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    • 专利摘要:
    The invention relates to a composition comprising solid lipid nanoparticles (NLS), wherein the NLS comprise an aminoalkyl glucosaminide phosphate (AGP).
    公开号:BE1024228B1
    申请号:E2016/5904
    申请日:2016-12-06
    公开日:2017-12-21
    发明作者:Hardeep Oberoi；David Burkhart；Jay T. Evans；Yvonne M. Yorgensen
    申请人:Glaxosmithkline Biologicals Sa；
    IPC主号:
    专利说明:

    NOVEL ADJUVANT FORMULATIONS Field of the invention
    The present invention relates to novel adjuvant formulations and methods for their preparation and uses. In particular, the invention relates to novel formulations of aminoalkyl glucosaminide phosphates (AGP). Statement on Federally Funded Research
    Aspects of this invention have been made with the support of the United States Government pursuant to the National Institutes of Health's contract # HHSNHHSN272200900008C; the United States Government may have certain rights in the invention.
    Context of the invention
    Toll-like receptors (TLRs) are linked to the innate immune response and recognize distinct structural components that are unique to pathogens. TLR agonists are currently used in medicine, for example as adjuvants in vaccines to potentiate immune responses. The first microbial product found to be a Toll receptor agonist was LPS, a component of the bacterial membrane specific for Gram-negative bacteria, which activates TLR4. Although LPS is a potent immunomodulatory agent, its medicinal use is limited because of its extreme toxicity.
    AGP is a different class of compounds that interact with TLR4 as agonists or antagonists. AGPs include both acyclic and cyclic compounds and have been described, for example, in US 6,113,918, US 6,303,347, WO 98/50399, WO 01134617, WO 01190129, WO 02112258 and WO 2004062599. These compounds have been shown to retain significant adjuvant characteristics when formulated with antigens in vaccine compositions. PFAs also demonstrate mucosal adjuvant activity and are effective in the absence of antigen, making them attractive for prophylactic and / or therapeutic use.
    While AGPs exhibit improved toxicity profiles when compared to LPS or its derivatives, their use in pharmaceutical configurations will benefit from further improvement in toxicity profile, including less pyrogenic formulations. Summary of the invention
    In a first aspect, the invention relates to a composition comprising solid lipid nanoparticles (NLS), wherein the NLS comprise an aminoalkyl glucosaminide phosphate (AGP).
    In a second aspect, the invention relates to an immunogenic composition comprising an NLS composition as described herein and an antigen.
    In another aspect, the invention relates to a composition as described herein, or an immunogenic composition as described herein, for use in medicine, as for use in the immunization of a human subject.
    In another aspect also, the invention relates to a method for preparing a composition comprising NLS as described herein, said method comprising: a. Dissolving and mixing AGP and a lipid in an organic solvent b. Removal of the organic solvent from the mixture c. Heating the resulting mixture and mixing with an aqueous solution containing a surfactant to produce an oil-in-water mixture, and d. Performing a size reduction step, for example, using microfluidization or sonication, and e. The step allowing the composition to cool, the process optionally comprising a filtration step.
    figures
    Figure 1: cationic lipids / surfactants used in the preparation of cationic NLS.
    Figure 2: Post-secondary IgG serum titers (top), post-tertiary IgG serum titers (middle), and IgA titers in post-tertiary tracheal / vaginal lavage fluids (bottom, vaginal lavage fluid) - black bars, tracheal lavage fluid - gray bars) from study 1 of the formulation of NLS CRX-601. NLS CAT: cationic NLS, NLS-1.5%: NLS prepared at 1.5% w / v of the support lipid; NLS-5%: NLS prepared at 5% w / v of the support lipid. The values of 1 μg or 5 μg refer to the dosage / animal / vaccination of CRX-601.
    Figure 3: Post-tertiary serum IHA titers derived from the sublingual evaluation of Study 1 of NLS / chitosan formulations CRX-601. All formulations tested were able to produce functional antibodies. The NLS-5% / CRX-601 and NLS / CRX-601 + chitosan groups were able to produce antibodies at levels similar to the IM control group. Statistical analysis: One-way ANOVA analysis of variance and Tukey post-hoc test.
    Figure 4: Post-secondary IgG serum titers (top), post-tertiary IgG serum titers (in the middle), and IgA titers in post-tertiary tracheal / vaginal washings (bottom, vaginal lavage fluid) - black bars, tracheal lavage fluid - gray bars) from study 2 of the formulation of NLS CRX-601. NLS CAT: cationic NLS, NLS-1.5%: NLS prepared at 1.5% w / v of the support lipid; NLS-5%: NLS prepared at 5% w / v of the support lipid. The dose of CRX-601 is 5 μg / animal / vaccination in all SL treatment groups and 1 μg / animal / vaccination in the control IM group.
    Figure 5: Post-tertiary serum IHA titers derived from the sublingual evaluation of Study 2 of NLS / chitosan formulations CRX-601. All tested formulations were able to produce functional antibodies at levels similar to the control IM groups (except for the old NLS CAT (SA - 0.5%) BLC and NLS CAT BLC (SA - 1%). Statistical analysis: One-way ANOVA analysis of variance and Tukey post-hoc test.
    Detailed description of the invention Definitions
    The term "solid lipid nanoparticle" (NLS) when used herein refers to a carrier in the submicron size range comprising lipids and surfactants and having a solidified lipid core. Lipids are generally biocompatible and biodegradable and solid at room and body temperature.
