred.
  • “Pharmaceutical composition” refers to a composition given to a mammal intended to treat or prevent a disease, or in the case of a vaccine composition, to produce an immunogenic response that treats or prevents a disease, reduce symptoms, or provides some type of therapeutic benefit, or in the case of an adjuvant composition, to enhance an immune response to one or more antigens.
  • Phosphate buffer or “phosphate” refers to a phosphate buffer selected from the following group: sodium phosphate dibasic, sodium phosphate monobasic, potassium phosphate monobasic, and potassium phosphate dibasic, or some combination thereof.
  • phosphate consists of sodium phosphate dibasic, sodium phosphate monobasic, and potassium phosphate monobasic. Unless otherwise noted, reference to phosphate buffer specifically excludes ammonium phosphate.
  • PBS refers to phosphate-buffered saline of a general composition of phosphate buffer (Na2HP04 and / or KH 2 PO 4 ), potassium chloride and sodium chloride.
  • a typical PBS composition is comprised of about 10 mM phosphate buffer (Na2HPC>4 and / or KH2PO4), 2.7 mM potassium chloride and 0.14 M sodium chloride, pH 7.4, at 25 °C.
  • Phosphate citrate buffer refers to a phosphate buffer containing citric acid and sodium phosphate where the pH is maintained by citrate/citric acid and phosphate/hydrogen phosphate equilibrium.
  • the phosphate may include, for example Na2HPC>4 and / or KH2PO4 and trisodium citrate may be used.
  • Phosphatidylcholine refers to lipids containing choline. Examples include, but not limited to, DMPC (1,2-dimyristoyl- sn-glycero-3-phosphocholine), DPPC (l,2-dipalmitoyl-sn-glycero-3-phosphocholine), DSPC (l,2-distearoyl-sn-glycero-3-phosphocholine), DOPC (l,2-dioleoyl-sn-glycero-3- phosphocholine), and POPC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine).
  • Phosphatidylcholines are available from Avanti Polar Lipids.
  • Phosphatidylethanolamine refers to lipids containing a phosphate group attached to an ethanolamine, e.g. l,2-dioleoyl-sn-glycero-3-phosphoethanolamine.
  • Phosphatidylglycerol refers to lipids containing glycerol.
  • Examples include, but are not limited to,l,2-dimyristoyl-sn-glycero-3-phospho-(l'-rac- glycerol) (DMPG), l,2-dipalmitoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DPPG), and l,2-distearoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DSPG).
  • DMPG diimyristoyl-sn-glycero-3-phospho-(l'-rac- glycerol)
  • DPPG l,2-dipalmitoyl-sn-glycero-3-phospho-(l'-rac-glycerol)
  • DSPG l,2-distearoyl-sn-glycero-3-phospho-(l'-rac-glycerol)
  • POPC refers to l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine.
  • Refrigerated refers to a temperature range from 2°C to 8°C.
  • Root temperature refers to a range of temperature from 19°C
  • Saline refers to about 125 mM to about 155 mM of NaCl in buffered aqueous solutions.
  • PBS generally contains 137 mM NaCl and Tris buffered saline may contain 150 mM.
  • “Severe injection site reaction” refers to one or more of the following: pain requiring narcotic pain reliever or that prevents daily activity; tenderness causing significant discomfort at rest; redness of more than 10 cm; and swelling of more than 10 cm or prevents daily activity.
  • “Severe systemic reaction” refers to one or more of the following: nausea / vomiting which prevents daily activity or requires outsubject IV hydration;
  • diarrhea consisting of 6 or more watery stools or > 800 grams with 24 hours or requires outsubject IV hydration; headache consisting of significant use of narcotic pain reliever or prevents daily activity; fatigue consisting of significant or prevents daily activity; and myalgia consisting of significant or prevents daily activity.
  • Substantially free in referring to cholesterol means that cholesterol, if present, is at 0.3 mM or less.
  • monoacylglycerol if present, is at 0.5 mM or less.
  • An example of monoacylglycerol is monopalmitoyl glycerol.
  • phosphatidylglycerol or phosphatidylethanolamine means these substances, if present, are at 0.1 mM or less.
  • Substantially free in referring to lyoprotectants means these substances, if present, are at a concentration of the composition or formulation of 0.5% or less.
  • substantially free of saline means less than 30 mM NaCl in the composition or formulation of the invention.
  • Systemic reactions refers to nausea / vomiting, diarrhea, headache, fatigue, and / or myalgia.
  • succinate buffer or “succinate” refers to disodium succinate or sodium succinate dibasic. Potassium succinate may be used, but is less preferred.
  • Stability in reference to adjuvants, actives, proteins, antigens, or drugs refers to the quality of the substance or product to remain acceptable for its intended use throughout a certain time period beginning from its date of manufacture while under the influence of such variables as temperature and/or humidity. The stability of a substance is often demonstrated by analytical data (or other equivalent evidence).
  • Trisodium citrate refers to citrate buffers (also referred to as
  • citrate such as, for example, trisodium citrate dihydrate, sodium citrate, sodium citrate tribasic hydrate, or citric acid trisodium salt dihydrate as referred to by suppliers including Sigma-Aldrich and BDH Chemicals. Potassium citrate, sodium citrate monobasic, and sodium citrate dibasic may be used, but they are less preferred.
  • the product imiglucerase for injection uses a combination of trisodium citrate and disodium hydrogen citrate. These types of combinations are acceptable.
  • Citric acid, CAS 77-92-9 may be used to adjust the pH of the buffer, but it cannot substitute for the listed buffers herein.
  • Truncated FimH refers to the FimH protein truncated to include at least about 25 to about 175 amino acid residues from the first 175 amino acids of FimH. With reference to truncated FimH, the FimH protein truncated to include preferably at least 9% of the FimH protein, more preferably at least 30% of the FimH protein, and most preferably at least 60% of the FimH protein.
  • Urinary Tract Infections refers to a medical diagnosis characterized by 1 or more of the following signs and symptoms: irritative voiding such as frequency, urgency, and dysuria; gross hematuria; or elicited suprapubic tenderness upon examination; and/or 1 or more of the following laboratory results: positive urine dipstick test from clean catch or catheter urine specimen; microscopic urinalysis from clean catch or catheter urine specimen (leukocytes, bacteria, and casts may be present); or urine culture from clean catch or catheter urine specimen for ?, coli at >10 3 CFU/mL.
  • Vaccine or "vaccine composition” refers to a composition that improves immunity to a disease.
  • the vaccine compositions are immunogenic
  • compositions that elicit immune responses and antibody production toward the antigen of the composition.
  • compositions [00125] In one embodiment a pharmaceutical composition or
  • compositions or pharmaceutically acceptable carriers are comprised of phosphorylated hexaacyl disaccharide or its derivative, 3-deacyl-phosphorylated hexaacyl disaccharide, and a buffer (referred to as " phosphorylated hexaacyl disaccharide composition” or " phosphorylated hexaacyl disaccharide containing composition” or " phosphorylated hexaacyl disaccharide containing composition and carriers” and these also refer separately to its derivative, 3- deacyl-phosphorylated hexaacyl disaccharide ).
  • phosphorylated hexaacyl disaccharide composition or " phosphorylated hexaacyl disaccharide containing composition” or " phosphorylated hexaacyl disaccharide containing composition and carriers”
  • phosphorylated hexaacyl disaccharide composition or " phosphorylated hexaacyl disaccharide containing composition” or " phosphorylated hexaacyl disacc
  • disaccharide compositions of the invention are preferably aqueous buffered suspensions.
  • the buffer is selected from the group consisting of citrate, succinate, and phosphate at about 25 mM to about 50 mM, more preferably 28 mM to about 50 mM, and most preferably 30 mM to about 50 mM.
  • the pH is in a range of about 4.0 to about 7.5, preferably about 4.5 to about 6.5, more preferably about 5.0 to about 6.0.
  • the pharmaceutical composition or carrier with this combination demonstrates excellent stability as a base for pharmaceutical compositions and improves the overall stability of phosphorylated hexaacyl disaccharide compositions. Specifically these phosphorylated hexaacyl disaccharide containing compositions and carriers achieve stability at room temperature and up to about 37°C.
  • compositions and carriers of the present invention also exhibit excellent long-term stability at refrigerated temperatures to room temperature.
