PARTICLES FOR DRUG DELIVERY

专利摘要:
The present invention relates to drug delivery particles that can prevent interaction between a biologically active cargo within the particles and components of an aqueous environment in which said particles are present. The particles are pH sensitive so that above a threshold pH level the biologically active cargo becomes accessible to the immediate environment. Therefore, such particles are useful for stably storing a biologically active cargo in an aqueous composition containing components that otherwise deleteriously interact with the cargo, and releasing the cargo to mediate a biological effect in the cargo. body of an animal, such as a human being, to which the composition is administered. Also provided are compositions comprising such particles, as well as methods of making and using such particles and compositions.
公开号:BE1024210B1
申请号:E2017/5134
申请日:2017-03-06
公开日:2017-12-18
发明作者:Abdelatif Elouahabi；Patrick Pohlhaus；Laurent Strodiot；Ashley Galloway；Jin Lee；Michele R. Stone
申请人:Glaxosmithkline Biologicals Sa；Liquidia Technologies Inc.；
IPC主号:

专利说明:

PARTICLES FOR THE DELIVERY OF MEDICAMENTS Field of the invention
The present invention relates to (micro- or nano-) particles which comprise a biologically active cargo, which cargo is, as a consequence of its presence within such particles, protected from undesirable interactions with substances co-formulated with the particles as in a parenteral formulation. Cargo, such as a drug or other therapeutically relevant agent, can in this way be stably stored in a parenteral formulation, with the release of the cargo from particles occurring only after administration. These particles have particular utility in vaccine compositions.
Context of the invention
Many medicinal compositions, such as (therapeutic) drugs or (prophylactic) vaccines, are combination products that contain two or more biologically active components, for example, pharmaceutical components or active antigens. Such compositions may have a synergistic effect, or they may offer advantages such as increased adherence to a therapeutic regimen, for example, through a reduction in the total number of administrations, especially in the case of calendars. immunization in pediatrics.
The respective biologically active components may have associated with them other components such as, in the case of vaccines, adjuvants, and the composition as a whole will contain pharmaceutically acceptable formulation excipients, often in an aqueous formulation. It is known that when they are co-formulated (i.e., formulated together in a single composition as a parenteral formulation), a biologically active constituent can interact with another biologically active component, or with an associated component such as an adjuvant or an excipient or even the water present in the formulation. Such an interaction may have a deleterious impact on the biological effect mediated by at least one of the interacting biologically active constituents (such an impact being "deleterious" with respect to the biological effect that such a biologically active constituent mediates. it is formulated alone, that is to say, as the only biologically active constituent).
In the case of vaccines, such deleterious interaction may be manifested as a physical or biochemical incompatibility, such as an effect on the stability of the biologically active constituent, and / or as an in vivo phenomenon having an impact. negative on the immune response triggered by the constituent ("immunological interference"). For example, in the case of combined pediatric vaccines containing Haemophilus influenzae type b ("Hib") polysaccharide conjugated to a carrier protein (such as tetanus toxoid, "TT"), together with other antigens adsorbed on an aluminum hydroxide adjuvant (such as diphtheria toxoid, tetanus toxoid and cell-free pertussis antigens, "DTPa"), the Hib antigen is lyophilized and packaged separately from the aqueous liquid formulation containing DTPa / aluminum hydroxide - this is the case, for example, in Infanrix ™ Hexa (GSK Vaccines). There are two reasons for this: the first, because the part of the polysaccharide derived from Hib (polyribosylribitol, "PRP"), Conjugated Hib antigen is labile to degradation by hydrolysis when in an aqueous formulation (i.e. Hib undergoes a physical / biochemical interaction with water molecules; duisant stability); and the second, because PRP can interact with aluminum hydroxide to form a network of particles ("flocculation") that is thought to mask epitopes in the recipient's immune system (i.e. Hib has immunological interference when formulated in the presence of aluminum hydroxide). In the case of vaccines such as Infanrix ™ Hexa, partitioning the vaccine components between an aqueous liquid component and a lyophilized component, which are reconstituted extemporaneously at the time of administration, solves the above problems. However, this leads to a two-part vaccine requiring a reconstitution step by the medical staff administering the vaccine. A one-part liquid vaccine with all components in a single container would provide benefits such as simplification of filling / conditioning, transport / storage, and administration.
Therefore, there is a desire for solutions to the problem of how to physically, biochemically, and / or immunologically combine incompatible components of combined ("multivalent") medicinal compositions into one-part aqueous liquid compositions that can be packaged and stored in containers, while avoiding the deleterious consequences of such incompatibilities. SUMMARY OF THE INVENTION The invention is based on the inventors' discovery that particles for drug delivery can be made, which particles contain a "cargo" (e.g., a biologically active constituent of a medicinal composition). In the context of a composition comprising such particles, the particles may protect the cargo contained within potentially deleterious interactions with the substances constituting the composition external to the particle during storage. In addition, the particles may "release" said cargo in response to the administration such that the cargo is then free to exert its effect within the body of the recipient subject. In particular, the particles are created to be pH sensitive so that the matrix of particles is insoluble at the pH of the final medicinal composition in which the particles are stored but soluble at the relatively high pH of the tissue of the site. injection of the subject.