    When used herein, the term "AGP" refers to aminoalkyl glucosaminide phosphates. This application discloses a number of examples of PMAs and includes references to other documents describing PMAs. Still other AGPs and information on AGP synthesis can be found, for example, in Johnson et al. (1999) Bioorg Med Chem Lett 9: 2273.
    The AGPs used in the present invention are preferably TLR4 agonists, i.e. compounds that activate a TLR4 receptor to produce a biological response.
    The term "mucoadhesive" when used herein refers to a tendency to adhere to mucus or mucous membranes. "Transmucosal administration" refers to the administration of a substance through mucus or mucous membranes.
    Other aspects and embodiments of the invention
    As explained above, in one aspect, the invention relates to a composition comprising solid lipid nanoparticles (NLS), wherein the NLS comprises aminoalkyl glucosaminide phosphates (AGP). AGP for use in the invention
    In preferred embodiments, the AGP used in the invention is a TLR4 agonist.
    Suitable AGPs for use in the invention are disclosed, for example, in WO 9850399, WO 0134617, WO 0212258, WO 03065806, WO 04062599, WO 06016997, WO 0612425, WO 03066065, WO 0190129 and WO 2012055981, US 2003/0199460 and US 2005/0227943, the disclosure of which is incorporated herein by reference. Such molecules have also been described in the scientific literature and patents as lipid A mimetics.
    In one aspect, AGP is an AGP in which an aminoalkyl (aglycone) group is linked by glycosidic linkages to a 2-deoxy-2-amino-α-D-glucopyranose (glucosaminide). The compounds are phosphorylated at carbon 4 or 6 on the glucosaminide ring. In addition, the compounds have three 3-alkanoyloxyalkanoyl residues comprising a primary and secondary fatty acyl chain, each carbon chain consisting of 2 to 24 carbon atoms, and preferably 7 to 16 carbon atoms. In a preferred embodiment, each primary chain contains 14 carbon atoms and each secondary chain has between 10 and 14 carbon atoms.
    In one embodiment, the AGP compounds are described by the general formula:
    Such compounds include a 2-deoxy-2-amino-α-D-glucopyranose (glucosamine) in glycosidic bond with an aminoalkyl (aglycone) group. The compounds are phosphorylated at carbon 4 or 6 on the glucosamine ring and have three alkanoyloxyalkanoyl residues. The compounds are generally described by formula I, wherein X represents an oxygen or sulfur atom, Y represents an oxygen atom or an NH group, "n", "m", "p" and " q "represent integers from 0 to 6, R1, R2, and R3 represent normal fatty acyl residues of 7 to 16 carbon atoms, R4 and R5 represent a hydrogen atom or a methyl group, R6 and R7 represent a hydrogen atom, a hydroxy, alkoxy, phosphono, phosphono-oxy, sulpho, sulpho-oxy, amino, mercapto, cyano, nitro, formyl or carboxy group and their esters and amides; R8 and R9 represent a phosphono group or a hydrogen atom. The configuration of the 3 'stereogenic centers to which normal fatty acyl residues are attached is R or S, but preferably R. The stereochemistry of the carbon atoms to which R4 or R5 are attached may be R or S.
    In a preferred embodiment, X represents an oxygen atom. The number of carbon atoms between the X heteroatom and the nitrogen atom of the aglycone is determined by the variables "n" and "m". The variables "n" and "m" can be integers from 0 to 6. In a preferred embodiment, the total number of carbon atoms between the heteroatom X is the nitrogen atom of the aglycone is from about 2 to about 6 and most preferably from about 2 to about 4. In another preferred embodiment, R8 is phosphono and R9 is hydrogen.
    The compounds are hexa-acylated, i.e., they contain a total of six fatty acid residues. The aminoalkyl glucosamine radical is acylated at the 2-amino and 3-hydroxyl groups of the glucosamine unit and at the amino group of the aglycone unit with 3-hydroxyalkanoyl residues. In formula I, these three positions are acylated with 3-hydroxytetradecanoyl radicals. The 3-hydroxytetradecanoyl residues are, in turn, substituted by normal fatty acids (R1 to R3), providing three 3-n-alkanoyloxytetradecanoyl residues or six total fatty acid groups.
    The length of the normal fatty acid chain R 1 to R 3 may be from about 7 to about 16 carbons. Preferably, R 1 to R 3 are from about 9 to about 14 carbons. The chain lengths of these normal fatty acids may be the same or different. Although only normal fatty acids are described, it is expected that unsaturated fatty acids (i.e., fatty acid radicals with double or triple bonds) substituted at R1 to R3 on the compounds produce biologically active molecules.
    Specific examples of suitable AGPs for use in the invention include CRX-527 which is disclosed in Stöver et al., (2004) J. Biol Chem 279, 6, page 4440. WO 0212258 and WO 03065806 disclose other suitable embodiments of AGP having a cyclic aminoalkyl group (aglycone) linked to a 2-deoxy-2-amino-α-D-glucopyranose (glucosaminide), commonly referred to as "cyclic AGP". The general reference to AGP here includes both cyclic and non-cyclic AGP.
    The cyclic PFAs have three 3-alkanoyloxyalkanoyl residues comprising a primary and secondary fatty acyl chain, each carbon chain consisting of 2 to 24 carbon atoms, and preferably 7 to 16 carbon atoms. In a preferred aspect, each primary chain contains 14 carbon atoms and each secondary carbon chain has between 10 and 14 carbon atoms per chain.