  • compositions and pharmaceutical carriers may optionally include other ingredients typical to vaccines, adjuvant formulations, and other pharmaceutical compositions such as excipients, modifiers, surfactants, and additives.
  • phosphatidylcholines may optionally be added, alone or in combination with other lipid carriers.
  • naturally derived phosphatidylcholines from soy or egg or hydrogenated phosphatidylcholines from soy or egg or synthetic or natural mixed acyl phosphatidylcholines may be added.
  • one or more vaccine antigens are added to the phosphorylated hexaacyl disaccharide containing compositions to form a vaccine.
  • the vaccine antigens may be any antigen used in vaccines including, but not limited to diphtheria, tetanus, pertussis, poliomyelitis, hepatitis, and or antigenic preparations of the influenza virus.
  • the vaccine antigen is a FimCH protein complex as described in detail below.
  • the phosphorylated hexaacyl disaccharide containing compositions and carriers can be prepared at any concentration but are typically prepared at about 0.005 to about 1.0 mg/ml of phosphorylated hexaacyl disaccharide, preferably about 0.05 to 1.0 mg/ml of phosphorylated hexaacyl disaccharide, but usually not more than about 2.5 mg/ml of phosphorylated hexaacyl disaccharide.
  • the phosphorylated hexaacyl disaccharide containing compositions may be administered to animals or humans as an adjuvant of vaccines to preferably deliver about 10 microgram of phosphorylated hexaacyl disaccharide per dose to about 50 micrograms of phosphorylated hexaacyl disaccharide per dose.
  • the exact dose may be modified according to the antigen used. More preferably about 20 micrograms of phosphorylated hexaacyl disaccharide per dose to about 50 micrograms of phosphorylated hexaacyl disaccharide per dose is administered. Even more preferably about 40 micrograms of phosphorylated hexaacyl disaccharide per dose to about 50 micrograms of phosphorylated hexaacyl disaccharide per dose is administered.
  • the unexpected stability of the phosphorylated hexaacyl disaccharide containing composition at room temperature and up to 37°C is absent when formulated in water, acetate buffer, PBS, or citrate or phosphate buffers at or greater than 100 mM.
  • the phosphorylated hexaacyl disaccharide compositions contain citrate, succinate, or phosphate buffer to produce the unexpected stability, preferably about 25 mM to about 50 mM, more preferably 28 mM to about 50 mM, and most preferably 30 mM to about 50 mM.
  • the concentrations of the buffers are selected from the group consisting of about 25 mM to about 50 mM; 25 mM to about 50 mM; about 30 mM to about 50 mM; 28 mM to about 50 mM; 30 mM to about 50 mM, about 30 mM; about 40 mM to about 50 mM; 40 mM to about 50 mM; about 40 mM; and about 50 mM.
  • the phosphorylated hexaacyl disaccharide compositions of the invention are preferably essentially free of squalene. [00134] The phosphorylated hexaacyl disaccharide compositions of the invention are preferably essentially free of metabolizable oil used as an adjuvant.
  • the phosphorylated hexaacyl disaccharide compositions of the invention are preferably essentially free of metabolizable oil.
  • the phosphorylated hexaacyl disaccharide compositions of the invention are preferably aqueous buffered suspensions, and preferably these aqueous buffered suspensions have particle sizes less than 150 nm, even more preferably less than 130 nm, and preferably these phosphorylated hexaacyl disaccharide compositions are not oil-in-water emulsions.
  • the phosphorylated hexaacyl disaccharide compositions of the invention are preferably essentially free of a second adjuvant including alum, squalene, QS21 , MF59, Toll-like receptor 9 agonists, and other adjuvants including squalene-based adjuvants.
  • Second adjuvants have the potential to generate more severe local and systemic reactions in humans yet without the benefit of further increasing an immune response to improve therapeutic outcomes.
  • the phosphorylated hexaacyl disaccharide compositions preferably contain less than 5 mM of cholesterol, more preferably less than 1 mM of cholesterol, and even more preferably substantially free of cholesterol, and most preferably essentially free of cholesterol.
  • the phosphorylated hexaacyl disaccharide compositions are substantially free of phosphatidylglycerol, and more preferably are essentially free of phosphatidylglycerol.
  • the phosphorylated hexaacyl disaccharide compositions are substantially free of phosphatidylethanolamine, and more preferably essentially free of phosphatidylethanolamine.
  • the phosphorylated hexaacyl disaccharide compositions are substantially free of monoacylglycerol, and more preferably essentially free of monoacy lgly cerol .
  • the phosphorylated hexaacyl disaccharide compositions are preferably substantially free of saline, preferably contain less than 20 mM NaCl, and more preferably contain less than 10 mM NaCl, and even more preferably essentially free of NaCl.
  • the phosphorylated hexaacyl disaccharide compositions are preferably substantially free of lyoprotectants, and even more preferably essentially free of lyoprotectants.
  • the phosphorylated hexaacyl disaccharide compositions do not require lyophilization, or equivalent process, to preserve the concentration of
  • the phosphorylated hexaacyl disaccharide compositions are preferably not lyophilized or lyophilization does not occur; the phosphorylated hexaacyl disaccharide compositions are preferably not dried after preparation; the phosphorylated hexaacyl disaccharide compositions preferably do not require reconstitution from dried material with liquid or buffer after preparation; and the phosphorylated hexaacyl disaccharide compositions preferably remain as an aqueous buffered suspension after manufacturing prior to administration.
  • the phosphorylated hexaacyl disaccharide compositions are substantially free of cholesterol, phosphatidylglycerol, and phosphatidylethanolamine, and essentially free of metabolizable oils and a second adjuvant.
  • the phosphorylated hexaacyl disaccharide compositions are essentially free of cholesterol, phosphatidylglycerol, and
  • the phosphorylated hexaacyl disaccharide compositions of the invention are substantially free of materials or excipients as described herein that are not required to achieve the novel characteristics of the invention, but even more preferably the phosphorylated hexaacyl disaccharide compositions of the invention are essentially free of materials or excipients as described herein that are not required to achieve the novel characteristics of the invention.
  • the phosphorylated hexaacyl disaccharide compositions of the invention are substantially free of one, two, three, or more of materials or excipients per composition as described herein, and this limitation is not exclusive to only one material or excipient per composition.
  • the phosphorylated hexaacyl disaccharide compositions of the invention are essentially free of one, two, three, or more materials or excipients per composition as described herein, and this limitation is not exclusive to only one material or excipient per composition.
  • a phosphorylated hexaacyl disaccharide containing composition or formulation of the invention is stored, shipped, held, or administered at room temperature or up to about 37°C, it is most preferred to have been sterile filtered or prepared using sterile techniques, more preferably the sterile
  • phosphorylated hexaacyl disaccharide compositions and formulations are contained in a sterile syringe.
  • novel adjuvant formulations are provided.
  • the adjuvant formulations include one synthetically produced phosphorylated hexaacyl disaccharide or its derivative, 3-deacyl-phosphorylated hexaacyl disaccharide, a buffer selected from the group consisting of citrate, succinate, and phosphate at about 10 mM to about 50 mM, and preferably one synthetically produced phosphatidylcholine (referred to as " phosphorylated hexaacyl disaccharide formulations" or " phosphorylated hexaacyl disaccharide containing adjuvant formulations" or " phosphorylated hexaacyl disaccharide containing formulations” and these also refer separately to its derivative, 3-deacyl- phosphorylated hexaacyl disaccharide).
  • phosphorylated hexaacyl disaccharide formulations or " phosphorylated hexaacyl disaccharide containing adjuvant formulations” or " phosphorylated hexaacyl disaccharide containing formulations
  • the phosphatidylcholine and phosphorylated hexaacyl disaccharide, or its derivative, 3-deacyl-phosphorylated hexaacyl disaccharide are present at a molar ratio of about 1 (PC) to 1 ( phosphorylated hexaacyl disaccharide) to about 40 (PC) to 1 ( phosphorylated hexaacyl disaccharide), preferably about 2.5 (PC) to 1 ( phosphorylated hexaacyl disaccharide).
  • Reference to phosphorylated hexaacyl disaccharide formulations apply to the adjuvant formulations unless otherwise noted.
  • These novel phosphorylated hexaacyl disaccharide formulations are preferably aqueous buffered suspensions.
  • the adjuvant formulations have excellent long-term stability when stored at refrigerated temperatures to room temperatures, and up to about 37°C.
  • the adjuvant formulations can be produced at low costs.
  • the adjuvant formulations include preferably one synthetically produced phosphatidylcholine, preferably DPPC, and one synthetically produced adjuvant phosphorylated hexaacyl disaccharide or its derivative, 3- deacyl-phosphorylated hexaacyl disaccharide, in a molar ratio of about 1 : 1 to 40: 1 (DPPC: phosphorylated hexaacyl disaccharide), and citrate, succinate, or phosphate buffers at about 10 mM to 50 mM, but preferably about 25 mM to 50 mM, more preferably 28 mM to about 50 mM, and most preferably 30 mM to about 50 mM.
  • DPPC phosphorylated hexaacyl disaccharide
  • the pH is in a range of about 4.0 to about 7.5, preferably about 4.5 to about 6.5, more preferably about 5.0 to about 6.0.
  • the phosphorylated hexaacyl disaccharide adjuvant formulations can be produced with only one adjuvant phosphorylated hexaacyl disaccharide, or its derivative, 3-deacyl-phosphorylated hexaacyl disaccharide and preferably one phosphatidylcholine, thereby enabling them to be produced at low cost.
  • the adjuvant formulations contain phosphorylated hexaacyl disaccharide, or its derivative, 3-deacyl-phosphorylated hexaacyl disaccharide as the sole adjuvant, however additional adjuvants may be used.
  • the long-term stability attribute of the invention further reduces costs of using these phosphorylated hexaacyl disaccharide containing adjuvant formulations.
  • the phosphorylated hexaacyl disaccharide containing adjuvant formulations of the invention are preferably essentially free of squalene.
  • the phosphorylated hexaacyl disaccharide containing adjuvant formulations of the invention are preferably essentially free of metabolizable oil used as an adjuvant.
  • the phosphorylated hexaacyl disaccharide containing adjuvant formulations of the invention are preferably essentially free of metabolizable oils.
  • the phosphorylated hexaacyl disaccharide containing adjuvant formulations of the invention are preferably aqueous buffered suspensions, and preferably these aqueous buffered suspensions have particle sizes less than 150 nm, even more preferably less than 130 nm, and preferably these phosphorylated hexaacyl disaccharide containing adjuvant formulations are not oil-in-water emulsions.
  • the phosphorylated hexaacyl disaccharide containing adjuvant formulations of the invention are preferably essentially free of a second adjuvant including alum, squalene, QS21, MF59, Toll-like receptor 9 agonists, and other adjuvants including squalene based adjuvants.
  • the phosphorylated hexaacyl disaccharide formulations are preferably essentially free of other or additional adjuvants because they have the potential to generate more severe local and systemic reactions in humans without the benefit of further increasing an immune response or substantially improving therapeutic outcomes.
  • the phosphorylated hexaacyl disaccharide containing adjuvant formulations preferably contain less than 5 mM of cholesterol, more preferably less than 1 mM of cholesterol, even more preferably substantially free of cholesterol, and most preferably essentially free of cholesterol.
  • the phosphorylated hexaacyl disaccharide containing adjuvant formulations are substantially free of phosphatidylglycerol, and more preferably essentially free of phosphatidylglycerol.
  • the phosphorylated hexaacyl disaccharide containing adjuvant formulations are substantially free of phosphatidylethanolamine, and more preferably essentially free of phosphatidylethanolamine.
  • the phosphorylated hexaacyl disaccharide containing adjuvant formulations are substantially free of monoacylglycerol, and more preferably essentially free of monoacylglycerol.
  • the phosphorylated hexaacyl disaccharide containing adjuvant formulations of the invention are preferably substantially free of saline, preferably contain less than 20 mM NaCl, and more preferably, the phosphorylated hexaacyl disaccharide formulations contain less than 10 mM NaCl, and even more preferably are essentially free ofNaCl.
  • the phosphorylated hexaacyl disaccharide containing adjuvant formulations are substantially free of lyoprotectants, and more preferably essentially free of lyoprotectants.
  • the phosphorylated hexaacyl disaccharide containing adjuvant formulations are substantially free of cholesterol, phosphatidylglycerol, and phosphatidylethanolamine, and essentially free of metabolizable oils and a second adjuvant.
  • the phosphorylated hexaacyl disaccharide containing formulations are essentially free of cholesterol, phosphatidylglycerol and phosphatidylethanolamine, and essentially free of metabolizable oils and a second adjuvant.
  • the phosphorylated hexaacyl disaccharide containing formulations of the invention are substantially free of materials or excipients as described herein that are not required to achieve the novel characteristics of the invention, but even more preferably the phosphorylated hexaacyl disaccharide containing formulations of the invention are essentially free of materials or excipients as described herein that are not required to achieve the novel characteristics of the invention.
  • the phosphorylated hexaacyl disaccharide containing formulations of the invention are substantially free of one, two, three, or more of materials or excipients per phosphorylated hexaacyl disaccharide containing formulation as described herein, and this limitation is not exclusive to only one material or excipient per phosphorylated hexaacyl disaccharide containing formulation.
  • the phosphorylated hexaacyl disaccharide containing formulations of the invention are essentially free of one, two, three, or more materials or excipients per phosphorylated hexaacyl disaccharide containing formulation described herein, and this limitation is not exclusive to only one material or excipient per phosphorylated hexaacyl disaccharide containing formulation.
  • vaccines, and methods of treating and preventing disease with vaccines are provided.
  • one or more vaccine antigens are added to the phosphorylated hexaacyl disaccharide containing adjuvant formulations described above.
  • the antigens may be a FimCH protein complex as described herein or other antigens including, but not limited to antigens associated with diphtheria, tetanus, pertussis, poliomyelitis, hepatitis, and or antigenic preparations of the influenza virus.
  • a vaccine prepared using the phosphorylated hexaacyl disaccharide formulation does not require lyophilization to preserve the concentration of
  • Lyophilization is a dehydration process or freeze-drying process primarily utilized to preserve materials. Equivalent processes exist to accomplish the same goal to preserve materials.
  • a vaccine or adjuvant formulation that does not need lyophilization is an unexpected and significant advantage in the preparation of vaccines and the phosphorylated hexaacyl disaccharide compositions described herein. By eliminating the need for lyophilization, many costly steps are eliminated. First, the lyophilization step itself is eliminated, which not only removes a costly manufacturing step that must occur under well-monitored sterile conditions, but also eliminates the validation and review of this manufacturing step.
  • the removal of the step represents an ongoing cost saving. For example, each time a lot is prepared a lyophilization step is saved. In addition, removing this step saves extra validation steps when larger batches are prepared or the manufacturing procedure is transferred to another facility.
  • lyophilization requires that a sterile vial of diluent be manufactured or procured, shipped, and stored with the lyophilized product for reconstitution. Eliminating the reconstitution step removes the cost of this diluent vial and its supply chain management.
  • the reconstitution of adjuvant formulations can compromise its sterility, necessitating its immediate use or waste of the product if not used in a pre-established amount of time.
  • formulations are preferably not lyophilized or lyophilization does not occur; the phosphorylated hexaacyl disaccharide containing formulations are preferably not dried after preparation; the phosphorylated hexaacyl disaccharide containing formulations preferably do not require reconstitution from dried material with liquid or buffer after preparation; and the phosphorylated hexaacyl disaccharide containing formulations preferably remain as an aqueous buffered suspension after manufacturing prior to administration.
  • the phosphorylated hexaacyl disaccharide compositions or phosphorylated hexaacyl disaccharide containing formulations of the invention can be efficiently and cost effectively packaged into syringes immediately after manufacturing.
  • the compositions and formulations are sterile and preferably the syringes are sterile.
  • These prefilled syringes can be shipped, stored, delivered, or transferred at refrigerated temperatures to room temperatures and up to about 37°C.
  • This advantage provides the most efficient and cost effective means to get phosphorylated hexaacyl disaccharide compositions and phosphorylated hexaacyl disaccharide containing formulations to a site for administration.
  • a non-ionic surfactant is added to the adjuvant formulation.
  • the non-ionic surfactant is polysorbate 80 although others may be used.
  • the non-ionic surfactant is typically added at a concentration of about 0.001 % to 1.0%, preferably 0.01 % to 0.1 %, to the adjuvant composition. This addition can prevent slight aggregation or slight increase in mean particle size of the phosphorylated hexaacyl disaccharide formulations while being stored at room temperatures and up to about 37°C.