    Cyclic PFAs are described by the general formula II:
    These compounds include 2-deoxy-2-amino-β-D-glucopyranose (glucosamine) linked by glycosidic linkages to a cyclic aminoalkyl group (aglycone). The compounds are phosphorylated at the 4 or 6 position of the glucosamine ring and acylated with alkanoyloxytetradecanoyl residues on the aglycone nitrogen and the 2 and 3 positions of the glucosamine ring. The compounds are generally described by formula (II): and their pharmaceutically acceptable salts, wherein X is -O- or NH- and Y is -O- or -S-; R1, R2, and R3 each independently represent a C2-C24 acyl group, including saturated, unsaturated and branched acyl groups; R4 is -H or -PO3R7R8, where R7 and R8 are each independently H or C1-C4 alkyl; R5 is -H, -CH3 or -PO3R9R10, wherein R9 and R10 are each independently selected from -H and C1-C4alkyl; R6 is independently selected from H, OH, C1-C4 alkoxy, -PO3R11R12, -OPO3R11R12, -SO3R11, -OSO3R11, -NR11R12, -SR11, -CN, -NO2, -CHO, -CO2R11, and -CONR11R12 wherein R11 and R12 are each independently selected from H and C1-C4 alkyl, wherein "1-3" and "**" represent chiral centers; wherein the indices n, m, p and q each independently represent an integer from 0 to 6, provided that the sum of p and m is from 0 to 6.
    In some embodiments, the cyclic AGP compound contains -O- in X and Y, R4 is PO3R7R8, R5 and R6 are H, and n, m, p, and q are integers from 0 to 3. In a more preferred embodiment, R7 and R8 are -H. In an even more preferred embodiment, the index n is 1, the index m is 2, and the indices p and q are 0. In an even more preferred embodiment, R 1, R 2, and R 3 represent tetradecanoyl residues. In an even more preferred embodiment, * 1 to 3 are in the R configuration, Y is in the equatorial position, and ** is in the S (N - [(R) -3-tetradecanoyloxytetradecanoyl] - (S) configuration. 2-pyrrolidinomethyl-2-deoxy-4-O-phosphono-2 - [(R) -3-tetradecanoyloxytetradecanoylamino] -3-O - [(R) -3-tetradecanoyloxytetradecanoyl] -β-glucopyranoside and its pharmaceutically acceptable salts) .
    Preferred cyclic structures include: [Ill]
    [IV-a]
    M
    Formula V represents CRX 590.
    In other embodiments, the AGP comprises one or more primary and / or secondary lipid groups linked to an ether rather than an ester. In such embodiments, R1-R3 represent straight chain alkyl groups and not acyl groups, making R10-, R2O- and R30- alkoxy rather than alkanoyloxy groups and attachment of the primary acyl chain a ether bond rather than ester. In the case of a primary ether-linked lipid group, the 3-alkanoyloxyalkanoyl residue attached to the 3-hydroxy group of the glucosamine unit is replaced by either a 3-alkanoyloxyalkyl radical or a 3-alkoxyalkyl radical, making the fixation of the primary lipid group at position 3 of glucosamine an ether bond rather than ester. A general formula for ethers is that of formula IV of WO 2006016997.
    An example of a preferred compound is CRX-601 (formula VI). In preferred embodiments, the composition comprises between 0.1 and 1.5% (w / v) CRX-601, more preferably between 0.1 and 1%, even more preferably between 0.2. and 0.5%, such as between 0.2 and 0.25%, or between 0.5 and 1%.
    [VI]
    In other embodiments, the AGP molecule may have a different number of carbons in the primary and / or secondary chains of the molecule. Such compounds are disclosed in WO 04062599 and WO 06016997. As with the other AGP, each carbon chain can be comprised of 2 to 24 carbon atoms, and preferably 7 to 16 carbon atoms. In a preferred embodiment, each primary chain contains 14 carbon atoms and each secondary carbon chain has between 10 and 14 carbon atoms per chain.
    Such compounds are represented by the following structures VII, VIII, IX and X: wherein X is selected from the group consisting of O and S at the axial or equatorial position; Y is selected from the group consisting of O and NH; n, m, p and q represent integers from 0 to 6; R1, R2 and R3 are the same or different and represent fatty acyl residues having from 1 to 20 carbon atoms and wherein one of R1, R2 or R3 optionally represents a hydrogen atom; R4 and R5 are the same or different and are selected from the group consisting of H and a methyl group; R6 and R7 are the same or different and are selected from the group consisting of H, hydroxy, alkoxy, phosphono, phosphono-oxy, sulfo, sulfo-oxy, amino, mercapto, cyano, nitro, formyl and carboxy and their esters and amides; R8 and R9 are the same or different and are selected from the group consisting of phosphono and H, and at least one of R8 and R9 is phosphono; R10, R11 and R12 are independently selected from unsaturated linear chain saturated aliphatic groups having 1 to 10 carbon atoms; or a pharmaceutically acceptable salt thereof.
    [VIII]
    wherein X is selected from the group consisting of O and S at the axial or equatorial position; Y is selected from the group consisting of O and NH; n and m are 0; R1, R2 and R3 are the same or different and represent fatty acyl residues having from 1 to 20 carbon atoms and wherein one of R1, R2 or R3 optionally represents a hydrogen atom; R4 is selected from the group consisting of H and a methyl group; p is 1 and R6 is COOH or p is 2 and R6 is OPO3H2; R8 and R9 are the same or different and are selected from the group consisting of phosphono and H, and at least one of R8 and R9 is phosphono; and R10, R11 and R12 are independently selected from unsaturated linear chain saturated aliphatic groups having from 1 to 10 carbon atoms; or a pharmaceutically acceptable salt thereof.