  • non-ionic surfactants are derived from polyethoxylated sorbitan and include but are not limited to polysorbate 20 and polysorbate 80.
  • Preferred adjuvant formulations comprise a specific buffer selected from the group consisting of citrate, succinate, and phosphate at about 25 mM to about 50 mM, more preferably 28 mM to about 50 mM, and most preferably 30 mM to about 50 mM, and preferably one synthetically produced phosphatidylcholine selected from the group consisting of DMPC, DPPC, DSPC, DOPC, and POPC, preferably DPPC, and one synthetically produced adjuvant, phosphorylated hexaacyl disaccharide or its derivative, 3-deacyl-phosphorylated hexaacyl disaccharide, in a molar ratio of about 1 : 1 to 40: 1 (phosphatidylcholine: phosphorylated hexaacyl disaccharide), preferably about 1 : 1 to 20: 1 (phosphatidylcholine: phosphorylated hexaacyl disaccharide), more preferably about 2: 1 to 5: 1
  • citrate or succinate buffers are used in the phosphorylated hexaacyl disaccharide formulations at about 10 mM to about 50 mM, preferably at about 25 mM to about 50 mM, more preferably 28 mM to about 50 mM, and most preferably 30 mM to about 50 mM.
  • the concentrations of the buffers are selected from the group consisting of about 10 mM to about 50mM; 15 mM to about 50 mM; 20 mM to about 50 mM; about 25 mM to about 50 mM; 25 mM to about 50 mM; about 30 mM to about 50 mM; 28 mM to about 50 mM; 30 mM to about 50 mM, about 30 mM; about 40 mM to about 50 mM; 40 mM to about 50 mM; about 40 mM to about 50 mM; about 40 mM; and about 50 mM.
  • the phosphorylated hexaacyl disaccharide containing formulations described herein are preferably substantially free of saline, preferably contain less than 20 mM NaCl, and more preferably, the phosphorylated hexaacyl disaccharide compositions contain less than 10 mM NaCl and even more preferably essentially free of NaCl.
  • the phosphorylated hexaacyl disaccharide formulations are preferably prepared by selecting a single phosphatidylcholine prepared synthetically with high purity.
  • naturally derived phosphatidylcholines from soy or egg or hydrogenated phosphatidylcholines from soy or egg or synthetic or natural mixed acyl phosphatidylcholines may also be used to prepare the adjuvant formulations.
  • the phosphorylated hexaacyl disaccharide formulations can be prepared at any concentration but are typically prepared at about 0.005 to about 1.0 mg/ml of phosphorylated hexaacyl disaccharide, preferably 0.05 to about 1.0 mg/ml of phosphorylated hexaacyl disaccharide, but preferably not more than about 2.5 mg/ml of phosphorylated hexaacyl disaccharide.
  • the phosphatidylcholine of the phosphorylated hexaacyl disaccharide formulation can be prepared at any concentration, but is preferably prepared at about 0.005 to about 16 mg/mL (0.007 mM to 22 mM), more preferably about 0.05 to about 8 mg/ml (0.07 mM to 11 mM), and even more preferably about 0.05 to about 0.8 mg/ml (0.07 mM to 1 mM).
  • the phosphorylated hexaacyl disaccharide formulations of the invention are prepared as follows to produce a molar ratio of about 40: 1 to about 1 : 1 of phosphatidylcholine to phosphorylated hexaacyl disaccharide, preferably DPPC to phosphorylated hexaacyl disaccharide, preferably about 2.5: 1 DPPC to phosphorylated hexaacyl disaccharide.
  • Phosphorylated hexaacyl disaccharide is weighed out into an appropriate glass vial, such as a Type 1 Plus Schott glass vial.
  • An appropriate amount of phosphatidylcholine, preferably DPPC, in ethanol is added.
  • This preparation is sonicated for approximately 1 minute while gently swirling the preparation, and then the ethanol is appropriately removed via evaporation.
  • the film is reconstituted with citrate, succinate or phosphate buffer, preferably 10 mM to about 50 mM trisodium citrate, pH 6.0, and sonicated at approximately 50°C to 65°C, preferably 55°C, for approximately 30 minutes, and more than one cycle of sonication can be performed, as preferred.
  • the prepared phosphorylated hexaacyl disaccharide formulation typically has particles sizes from about 60 nm to about 500 nm.
  • the phosphorylated hexaacyl disaccharide formulation is preferably further processed to achieve a reduced and appropriate homogenous particle size, preferably between about 70 nm to 130 nm.
  • the phosphorylated hexaacyl disaccharide formulations are extruded through an 80 nm pore polycarbonate membrane (Avestin, LFLM-80) with an Avestin extruder, or equivalent, for about 7 to about 12 passes at about 45°C to 65°C, preferably 55°C to achieve a homogenous particle size below about 130 nm.
  • the particle sizes of the phosphorylated hexaacyl disaccharide formulations are 150 nm or less, but even more preferably 130 nm or less.
  • the phosphorylated hexaacyl disaccharide formulations may optionally then be diluted with citrate, succinate or phosphate buffer at concentrations described herein, but preferably at about 25 mM to about 50 mM, more preferably 28 mM to about 50 mM, and most preferably 30 mM to about 50 mM, pH 6.0, containing an appropriate amount of polysorbate 80 to achieve a final concentration of preferably 0.02% of polysorbate 80, but 0.01% to 0.1 % polysorbate 80 is acceptable.
  • the phosphorylated hexaacyl disaccharide formulations will be diluted to about a
  • the phosphorylated hexaacyl disaccharide formulations are then sterile filtered through a 0.2 um filter, preferably Sartorius.
  • the phosphorylated hexaacyl disaccharide and adjuvant formulations of the invention described herein exhibit zeta potentials of about -20 mV to -80 mV.
  • the phosphorylated hexaacyl disaccharide formulations described herein are used in the preparation of vaccines and are administered to animals and humans as an adjuvant of vaccines.
  • the formulations are designed to deliver about 10 micrograms of phosphorylated hexaacyl disaccharide per dose to about 50 micrograms of phosphorylated hexaacyl disaccharide per dose, preferably about 20 micrograms of phosphorylated hexaacyl disaccharide per dose to about 50 micrograms of phosphorylated hexaacyl disaccharide per dose, and even more preferably about 40 micrograms of phosphorylated hexaacyl disaccharide per dose to about 50 micrograms of phosphorylated hexaacyl disaccharide per dose.
  • phosphorylated hexaacyl disaccharide is provided as a single compound of approximately 98% purity with a molecular weight of 1763 Daltons (the structure of which is shown in FIG. 1.
  • One source for the preferred phosphorylated hexaacyl disaccharide is Avanti Polar Lipids (Alabaster, Alabama, USA) (PHAD).
  • the phosphorylated hexaacyl disaccharide compositions of the present invention encompass phosphorylated hexaacyl disaccharide or pharmaceutically acceptable salts of phosphorylated hexaacyl disaccharide.
  • the phosphorylated hexaacyl disaccharide used in the compositions may be fully or partially synthetic or non-synthetic, although fully synthetic is preferred.
  • derivatives of phosphorylated hexaacyl disaccharide such as 3-deacyl-phosphorylated hexaacyl disaccharide (available from Avanti Polar Lipids; referred to as 3D-PHAD) are used alone or in combination with phosphorylated hexaacyl disaccharide in the compositions and formulations of the present invention.
  • 3-deacyl-phosphorylated hexaacyl disaccharide is provided with approximate purity of greater than 98% with a molecular weight of 1537 Daltons (the structure of which is shown in FIG. 1A).
  • PHAD is superior to GSK's monophosphoryl lipid A because PHAD's manufacturing process, supply, use, and stability can be closely monitored and controlled as a pure compound.
  • the concentration of citrate, succinate, or phosphate buffer is used to improve stability of the phosphorylated hexaacyl disaccharide formulation at room temperature and up to about 37°C. While not bound by theory, it is believed that a synergistic effect results from composition of molar ratio of PC: phosphorylated hexaacyl disaccharide or its derivative, 3-deacyl-phosphorylated hexaacyl disaccharide, which is a preferred combination, and citrate, succinate, or phosphate buffers within a specific concentration range as described herein.
  • formulations are preferably formulated to be substantially free of various excipients or chemicals. Therefore, the addition of cholesterol or two or more phosphatidylcholines or one or more phosphatidylglycerols is not required, and preferably not included in the formulations of the invention. Adjuvant formulations essentially free of metabolizable oils, including squalene, and substantially free of cholesterol are preferred. In addition, while the prior art suggests cholesterol is a necessary chemical required for liposomes or adjuvant formulations, the adjuvant formulations described herein do not require cholesterol for the adjuvant to provide all the significant advantages over prior art formulations, and preferably no cholesterol is added to the adjuvant formulations. As shown in the Examples, two or more phosphatidylcholines or one or more
  • phosphatidylglycerols (for a total of two or more phosphatidylcholines or
  • phosphatidylglycerols may optionally be added to the formulations at minor
  • Another embodiment of this invention further describes novel vaccine compositions comprising the adjuvant or phosphorylated hexaacyl disaccharide formulations and FimCH or truncated FimH.
  • Such vaccines are used to treat and prevent urinary tract infections caused by gram-negative bacteria including Escherichia coli and multi-drug resistant E. coli.
  • FimCH is a non-covalent complex of FimC and FimH recombinant proteins.
  • a vaccine of FimCH and phosphorylated hexaacyl disaccharide formulation is prepared by adding a predetermined volume of a phosphorylated hexaacyl disaccharide formulation to a vial of FimCH.
  • a vaccine prepared in accordance to the invention is an example of a vaccine prepared in accordance to the invention.
  • an effective amount of an antigen of FimCH or truncated FimH is combined with an adjuvant formulation containing about 0.005 mg/ml to about 0.5 mg/ml of phosphorylated hexaacyl disaccharide to administer about 10 ⁇ g to about 50 ⁇ g of phosphorylated hexaacyl disaccharide per injection to a human.
  • a vial of FimCH vial is removed from storage of about -20°C and allowed to stand at room temperature for approximately twenty minutes to reach approximate room temperature. After the FimCH vial reaches approximately room temperature, the vial is inverted a number of times to mix the contents.
  • a vial containing the phosphorylated hexaacyl disaccharide formulation is removed from a storage container from 2°C to 8°C storage. The vial of phosphorylated hexaacyl disaccharide formulation is inverted a number of times to mix the contents, and then about 0.2 mL is withdrawn with a sterile 1.0 mL syringe and injected into the FimCH vial through the stopper.
  • Sterile water for injection (WFI) or more preferably preferred sterile buffer of the invention is withdrawn in an amount of 0.2 mL using a sterile 1.0 mL syringe and injected into the FimCH/ phosphorylated hexaacyl disaccharide vial through the stopper. Again the vial is inverted a number of times. Finally, about 0.3 mL of prepared FimCH / phosphorylated hexaacyl disaccharide vaccine using a sterile 1.0 mL syringe is withdrawn. The prepared vaccine contains 50 ⁇ g of FimCH and 20 ⁇ g of phosphorylated hexaacyl disaccharide per a 0.3 mL dose. This prepared vaccine can be stored at refrigerated or room temperatures prior to administration.
  • FimCH is stored at -70°C, -20°C, or 2°C to 8°C for long time periods, or even room temperature for a short time period of about 4 days to two to three weeks.
  • the vaccine is preferably administered by intramuscular injection.
  • about 5 micrograms of FimCH to about 200 micrograms of FimCH would be administered to a human, preferably about 20 micrograms to about 110 micrograms.
  • micrograms of phosphorylated hexaacyl disaccharide per dose to about 50 micrograms of phosphorylated hexaacyl disaccharide per dose would be administered with FimCH, more preferably about 20 micrograms of phosphorylated hexaacyl disaccharide per dose to about 50 micrograms of phosphorylated hexaacyl disaccharide per dose, even more preferably about 40 micrograms of phosphorylated hexaacyl disaccharide per dose to about 50 micrograms of phosphorylated hexaacyl disaccharide per dose although more or less may be used.
  • FimCH with the phosphorylated hexaacyl disaccharide formulation is administered to a patient in need. These doses typically occur at day 0 and then about days 30 to 60, then about 90 to 180 days, and then, if preferred, about 180 to 360 days from the first administration. As needed, additional injections may occur 12 to 36 months after the initial vaccination.
  • the more preferable aspect of the invention is that the phosphorylated hexaacyl disaccharide compositions and formulations are stored separately from the antigen or FimCH because the phosphorylated hexaacyl disaccharide compositions and formulations have excellent stability, and formulated or mixed appropriately with the antigen or FimCH sometime before administration to a patient or human.
  • other methods of mixing, preparing and administering vaccines are possible and will function effectively.
  • the data in the following sections demonstrates that the adjuvant formulations of the invention enhance the immune response of other antigens including bacterial and viral antigens.
  • One or more vaccine antigens may be added to the phosphorylated hexaacyl disaccharide formulations prepared in accordance with the invention. These antigens may be the FimCH protein complex as described herein or other antigens including, but not limited to diphtheria, tetanus, pertussis, poliomyelitis, hepatitis, and or antigenic preparations of the influenza virus.
  • methods of administration of the novel vaccine compositions comprising adjuvant or phosphorylated hexaacyl disaccharide formulations and FimCH or truncated FimH are provided.
  • methods of treatment to prevent and treat urinary tract infections caused by gram-negative bacteria including E. coli and multi-drug resistant E. coli are provided.
  • about 5 micrograms of FimCH to about 200 micrograms of FimCH would be administered to a human, preferably about 20 micrograms to about 1 10 micrograms.
  • micrograms of phosphorylated hexaacyl disaccharide per dose to about 50 micrograms of phosphorylated hexaacyl disaccharide per dose would be administered, preferably about 20 micrograms of phosphorylated hexaacyl disaccharide per dose to about 50 micrograms of phosphorylated hexaacyl disaccharide per dose, even more preferably about 40 micrograms of phosphorylated hexaacyl disaccharide per dose to about 50 micrograms of phosphorylated hexaacyl disaccharide per dose.
  • Other dosage amounts and regimens may be used dependent on the antigen used and the condition being treated.
  • vaccine compositions that induce the production of antibodies against FimH in a human with recurrent urinary tract infections are provided.
  • compositions containing phosphorylated hexaacyl disaccharide or its derivative, 3-deacyl-phosphorylated hexaacyl disaccharide are provided; preferably these compositions of aqueous buffered suspensions have particle sizes less than 150 nm, even more preferably less than 130 nm.
  • the sterile phosphorylated hexaacyl disaccharide compositions and formulations are stored in a pharmaceutical container which is in direct contact to the phosphorylated hexaacyl disaccharide compositions and formulations.
  • Examples of these pharmaceutical containers are vials or syringes, more preferably the sterile phosphorylated hexaacyl disaccharide compositions and formulations are contained in a sterile syringe.
  • These pharmaceutical containers holding the phosphorylated hexaacyl disaccharide composition or formulation can be stored in a temperature-validated container, e.g. incubator, at room temperature.
  • These pharmaceutical containers holding the sterile phosphorylated hexaacyl disaccharide composition formulation can be added to a shipping container assembled to be transferred to another location at room temperature or up to about 37°C.
  • These shipping containers can be transferred via a government postal service or a commercial shipping service. Due to the remarkable stability of the inventions described herein, the location receiving said phosphorylated hexaacyl disaccharide compositions and formulations may be a location without refrigeration or intermittent access to refrigeration or a location without electricity or with intermittent electricity.
  • a vaccine kit comprising the
  • kits may optionally include methods of preparing and administering the vaccine and/or instructions for storage and exposure of the
  • kits for phosphorylated hexaacyl disaccharide compositions or formulations at room temperature and up to and about 37°C.
  • kit components is the phosphorylated hexaacyl disaccharide compositions or formulations in a syringe.
  • the phosphorylated hexaacyl disaccharide compositions or phosphorylated hexaacyl disaccharide containing formulations are sterile and are in a sterile syringe.
  • the kit may include a label for the phosphorylated hexaacyl disaccharide or adjuvant compositions or formulations of the invention providing instructions or limitations for storage and exposure of the phosphorylated hexaacyl disaccharide compositions or formulations at room temperature and up to and about 37°C.
  • the labels or instructions describing storage, shipping, and exposure temperatures may be approved by a government regulatory authority including the US FDA or European Medicines Agency.