    [IX]
    wherein X is selected from the group consisting of O and S at the axial or equatorial position; Y is selected from the group consisting of O and NH; n, m, p and q represent integers from 0 to 6; R 1, R 2 and R 3 are the same or different and represent linear chain saturated aliphatic groups (i.e., straight chain alkyl groups) having from 1 to 20 carbon atoms and wherein one of R 1, R2 or R3 optionally represents a hydrogen atom; R4 and R5 are the same or different and are selected from the group consisting of H and a methyl group; R6 and R7 are the same or different and are selected from the group consisting of H, hydroxy, alkoxy, phosphono, phosphono-oxy, sulfo, sulfo-oxy, amino, mercapto, cyano, nitro, formyl and carboxy and their esters and amides; R8 and R9 are the same or different and are selected from the group consisting of phosphono and H, and at least one of R8 and R9 is phosphono; R10, R11 and R12 are independently selected from unsaturated linear chain saturated aliphatic groups having 1 to 11 carbon atoms; or a pharmaceutically acceptable salt thereof.
    The general formula may also include a group R 5, at the same position as that shown in formula VII above, wherein R 5 is selected from the group consisting of H and a methyl group.
    (IV)
    Yet another type of compound of this invention has formula (IV) above: wherein Y is now attached in the form of oxygen; X is selected from the group consisting of O and S at the axial or equatorial position; n and m are 0; R1, R2 and R3 are the same or different and represent fatty acid residues having from 1 to about 20 carbon atoms and wherein one of R1, R2 or R3 optionally represents a hydrogen atom; R4 is selected from the group consisting of H and a methyl group; p is 0 or 1 and R6 is COOH, or p is 1 or 2 and R6 is OPO3H2; R8 and R9 are the same or different and are selected from the group consisting of phosphono and H, and at least one of R8 and R9 is phosphono; and R10, R11 and R12 are independently selected from unsaturated linear chain saturated aliphatic groups having from 1 to 10 carbon atoms; or a pharmaceutically acceptable salt thereof. These compounds thus comprise two acylated chains and an unacylated ether chain. Other suitable AGP structures such as CRX-524 are disclosed in Cluff et al. (2005) Infection and Immunity 73: 3044.
    Methods of making AGP are also disclosed, for example, in WO 0612425.
    Lipids and surfactants suitable for use in the invention
    As explained above, NLSs are supports in the submicron size range comprising lipids and surfactants and having a solidified lipid core. The lipids used are generally biocompatible and biodegradable and solid at room and body temperature.
    Many lipids can be used for the preparation of NLS, and examples of suitable lipids are given, for example, in Table 8.1 of Svilenov and Tzachev (2014) Nanomedicine Chapter 8, page 187 (ISBN (eBook): 978-1 -910086-018: Publisher: One Central Press (OCP)) (incorporated herein by reference).
    Preferred lipids for use in the present invention are glycerol behenates, i.e., glycerol monobhenate, glycerol dibehenate, and glycerol tribehenate. Also preferred are mixtures of these three types of behenates. Preferred concentrations are 1 to 5% (w / v), such as 1 to 4%, for example, 1.5% to 3%. A preferred lipid is Compritol 888 ATO (glyceryl dibehenate, European Pharmacopoeia [EP], glyceryl behenate, National Formulary [NF]), which is a hydrophobic mixture of mono- (12-18% w / w), di- ( 45 to 54% w / w) and tri- (28 to 32% w / w) glycerol behenate with a melting point in the range of 69 to 74 ° C and with a hydrophilic lipophilic balance (BHL) "2.
    In addition, many surfactants can be used for the preparation of NLS, and examples of suitable surfactants are given, for example, in Table 8.2 of Svilenov and Tzachev (2014).
    Nanomedicine Chapter 8, page 187 (ISBN (eBook): 978-1910086-01-8: Publisher: One Central Press (OCP)) (incorporated herein by reference). A preferred surfactant is polysorbate 80 (also known as Tween 80). Preferably, the composition comprises from 0.5 to 3% (v / v) of polysorbate 80.
    mucoadhesion
    In preferred embodiments, the compositions of the invention are mucoadhesive. NLS can be made more mucoadhesive by combination with or incorporation of mucoadhesive compounds.
    In some embodiments, the NLSs are cationic. The cationic NLSs may, for example, potentially allow mucoadherence through their electrostatic interaction with a polyanionic mucin coating on the sublingual mucosa. Preferably, the zeta potential, for example, as measured by dynamic light scattering, is 20 mV or greater, for example 20 to 30 mV, or greater. NLS can be made more cationic by combination with or incorporation of cationic lipids and / or cationic surfactants. Thus, in certain embodiments, the NLSs in the composition of the invention comprise a cationic lipid and / or a cationic surfactant. In one embodiment, the NLSs comprise a compound selected from the group consisting of stearylamine (octadecylamine) (SA), dimethyl-dioctadecylammonium bromide (DDAB), or cetyltrimethylammonium bromide (CTAB). In another embodiment, the composition comprises 0.25 to 1.5% of SA, DDAB or CTAB, such as 0.25 to 0.55%, for example, 0.5% of SA, DDAB or from CTAB.
    In some embodiments, the NLS are made mucoadhesive by addition of a mucoadhesive agent. Preferred mucoadhesive agents are chitosan derivatives, such as alkylated chitosans. A particularly preferred mucoadhesive agent is methylglycol chitosan, which is preferably used in a concentration of 0.2 to 20 mg / ml, more preferably 2 to 10, such as 2 to 5 mg / ml.