  • the novel characteristics of the invention enable the phosphorylated hexaacyl disaccharide compositions and formulations to be manufactured, tested, analyzed, stored, shipped, held, moved, transferred, or administered at refrigerated temperatures, room temperature, up to and about 37°C, temperatures between refrigerated temperatures and room temperature for periods of time as described herein. Due to the remarkable stability of the inventions described herein, the location receiving said phosphorylated hexaacyl disaccharide compositions and formulations may be a location without refrigeration or intermittent access to refrigeration or a location without electricity or with intermittent electricity.
  • PHAD Avanti Polar Lipids (Alabaster, Alabama, USA), phosphorylated hexaacyl disaccharide, and DPPC can be obtained from Avanti Polar Lipids (Alabaster, Alabama, USA) as either non-GMP or GMP material (the Certificate of Analysis is provided in Table 1).
  • PHAD is a synthetic version of monophosphoryl lipid A.
  • PHAD is formulated with DPPC to prepare the adjuvant formulation of the invention.
  • DPPC's release specifications are provided in the table 2.
  • the transition temperature of DPPC is 41°C.
  • PHAD is weighed out into an appropriate glass vial, preferably a
  • Type 1 Plus Schott glass vial An appropriate amount of an approximately 2.3 mg/ml DPPC solution, or equivalent, in ethanol is added. This preparation is sonicated for approximately 1 minute while gently swirling the preparation, and then the ethanol is appropriately removed via evaporation using care.
  • the film is reconstituted with 10 mM trisodium citrate, pH 6.0, and sonicated at approximately 50°C to 65°C, preferably 55°C, for approximately 30 minutes.
  • the phosphorylated hexaacyl disaccharide formulations are typically prepared at about 0.5 to 1.0 mg/ml, but not more than about 2.5 mg/ml. (As described herein, molar ratios of for example about 13: 1 DPPCphosphorylated hexaacyl disaccharide can also be prepared using this same procedure by adjusting the amount of DPPC as needed.
  • the prepared PHAD formulations typically exhibit particles sizes from about 60 nm to about 500 nm. To enable sterile filtration, the PHAD formulations have to be further processed to achieve a reduced and appropriate homogenous particle size, typically between about 70 nm to 130 nm. Even though numerous literature references, including US Patent 6,630,161, report high-pressure homogenization is a preferred method to reduce the particle size of liposomes, high-pressure homogenization using an Avestin homogenizer of pressures up to 25,000 psi do not significantly or relevantly reduce the particle sizes of these PHAD formulations. This difficulty was unexpected.
  • PHAD formulations must be extruded through an 80 nm pore polycarbonate membrane (Avestin, LFLM-80) with an Avestin extruder, or equivalent, for about 7 to about 12 passes at about 45°C to 65°C, preferably 55°C to achieve a homogenous particle size below about 130 nm. Extruding at 45°C to 65°C is an important parameter.
  • the particle sizes of the phosphorylated hexaacyl disaccharide adjuvant formulations are preferably 150 nm or less, but more preferably 130 nm or less.
  • the phosphorylated hexaacyl disaccharide formulations may then be diluted with 10 mM trisodium citrate, pH 6.0 containing an appropriate amount of polysorbate 80 to achieve a final concentration of most preferably 0.02% of polysorbate 80, but 0.01 % to 0.1 % polysorbate 80 is acceptable.
  • phosphorylated hexaacyl disaccharide formulations will be diluted to about a concentration of 0.05 mg/ml to 0.5 mg/mL.
  • These phosphorylated hexaacyl disaccharide formulations are then sterile filtered through a 0.2 um filter, preferably Sartorius.
  • the adjuvant formulations described herein are suspensions.
  • the ethanol may be evaporated by rotary evaporation, or equivalent, or via a nitrogen stream, or equivalent.
  • the sonication step including a buffer of the invention may be repeated two or more times, the formulation may be cooled to room temperature or less between repeated sonications, and the formulation may be held at a temperature similar to the sonication step for one or more hours before, during, or after said sonication.
  • NMT 2000 ppm acetone None Detected NMT 200 ppm hexane None Detected NMT 2000 ppm cyclohexa ne None Detected NMT 500 ppm toluene None Detected NMT 50 ppm None Detected chloroform/ethyl acetate None Detected NMT 5000 ppm total resid ual
  • Antigens for the vaccine may be prepared as follows.
  • FimCH is a non-covalent complex of FimC and FimH recombinant proteins.
  • the recombinant proteins are derived from transgenic E. coli culture.
  • the FimC and FimH proteins are expressed separately in E. coli, and they spontaneously form a non- covalent complex.
  • the molecular weight of the FimCH complex is approximately 51,700 Daltons.
  • FimH protein (SEQ ID No: 1) of the complex has a molecular weight of 29,065 Daltons, and it consists of 279 amino acid residues represented by the sequence below:
  • FimC (SEQ ID No: 2) protein of the complex has a molecular weight of 22,700 Daltons, and it consists of 205 amino acid residues represented by the following sequence:
  • the FimC gene from E. coli strain J96 was amplified with primers SLC4-28-fimC5 and SLC4-28-FimC3 from purified J96 genomic DNA to give a 771 base pair product. This was digested with BamHI and EcoRI, purified, and ligated into pTRC99a cut with the same enzymes (BamHI and EcoRI). The ligation product was transformed into E. coli C600 cells, and selected on ampicillin, producing plasmid pSJH-32.
  • the ampicillin antibiotic resistance was switched to kanamycin using the following procedure: Primers pKD4-prl and pKD4-pr2 were used to amplify the kanamycin resistance gene from pKD4. This PCR product was phosphorylated with T4 polynucleotide kinase and gel purified. pSJH-32 was cut with Seal and Bgll, blunted with T4 DNA polymerase, dephosphorylated with calf intestinal alkaline phosphatase, then ligated with the phosphorylated kanamycin resistance gene PCR product. The ligation product was then transformed into E. coli C600 cells and selected on kanamycin, creating plasmid pSJH-319.
  • FimH gene from strain J96 was amplified with primers FimH5 and FimH3 from purified J96 genomic DNA to give a 978 base pair product. It was digested with Sad and Hindlll, purified, and ligated into pBAD33 digested with Sacl and Hindlll. Afterwards the construct was transformed into C600 cells, and selected on chloramphenicol.
  • Bioprocessing Provides to obtain antigens of the vaccine.
  • the bioprocessing step is initiated with inoculation of master cell bank (MCB) into shake flasks containing APS LB medium with kanamycin (50 ⁇ g/mL) and chloramphenicol (20 ⁇ g/mL).
  • MMB master cell bank
  • kanamycin 50 ⁇ g/mL
  • chloramphenicol 20 ⁇ g/mL
  • the cell culture is transferred aseptically into reactors for fed-batch fermentation.
  • the medium containing APS Super Broth, about 0.8% glycerol, and antibiotics is sterilized prior to inoculation.
  • FimH protein expression is induced at OD > 10 with IPTG. Five minutes after IPTG addition, FimC protein expression is induced with arabinose. Cells are harvested approximately one hour later. After harvesting, the cells are separated from the media components by continuous or batch centrifugation.
  • E. coli as a gram negative bacteria, possess an inner and an outer lipid bilayer membrane. The space between the lipid bilayers is the periplasm.
  • FimCH is recovered from the cell using a periplasm preparation. The cells are reacted with recombinant lysozyme in the presence of sucrose, Tris, and EDTA at 2-8°C. The mixture is then centrifuged, and the resulting periplasmic protein solution is collected.
  • the protein is then precipitated with ammonium sulfate, centrifuged, resuspended in 20 mM MES pH 5.9, and diafiltered via dialysis into 20 mM MES pH 5.9 using SpectraPor 2 Dialysis Membrane (Spectrum Labs 132680).
  • SpectraPor 2 Dialysis Membrane Spectrum Labs 132680
  • the two CEX steps use Source 15S (GE Healthcare 17-1273-02) in an XK26 column.
  • Buffer A 20 mM MES, pH 5.9
  • Buffer B 20 mM MES / 500 mM Sodium Chloride, pH 5.9;8 ml/min for all steps except loading for XK26/10 column, pre-equilibrate the column with 5 CV of Buffer B, equilibrate the column with 4 CV of Buffer A, load Dialyzed FimCH sample at 5 ml/min for XK26/10 using a sample pump and specifically not via the chromatography pump, wash the column with 4 CV of Buffer A, and elute the column with a linear 5 CV gradient from 0-25% Buffer B with fraction collection.