    Other Preferred Properties of the Compositions of the Invention
    In preferred embodiments, the composition of the invention has a size distribution below the filterability limit for sterilization, to avoid material loss during filtration sterilization. Thus, in a preferred embodiment, the average size of the NLS in the composition is between 30 and 200 nm, such as between 30 and 150 nm, as between 30 and 100 nm. In addition, in a preferred embodiment, the polydispersity index is less than 0.75, as less than 0.5, as less than 0.4. The polydispersity index and size can be measured, for example, using a Malvern Zetasizer.
    In another preferred embodiment, the composition remains a stable homogeneous composition and does not become a semi-solid gel for at least 24 hours following its preparation.
    Processes for preparing the compositions of the invention
    NLS preparation methods are described in the art. Such methods have been described, for example, in Svilenov and Tzachev (2014) Nanomedicine Chapter 8, page 187 (ISBN (eBook): 978-1-910086-01-8: Publisher: One Central Press (OCP)) (incorporated here in reference).
    As explained above, in yet another aspect, the invention relates to a method for preparing a composition comprising NLS as described herein, said method comprising: a. Dissolving and mixing AGP and a lipid in an organic solvent b. Removal of the organic solvent from the mixture c. Heating the resulting mixture and mixing with an aqueous solution containing a surfactant to produce an oil-in-water mixture, and d. Performing a size reduction step, for example, using microfluidization or sonication, and e. The step allowing the composition to cool.
    The process may comprise a filtration step.
    Uses of the compositions of the invention
    The compositions of the invention can be used in medicine. The compositions may be used by themselves or in combinations with other substances, for example, in combination with an antigen for use in immunization.
    Methods of preventing and treating diseases, for example, infectious diseases, autoimmune diseases or allergies, by administering AGP in the absence of exogenous antigen are disclosed in WO 03066065 and WO 0190129. The compositions of the invention can be used in such methods.
    As mentioned above, the compositions of the invention can also be used as adjuvants in a vaccination. Thus, in another aspect, the invention relates to an immunogenic composition comprising an NLS composition as described herein and an antigen. Vaccine preparation is generally described in Vaccine Design ("The Subunit and Adjuvant Approach" (Eds Powell M.F. & Newman M.J.) (1995)
    Plenum Press New York). The compositions of the invention are suitable for combination with many different antigens including, for example, proteins, nucleic acids and polysaccharides of various origins. In one embodiment, the antigen is an antigen of the influenza virus, such as hemagglutinin.
    The immunogenic compositions or compositions of the invention may be used to protect or treat a mammal by means of systemic or transmucosal administration. Such administrations may include intramuscular, intraperitoneal, intradermal or subcutaneous injection; or by a transmucosal administration to the oro-alimentary, respiratory, urogenital systems. Administration by a transmucosal route, such as sublingual administration is preferred. The composition of the invention may be administered as a single dose, or multiple doses.
    The subject doses of the composition described herein are generally in the range of about 0.1 μg to 10 mg of AGP per administration. Preferred mucosal or local doses are in the range of 1 μg to 10 mg of AGP per administration, and most typically 10 μg to 1 mg of AGP.
    The disclosure will be further described with reference to the following non-limiting examples.
    Examples
    Example 1 - Preparation of NLS with CRX-601
    CRX-601 (2% w / v) stock solutions and
    Compritol 888 ATO (glyceryl behenate: a mixture of glycerol mono-, di- and tri-behenate, 5% w / v) was prepared in tetrahydrofuran (THF). Variable amounts of these stock solutions were added to a glass vial to achieve the desired concentrations of CRX-601 and Compritol 888 ATO in the final aqueous preparation (shown in Table 1). The organic solvent was removed by evaporation on a rotary evaporator and further under vacuum at elevated pressure for 12 h. The lipid mixture of CRX-601 thus obtained was heated to> 80 ° C, and an aqueous solution containing Tween 80 (0.25 to 2% v / v) was added to it at the same temperature while mixing at speed. high (8000 to 12000 rpm). The resulting oil-in-water emulsion was further sonicated (15 to 25 min) using a sonicator with a probe on a hot plate maintained at a temperature> 80 ° C. The NLS composition was then allowed to solidify at room temperature and was aseptically filtered using a 0.22 μm filter in a sterile depyrogenic container. The representative mean particle sizes and zeta potential values of the resulting formulations measured by dynamic light scattering are shown in Table 1. The aminoalkyl glucosaminide 4-phosphate (AGP) CRX-601 used in this work was synthesized as it has been previously described (Bazin et al (2008) Bioorg & Med Chem Lett 18: 5350 (CRX-601 is the ether analogue of CRX-527)) and purified by chromatography (at purity> 95%). CRX-601, either in the starting material or in the final product was quantified by a conventional reverse phase HPLC analytical method.
    Formulations with more than 5% w / v Compritol and> 1% w / v CRX-601 produce nanoparticles with a size distribution approaching the filterability limit for sterilization, and are associated with a loss of material during sterilization by filtration.
    Representative Parameters of Formulations for CRX-601 NLS
    Comparative Example - Stearic acid was explored for the preparation of NLS (instead of glyceryl behenate). Stearic acid concentrations of 1.5%, 3.0% and 6.0% were used. CRX-601 has been targeted at 1, 2 and 3 mg / ml. Clear homogeneous formulations were observed initially just after preparation, but after overnight storage they turned into semi-solid gels.
    Example 2 - Preparation of cationic CRX-601 / NLS
    NLS formulations based on glyceryl behenate are anionic in zeta potential. The cationic NLSs were prepared by incorporation of cationic lipids / surfactants (shown in Figure 1) into the glyceryl behenate NLS. Cationic NLSs may potentially allow mucoadherence through their electrostatic interaction with the polyanionic mucin coating on the sublingual mucosa. The formulations were prepared as in Example 1, but with the addition of 0.1 to 1% w / v of stearylamine (octadecylamine) (SA), dimethyldioctadecylammonium bromide (DDAB), or cetyltrimethylammonium bromide ( CTAB). The representative mean particle sizes and zeta potential values of the resulting formulations measured by dynamic light scattering are shown in Table 2.
    Summary of formulation parameters for cationic NLS
    NT: not tested
    Only the highest cationic lipid concentration tested of 0.5% gave cationic NLS, and not the lower concentrations, probably because of the excess anionic charge contributed by CRX-601 compared to NLS. Higher% by weight of DDAB of 0.6, 0.8 and 1% was further tested but in all cases an IPD> 0.4 with a multimodal size distribution was obtained. Therefore, cation based NLSs with 1.5% glyceryl behenate were selected for initial analysis in a sublingual study in mice.
    Example 3 Preparation of CRX-601 / NLS Coated with Methylglycol Chitosan
    Chitosan methylglycol (trimethylammonium glycol chitosan iodide) was dissolved in 10 mM HEPES or 10 mM HEPES-physiological saline pH 7.2 to produce concentrations of 2 and 10 mg / ml. The solutions were aseptically filtered using a 0.22 μη filter in a sterile depyrogenous container. The formulations of Examples 1 and 2 were aseptically mixed with varying volumes of methylglycol chitosan solution to produce chitosan methylglycol concentrations in the range of 1 to 10 mg / ml. The representative mean particle sizes and zeta potential measured by dynamic light scattering are presented in Table 3. In some formulations combinations, where the IPD values exceed 0.3 (bimodal or multimodal size distribution), the Sonication of the formulation on a water bath sonicator for 10 min was used to reduce the IPD values and make the size distribution unimodal. Overall, the data indicate some increase in particle size and a reversal of the zeta potential (positive net potential from a net negative potential) exceeding approximately 1 mg / ml of chitosan methylglycol, consistent with the coating of the surface with methylglycol chitosan. At concentrations above a certain threshold, it is expected that methylglycol chitosan saturates the surface of the NLS, with a free excess in solution. Summary of Formulation Parameters for Methylglycol Chitosan-coated NLS-CRX-601
    NT: untested, U: unimodal, B: bimodal, M: multimodal
    Most combinations of formulations were unimodal in the size distribution. The 1.5% -NLS formulations were less than 100 nm, while the 6% -NLS formulations were> 200 nm. No precipitation / aggregation was observed at any of the tested MGC concentrations indicating incorporation of CRX-601 into the NLS (precipitation is generally observed with free aqueous CRX-601 in the neutral zone) .
    Comparative Example: An Alternative Formulation Process Based on the Sandri et al. Study, [J of Microencapsulation, 2010, 27 (8), 735-746], which describes the preparation of NLS directly in a solution of chitosan (instead of water used previously) was also attempted. Only NLS blanks (without CRX-601) were prepared, with representative mean particle size measurements and zeta potentials shown in Table 4.
    Table 4 Summary of formulation parameters for chitosan methylglycol-associated NLS prepared using the method of Sandri et al.
    U: unimodal, B: bimodal, M: multimodal, NT: not tested
    Most formulations had significantly higher IPD values with this method (Table 4), therefore this method was not considered a suitable method for the preparation of CRX-601 / NLS.
    Example 4 - Pyrogenicity test in rabbits
    The pyrogenicity test is used here as a proxy measure for the incorporation of CRX-601 into the NLSs from Examples 1 to 3 and as a measure of their stability in a biological medium. The test was conducted at Pacific Biolabs (Hercules, CA) according to their SOP (Standard Operating Procedure) 16E-02, which follows the procedures outlined in USP <151>. The 1.5% -NLS / CRX-601 formulation showed no pyrogenicity at a concentration of 25 ng CRX-601 / kg body weight of the animal. This lack of pyrogenicity up to 25 ng / kg corresponds to an improvement of at least 10-fold compared to free CRX-601 (maximal non-pyrogenic dose of 2 ng / kg), and indicates> 90% incorporation of CRX -601 in the NLS. Increases in individual temperature from three rabbits per test are shown in Table 5.
    Representative measures of the rabbit pyrogenicity test for some of the formulations described in Examples 1, 2, and 3. The values in parentheses represent the maximum temperature variation for three animals during the analysis period.
    NT: not tested; * NP: non-pyrogenic response, P: pyrogenic response. The test is considered NP if none of the rabbits show an individual temperature increase of 0.5 ° C or more above its respective control temperature at any point.
    Example 5 Compatibility of influenza virus antigen with NLS formulations
    The antigenic compatibility of the split influenza virus (A / Victoria / 210/2009 H3N2, lot # AS20APA-051-02) was tested by mixing with the NLS formulations at the concentrations required for dose administration to the animals. . The resulting combination was tested for any precipitation by visual observation and turbidimetry (no decrease in% of light transmitted at 600 nm). No significant precipitation or variation of% T was observed with any of the formulations.