  • FimCH is formulated in the concentration of 0.3 mg/mL in 20 mM MES pH 5.9 or 20 mM trisodium citrate, pH 5.4. It is then aseptically filtered through a 0.2 ⁇ sterile filter. FimCH is stable and can be stored at -20°C for about at least 2 years. [00234] EXAMPLE 4
  • the biological activity of the FimCH drug substance is determined by an in vitro mannose binding assay.
  • the FimH protein is a bacterial adhesin utilized by E. coli to bind mannose residues on glycosylated proteins.
  • the FimH adhesin binds mannosylated uroplakin proteins on bladder epithelial cells, which promotes the internalization of bound E. coli.
  • the binding of FimH to mannosylated uroplakin is essential for ?, coli to cause urinary tract infections.
  • HRP horseradish peroxidase
  • HRP is a glycosylated protein containing mannose residues, and has previously been used to study mammalian mannose- binding receptors. Complexes of HRP and the lectin ConA, which binds a-D-mannosyl and a-D-glucosyl groups, have also been generated and studied. These results demonstrate that HRP acts as a ligand for other known mannose-binding proteins. Using this potency assay as described below, HRP binding to FimH is shown to be concentration-dependent and to be inhibited by small molecules that the block the binding of mannose to FimH.
  • FimCH is "captured” by purified and qualified anti-FimH antisera bound to an ELISA plate.
  • the anti-FimH antisera used in this assay have demonstrated the ability to bind to FimH in both indirect ELISAs (as the detection antisera, Figure 3) and western blots. HRP is then added, excess HRP is rinsed away, and the activity of bound HRP is detected. The measured HRP activity is proportional to the concentration of FimCH added ( Figure 4).
  • FimH at positions 54, 133, 135, and 140 completely abolish mannose binding.
  • Hung et al. "... even the slightest change in the mannose-binding pocket, in an atom that does not bind directly to mannose, significantly reduces binding," suggesting that mutations that could occur in vitro could severely limit or abolish FimH mannose binding activity.
  • the lack of HRP binding to the Q133K mutant supports this assay's ability to assess the biological activity of FimH binding to mannosylated proteins.
  • CEX-HPLC is used for determination of the FimCH complex, unbound FimC and impurities in the final FimCH drug substance.
  • the protein is eluted from a GE Healthcare Mono S 5/50 GL column using a gradient of 0.3 M NaCl in 20 mM MES buffer, pH 6.2 (Buffer B) (Buffer A is 20 mM MES buffer, pH 6.2).
  • Buffer B Buffer A is 20 mM MES buffer, pH 6.2
  • the relative content of the unbound FimC and impurities is determined based on the peak areas.
  • a representative chromatogram is provided in Figure 6.
  • FimCH and PHAD formulation An 85-Day Intramuscular Toxicity
  • Three days following the fifth dose (Day 88) 6 rabbits per group were euthanized with the remaining 6 rabbits in the PHAD alone group and FimCH high dose groups euthanized following a 3-week recovery period (Day 106).
  • Toxicity was assessed based on clinical observation
  • ophthalmology body temperature, body weight, food consumption, clinical pathology, gross necropsy, organ weight, and histopathology data.
  • Body temperatures were obtained prior to and 2, 4, 6, 24, 48 and 72 hours after each injection.
  • C-reactive protein (CRP) and fibrinogen were evaluated 2 and 7 days after dosing.
  • Potential injection site reactions were scored for edema and erythema using the Draize scale and assessed for other manifestations of local toxicity (i.e. eschar, vesiculation, ulceration, and hematoma) 24, 48, and 72 hours post dose.
  • Anti-FimH antibody assessment was performed on serum samples collected prior dosing on Days 1, 22, 43, 64 and 85, and prior to necropsy on Days 88 and 106, on urine samples collected prior to dosing, Day 64, and prior to necropsy on Days 88 and 106, and on vaginal washings collected prior dosing on Days 1 and, and prior to necropsy on Days 88 and 106.
  • Antibody evaluation of the urine and vaginal wash samples was qualitative using a qualified MSD-ECL assay. Antibody levels in serum were determined using a validated MSD-ECL assay. Of 18 rabbits vaccinated with FimCH and PHAD, 17 demonstrated anti-FimH IgG titers at about 1 :3,200,000 at about Day 88.
  • DPPCPHAD at about a molar ratio of 2.4: 1 demonstrated anti-FimH IgG titers from 1 :400,000 to 3,200,000 at about Day 43. These immunogenic differences are consistent with the studies performed in mice described herein demonstrating that a molar ratio of DPPCPHAD of about 2.4: 1 is superior to a molar ratio of DPPCPHAD of about 1 :3.9.
  • C3H/HeN mice were infected with approximately 1 xlO 8 CFU of clinical cystitis E. coli isolate UTI89 via transurethral catheterization after IM immunization.
  • Female C3H/HeN mice were infected with approximately 1 xlO 8 CFU of clinical cystitis E. coli isolate UTI89 via transurethral catheterization after IM immunization.
  • Female C3H/HeN mice were infected with approximately 1 xlO 8 CFU of clinical cystitis E. coli isolate UTI89 via transurethral catheterization after IM immunization.
  • mice were injected via the intramuscular route (IM) in the right thigh under light isoflurane anesthesia (Henry Schein, Melville, NY) in a 50 ⁇ volume using a 30 gauge needle.
  • IM intramuscular route
  • mice were immunized with 12.5 ⁇ g PHAD / 15 ⁇ g FimCH.
  • Mice immunized using PHAD as the adjuvant and FimCH as antigen showed a statistically significant decrease of E. coli CFU in bladders one or two days after infection compared to mice immunized with adjuvant alone and to naive mice (experiment using PHAD shown in Figure 7).
  • Truncated FimH (FimHt) adjuvanted with phosphorylated hexaacyl disaccharide formulation or Freund's adjuvant An Immunogenicity Study in Rabbits.
  • Truncated FimH has a series of histi dines or histidine tag, and those skilled in the art understand that other truncated versions of FimH can also be used, most preferably requiring the mannose binding domain.
  • Truncated FimH in this example consists of FimH residues 1 to 175 with a C-terminal 6-histidine tag (SEQ ID No: 3). The sequence of FimH is described in Example 2 .
  • Anti-FimH antibody assessments were performed on serum samples collected on about day 30 or about days 35 and 56. Antibody levels in serum were determined using an ELISA as described herein. The capture antigen for this experiment was an equivalent truncated FimH without a histidine tag.
  • PHAD and DPPC concentrations in the PHAD formulation are analyzed by HPLC-ELSD using an Agilent Eclipse XBD CI 8, 1.8 um, 4.6 mm x 50 mm column.
  • the mobile phases are as follows: MP A: 20 mM ammonium acetate 11% acetic acid in water; MP B: 20 mM ammonium acetate 11% acetic acid in methanol; and MP C: 20 mM ammonium acetate l ⁇ % acetic acid in methanol/chloroform (50/50).
  • Methodhod 1 gradient begins at 5% MP A and 95% MP B, at 2 minutes is 100%MP B, and at 8 minutes is 100% MP C.
  • Methodhod 2 gradient begins at 5% MP A and 95% MP B, at 2 minutes is 100% MP B, and at 15 minutes is 100% MP C.
  • Sample Diluent 1 85: 15 (75: 15: 10 methanol: chloroform: water with 20 mM ammonium acetate 11% acetic acid):(l : 1 methanol: chloroform with 20 mM ammonium acetate 11% acetic acid) Sample and standards are diluted 1 :4 with sample diluent 1. Sample Diluent 2: (70:25:5)
  • sample and standards are diluted 1 : 10 with sample diluent 2.
  • the ELSD gain is at 8 with temperature at 60C and nitrogen flow set to approximately 3.7 bars.
  • An example chromatogram using method 2 and sample diluent 2 is shown in Figure 8.
  • citrate and phosphate buffers from about 30 mM to about 50 mM are superior to the other buffers examined.
  • the citrate and phosphate buffers provide stability at the listed temperatures for the listed time periods.