    Example 6 Sublingual Vaccination of Mice and Determination of Specific Antibody Responses
    Female BALB / c mice (6 to 8 weeks old) obtained from Charles River Laboratories (Wilmington, Mass.) Were used for these studies. In mice anesthetized by intraperitoneal (i.p.) administration of ketamine (100 mg / kg) and xylazine (10 mg / kg), the vaccine was administered sublingually (5-6 μg). All mice were vaccinated on days 0, 21 and 42 with the 5 gg of CRX-601 in the formulation of the NLS mixed with 1 or 1.5 g of HA / mouse using the influenza A antigen. Victoria / 210/2009 H3N2. The serum was harvested using a retro-orbital puncture at day 36 (14jp2) under anesthesia, at day 56 (14jp3) the mice were sacrificed and a final harvest of the vaginal washings, tracheal washings and serum was collected. All animals were used according to guidelines established by the U.S. Department of Health and Human Services Office of Animal Welfare Laboratory and the Institutional Animal Care and Use Committee at GSK Biologicals, Hamilton, Montana.
    The specific antibody responses were measured by two independent immunological tests, the enzyme linked immunosorbent assay (ELISA) and the influenza virus haemagglutinin (HI) inhibition test.
    The ELISA was performed using 96-well plates sensitized with the split influenza virus (Nunc Maxisorp) and detecting bound immunoglobulins from the added samples of serum or tracheal lavage fluid or vaginal lavage fluid. using goat anti-mouse IgG, IgG1, IgG2a or IgA bound to peroxidase. This was followed by the addition of a chromogen specific to the enzyme, which produced a color intensity directly proportional to the amount of IgG / IgA specific anti-influenza virus contained in the serum. The optical density was read at 450 nm.
    The HI test was performed by evaluating the inhibition of chicken or rooster erythrocytes after exposure to the influenza virus in the presence of mouse sera. The reverse of the last dilution of the influenza virus that produced complete or partial agglutination of erythrocytes was used to calculate the HI titre and expressed in HA units / 50 μl of serum.
    Example 7 Sublingual Vaccination of Mice with NLS Formulations (NIH # 157, 165, 170, 171)
    Based on the studies described above with various NLS formulations, the formulations described in Table 6 below were selected, prepared, characterized, sterilized by filtration, and quantified by RP-HPLC for a sublingual study in the mouse (study 1). Summary of formulations prepared for sublingual study in mice (Study 1)
    The mice were vaccinated using the procedure set forth in Example 6 with formulations from Table 6. Post-secondary antibody serum titers were superior with CRX-601 NLS formulations compared to vehicle controls or liposomal formulation of CRX-601 DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) (Figure 2, top panel), with the exception of cationic NLS CRX-601, which were not better than the witnesses in this particular test. Mice vaccinated with intramuscular administration of CRX-601 + fractional influenza virus antigen are also included as a positive control. For most of the groups, serum titers of post-tertiary antibodies were stimulated compared to post-secondary titers (Figure 2, middle panel). The pattern of titers was somewhat different compared to post-secondary, with 5 μg of CRX-601 in the 5% -NLS and 5 μg of CRX-601 in the 1.5% -NLS coated with chitosan providing the best global titles. Surprisingly, the white of the cationic NLS also provided a high humoral immunity that could be due to the adsorption of the anionic antigen of the influenza virus on the surface of these nanoparticles, helping their transport through the mucosa SL. Influenza virus IgA titers were readily detected in mucosal lavage fluids of SL vaccinated mice with split influenza virus and NLS formulations or CRX-601 controls (Figure 2).
    HI serum titers were highest in mice vaccinated with the 1.5% -NLS coated with chitosan in the 5 μg CRX-601 group and significantly higher than the CRP-601 DOPC liposome group (Figure 3). There was no significant difference between the other SL treatment groups. The study was followed by a 2 SL study, with formulations described in Table 7, with vaccinated mice using the procedure set forth in Example 6. The follow-up study included additional groups with pooled mice. Cationic NLS, an additional ratio of NLS-1.5% / CRX-601 + MGC, and an additional group with formulations of the formulation NLS-5% / CRX-601 + MGC.
    NLS formulations prepared for a sublingual study in mice. All formulations were prepared at a concentration of CRX-601> 1 mg / ml and were further diluted to 1 mg / ml prior to transfer to immunology.
    * Although the IPD value is much higher than previously observed, the formula was clear, indicated no aggregation, and therefore was found to be acceptable for another use. NT: not tested
    The results of Study 2 are summarized in Figure 4. In general, serum IgG responses were higher when chitosan was present in the NLS formulation, compared to the corresponding NLS formulation without chitosan (Figure 4). . 1.5% and 5% -NLS CRX-601 formulations with chitosan exhibited higher IgG responses than the IM control groups. Notably, NLS CAT formulations without CRX-601 triggered significant levels of IgG (Figure 4). Trends in post-secondary serum antibodies were similar to post-tertiary, although with lower rates. All sublingual vaccine groups demonstrated significantly higher tracheal and vaginal lavage fluid IgA titers versus IM positive controls, with titers in the vaginal lavage fluid surpassing the titers in the tracheal lavage fluid (Figure 4) . The 1.5% NLS CRX-601 with 5 mg / ml chitosan triggered the largest IgA response compared to other formulations of CRX-601. In particular, NLS CAT formulations without CRX-601 (BLC) also triggered significant levels of IgA.
    Based on the results of this study, NLS formulations, particularly with 5-10 mg / ml chitosan, are promising lead candidates for SL vaccine formulations. In addition, NLS CAT formulations without CRX-601 triggered significant levels of IgG and IgA.