  • the data demonstrate the remarkable benefit of stability of increasing the citrate and phosphate concentrations to about 25 mM to about 50 mM, more preferably 28 mM to about 50 mM, and most preferably 30 mM to about 50 mM.
  • the preferred use of succinate as a buffer is shown from all of the data collectively as described herein.
  • formulations prepared in water are stable short-term when stored at 2°C to 8°C; however, the long sought-after goal is to reduce cold chain storage and management.
  • inventive adjuvant formulation disclosed herein achieved this goal by providing an adjuvant formulation with extended stability at room temperature to about 37°C.
  • Table 6 clearly show that the PHAD concentration of these formulations prepared in citrate buffer will remain stable for at least approximately 6 or more months at about 25°C and potentially 2 to 3 years at 2°C to 8°C.
  • Phosphatidylcholine phosphorylated hexaacyl disaccharide formulations remarkably and unexpectedly enables storage at room temperature and exposure up to and at about 37°C.
  • Phosphatidylcholine phosphorylated hexaacyl disaccharide formulations prepared in water can produce equivalent immunogenic responses in mice and rabbits but are not stable at about 25 °C.
  • Particle sizes and zeta potentials were determined on PHAD formulations using Dynamic Light Scattering with the Malvern Zetasizer® ZS90 or Brookhaven Instruments Corp. using ZetaPlus Particle Sizing software. The
  • Table 7 provides representative data. Zeta potential values are used as one piece of qualitative data estimating the electrical charge at a bilayer and as described herein. Stability of the PHAD formulations is experimentally determined from particle size of the formulation and concentration of PHAD.
  • Example 4 Adjuvant formulation of Example 1, containing 202 -76 DMPCDLPC and not DPPC, in an approximate
  • DPPC alone has a significantly less zeta potential and much larger mean particle size compared to the formulations of the invention containing PHAD.
  • the critical micelle concentration of DPPC is about 0.46 nanomolar.
  • PHAD formulations over 12 to 36 months. Multiple batches of PHAD formulations were prepared and typically exhibited particles sizes between 70 to 100 nm immediately after extrusion.
  • the goal for these PHAD formulations is to ensure that their mean effective diameter remains preferably less than 150 nm over its shelf life, even more preferably less than 130 nm, which is predicted to be 2 to 3 years or longer based upon the data described herein. It is important to trend the mean effective diameter over a certain time period at intermediate / accelerated conditions, e.g. 25°C, to assist with predicting the particle size at 2°C to 8°C in approximately 2 or more years.
  • Adjuvant formulation of Example ] L without 1 month/ 25 °C 99 polysorbate 80
  • Adjuvant formulation of Example 1 1 month/ 2-8°C 79
  • Adjuvant formulation of Example 1 1 month/ 25 °C 82
  • Adjuvant formulation of Example ] L without 4 months/ 25 °C 114 polysorbate 80
  • Adjuvant formulation of Example 1 4 months/ 25 °C 86
  • Adjuvant formulation of Example 1 5 months/ 2-8°C 82
  • Adjuvant formulation of Example 1 6 months/ 25 °C 101
  • Adjuvant formulation of Example 1 6 months/ 2-8°C 94
  • Adjuvant formulation of Example ] L without 8 months/ 2-8°C 117 polysorbate 80
  • Aqueous formulation in this example refers to the molar ratio of lipid to adjuvant described in US Patent 6,491,919 and US Patent Application 20080131466) used below in this example were prepared as described in Example 1 with the following exceptions.
  • mice Female C3H/HeN mice (approximately 9 weeks old) were purchased from Charles River laboratories (Wilmington, MA). Mice were injected via the intramuscular route (IM) in the right thigh under light isoflurane anesthesia (Henry Schein, Melville, NY) in a 50 ⁇ volume using a 30 gauge needle. Mice were vaccinated with 12.5 ⁇ g PHAD, or its derivative 3-deacyl-phosphorylated hexaacyl disaccharide or Freund's adjuvant (subcutaneous administration), and 15 ⁇ g FimCH, on days 1 and 29. As known to those skilled in the art, when used, complete Freund's adjuvant was given on day 1 and incomplete Freund's adjuvant on day 29.
  • ELISA for serum antibody detection Sera was collected from the mice at sacrifice and analyzed by ELISA for anti-FimH antibodies. FimH truncate T3 was adhered to Immulon 4 HBX plates (ThermoFisher) at 2 ⁇ g/ml in PBS overnight at 4°C. After washing with PBS+0.05% Tween 20, open binding sites were blocked with 1.5% BSA (Sigma Aldrich) in PBS for lhr. After washing, dilutions of sample sera (in PBS with 0.05% Tween 20, 0.1% BSA, 0.5% methyl a-D-mannopyranoside) were incubated for 2hrs.
  • a molar ratio of DPPC to PHAD of about 2: 1 to about 13: 1 was superior at enhancing an immune response to FimH in mice when compared to no adjuvant, Freund's adjuvant, or the aqueous formulation of DPPC to PHAD at a molar ratio of 1:3.9.
  • Freund's adjuvant is considered a standard adjuvant used in animal experiments.
  • the data shows that the molar ratio of the invention described herein of about 2: 1 to 13: 1 is superior to this frequently used preclinical adjuvant. This is one aspect of the invention that demonstrates its superiority to the prior art formulations.
  • the data in Table 9 also demonstrate that adding DPPG into these formulations does not diminish the intended use of the PHAD formulations, i.e., to enhance an immune response to FimH.
  • Example 1 The procedure of Example 1 was used to prepare the PHAD formulation with a molar ratio of DPPC: PHAD at about 2.5 to 1, except polysorbate 80 was not added. The vial containing the PHAD formulation was gently inverted about three to five times. Then, the PHAD formulation was allowed to remain at room temperature for about 24 hours. After 24 hours and without inverting, shaking, or stirring the PHAD formulation, small aliquots were carefully removed from the top, middle, and bottom of the PHAD suspension. These aliquots were analyzed for PHAD concentration via HPLC as previously described herein. The results demonstrated that the aliquots from the top, middle, and bottom contained equivalent PHAD concentrations. These results demonstrate the PHAD formulation of the invention does not settle within 24 hours.
  • Example 1 The adjuvant formulation of Example 1 and FimCH were prepared under cGMP for use in a human clinical study involving females of about 21 to 64 years of age.
  • a vaccine of FimCH and PHAD formulation was prepared by adding a
  • a PHAD formulation predetermined volume of a PHAD formulation to a vial of FimCH as previously described herein to obtain the preferred concentrations of FimCH and PHAD per each vial.
  • An appropriate volume of the prepared vaccine was injected IM (intramuscular) to administer either 50 ⁇ g or about 107 ⁇ g of FimCH with either 10 ⁇ g, 20 ⁇ g, or about 40 ⁇ g of PHAD to each female subject.
  • the vaccine demonstrates that the adjuvant formulation of the invention with FimCH produces less severe injection site and systemic reactions in humans compared to certain other known adjuvant formulations used in humans . This is a remarkable aspect of the invention.
  • Injection Site and Systemic Reactions are shown below for number of injections as of the interim analysis per female.
  • the human study is ongoing and the females in the study are scheduled to receive 4 injections.
  • the adjuvant formulation of the invention overcomes this limitation with superior formulations that enable administration of up to 50 ⁇ g of phosphorylated hexaacyl disaccharide, or potentially more, with less severe injection site and systemic reactions.
  • These human data offer evidence that phosphorylated hexaacyl disaccharide can be administered to humans at 100 ⁇ g or more micrograms with the formulations described herein.
  • the adjuvant formulations of the invention are superior to those formulations previously attempted.

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Abstract

La présente invention concerne de nouvelles compositions et formulations d'adjuvants présentant une excellente stabilité en milieu réfrigéré et à température ambiante jusqu'à environ 37 °C, et pouvant être produites à des prix remarquablement bas. La présente invention concerne également de nouvelles compositions et formulations de vaccins pour traiter et prévenir des infections urinaires provoquées par des bactéries à Gram négatif, dont Escherichia coli et E. coli multi-résistant aux médicaments. La présente invention concerne en outre des méthodes d'administration desdites nouvelles compositions et formulations de vaccins et des méthodes thérapeutiques pour prévenir et traiter des infections urinaires provoquées par des bactéries à Gram négatif, notamment E. coli et E. coli multi-résistant aux médicaments.
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