    Post-tertiary HI titers corresponded to post-tertiary IgG titers. All tested formulations were able to produce functional antibodies at levels generally similar to the IM control groups (except for the old NLS CAT (SA-0.5%) Blc and NLS CAT (SA-1%) BLC). (Figure 5).
    权利要求:
    Claims (25)
    [1]
    A composition comprising solid lipid nanoparticles (NLS), wherein the NLS comprises an aminoalkyl glucosaminide phosphate (AGP).
    [2]
    The composition of claim 1, wherein the AGP is a TLR4 agonist.
    [3]
    3. The composition according to any one of the preceding claims, wherein the AGP is CRX-601.
    [4]
    4. Composition according to claim 3, the composition comprising from 0.45 to 2.5 mg / ml of CRX-601.
    [5]
    5. The composition according to any one of the preceding claims, wherein the NLS comprise glyceryl behenate.
    [6]
    The composition of claim 5, the composition comprising 1 to 5% (w / v) glyceryl behenate, such as 3% (w / v) glyceryl behenate.
    [7]
    The composition of any preceding claim, wherein the NLS comprises polysorbate 80.
    [8]
    8. Composition according to Claim 7, the composition comprising from 0.5 to 3% (v / v) of polysorbate 80.
    [9]
    9. A composition according to any one of the preceding claims wherein the NLS are cationic.
    [10]
    A composition according to any one of the preceding claims wherein the NLS comprises a cationic lipid and / or a cationic surfactant.
    [11]
    A composition according to any one of the preceding claims wherein the NLS comprises a compound selected from the group consisting of SA, DDAB or CTAB.
    [12]
    12. Composition according to claim 11, the composition comprising 0.25 to 1.5% of SA, such as 0.25 to 0.55% of SA, for example 0.5% of SA or 0.25 to 1, 5% DDAB, such as 0.25 to 0.55% DDAB, for example, 0.5% DDAB.
    [13]
    13. A composition according to any one of the preceding claims, wherein the zeta potential is 20 mV or greater, for example, between 20 and 30 mV.
    [14]
    14. A composition according to any one of the preceding claims, the composition further comprising a mucoadhesive agent, such as methylglycol chitosan.
    [15]
    15. Composition according to claim 14, the composition comprising from 0.2 to 5 mg / ml of methylglycol chitosan, preferably from 2 to 5 mg / ml of methylglycol chitosan.
    [16]
    16. A composition according to any one of the preceding claims, wherein the average size of the NLS in the composition is between 30 and 200 nm, such as between 30 and 150 nm, as between 30 and 100 nm.
    [17]
    17. A composition according to any one of the preceding claims, wherein the polydispersity index of the NLS is less than 0.75, as less than 0.5, as less than 0.4.
    [18]
    An immunogenic composition comprising an NLS composition according to any one of the preceding claims and an antigen.
    [19]
    The immunogenic composition of claim 18, wherein the antigen is an antigen of the influenza virus, such as haemagglutinin.
    [20]
    20. A composition according to any of claims 1 to 17, or an immunogenic composition according to claim 18 or 19 for use in medicine.
    [21]
    21. A composition according to any one of claims 1 to 17, or an immunogenic composition according to claim 18 or 19, for use in the immunization of a human subject.
    [22]
    22. An immunogenic composition or composition according to claim 21, wherein the composition is administered by a transmucosal route, such as sublingual administration.
    [23]
    A process for preparing a composition comprising NLSs according to any one of the preceding claims, said process comprising: a. Dissolving and mixing AGP and a lipid in an organic solvent b. Removal of the organic solvent from the mixture c. Heating the resulting mixture and mixing with an aqueous solution containing a surfactant to produce an oil-in-water mixture, and d. Performing a size reduction step, for example, using microfluidization or sonication, and e. The step allowing the composition to cool.
    [24]
    24. The method of claim 23, comprising the further step of mixing with a solution containing a mucoadhesive agent, such as a solution containing methylglycol chitosan.
    [25]
    25. The method of claim 23 or 24 comprising the additional step of mixing with an antigen.
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    同族专利:
    公开号 | 公开日
    BE1024228A1|2017-12-15|
    US20180318415A1|2018-11-08|
    EP3386540A1|2018-10-17|
    WO2017097783A1|2017-06-15|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US6113918A|1997-05-08|2000-09-05|Ribi Immunochem Research, Inc.|Aminoalkyl glucosamine phosphate compounds and their use as adjuvants and immunoeffectors|
    US6303347B1|1997-05-08|2001-10-16|Corixa Corporation|Aminoalkyl glucosaminide phosphate compounds and their use as adjuvants and immunoeffectors|
    AU5214200A|1999-05-20|2000-12-12|Pharmasol Gmbh|Stability, biocompatibility optimized adjuvant for enhancing humoral and cellular immune response|EP3820439B1|2018-09-11|2021-09-29|Lead Biotherapeutics Ltd.|Mucoadhesive dispersion nanoparticle system and method for production the same|
    EP3886901A1|2018-11-29|2021-10-06|GlaxoSmithKline Biologicals S.A.|Methods for manufacturing an adjuvant|
    法律状态:
    2018-02-15| FG| Patent granted|Effective date: 20171221 |
    2021-09-03| MM| Lapsed because of non-payment of the annual fee|Effective date: 20201231 |
    优先权:
    申请号 | 申请日 | 专利标题
    US201562264506P| true| 2015-12-08|2015-12-08|
    US62264506|2015-12-08|
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