Thus, in one aspect, the invention provides a plurality of pH sensitive drug delivery particles comprising a biologically active cargo within a matrix, wherein said particles are triggered to release said cargo when present in a carrier. an aqueous environment having a pH greater than the pH of an aqueous environment in which said particles are present (i.e., stored) before being so triggered.
In another aspect, the invention provides a plurality of pH sensitive drug delivery particles comprising a biologically active cargo within a matrix, wherein the amount of cargo released from said plurality of particles when they are present in an aqueous environment for at least 6 months at a sub-physiological pH of not more than 30% by weight of the total quantity of the cargo, and in which when the said particles are subjected to a physiological pH trigger (greater than a threshold pH) the amount of cargo released in 24 hours or less is not less than 50% by weight of the total quantity of the cargo.
In another aspect, the invention provides a composition, an immunogenic composition or a vaccine comprising such a plurality of pH-sensitive drug delivery particles. In another aspect, the invention provides a vial or parenteral delivery device containing such a composition (immunogen) or a vaccine or a plurality of particles for administering pH-sensitive drugs.
In another aspect, the invention provides the medical use of such a composition, such an immunogenic composition or vaccine or such particles for the administration of pH-sensitive drugs, particularly for the treatment or prevention of infection caused directly or indirectly by a pathogen, or a pathology associated with immunologically distinct host cells such as cancer. In another aspect, the invention provides a method of triggering an immune response against a pathogen or allergen causing an infection or pathology, or immunologically distinct host cells responsible for a condition such as cancer, comprising the step administering to an subject an effective amount of such a plurality of particles or a composition, an immunogenic composition or a vaccine.
In another aspect, the invention provides a method of preventing or reducing the interaction between a biologically active cargo and the components of an aqueous environment in which said cargo is present, comprising: forming such particles for the administering pH sensitive drugs comprising said cargo; and formulating said plurality of particles in said aqueous environment, including any adjustment necessary to render said subphysiological pH environment.
In another aspect, the invention provides a method of preventing or reducing the interaction between a biologically active cargo and the components of an aqueous environment of subphysiological pH wherein said cargo is present, comprising: forming a such plurality of pH sensitive drug delivery particles comprising said cargo; and formulating said plurality of particles in said aqueous environment.
In another aspect, the invention provides a method of manufacturing a plurality of drug delivery particles comprising a biologically active cargo within a matrix, comprising the step of manufacturing a solution of said cargo. and a polymer of the matrix and / or the use of a solution comprising a polymer and a cargo. In another aspect, the invention provides a method of manufacturing a plurality of drug delivery particles comprising a biologically active cargo within a matrix, comprising the steps of: at least partial deprotonation of a polymer, which is insoluble in its protonated state, in an aqueous environment such that the polymer has a net negative charge and is soluble in said aqueous environment; combining said polymer with said cargo to produce a stock solution; and forming the particles by molding said stock solution and removing the aqueous environment.
In another aspect, the invention provides a plurality of particles for drug delivery obtainable or obtainable by such methods of manufacturing a plurality of particles for drug delivery.
In another aspect, the invention provides a method of making a composition, including such methods of manufacturing a plurality of particles for drug delivery and formulating said particles in an aqueous environment. In another aspect, the invention provides a method of making a composition comprising a plurality of drug delivery particles comprising a biologically active cargo within a matrix, the matrix comprising a polymer, comprising the steps of the introduction of a plurality of particles made according to such methods of manufacturing a plurality of particles for administering drugs in an aqueous acidic environment such that the acidic environment protonates the polymer of the matrix making the insoluble polymer in said environment; elevating the pH of the aqueous acidic environment to a sub-physiological pH that is acceptable for parenteral administration while maintaining the insoluble state of the matrix polymer in said environment; and optionally formulating said particles in an aqueous environment of subphysiological pH.
In another aspect, the invention provides a composition obtainable or obtained by such methods of making a composition.
Brief description of the drawings
Figure 1 - Proportion of total Hib (unconjugated and TT conjugated) in the supernatant fraction as a percentage (% by weight) of all Hib present (supernatant + pellet) in the sample containing the particles 700-65 during storage at 4 ° C, detected by HPAEC-PAD.
Figure 2 - Proportion of "free" (unconjugated) Hib as a percentage (% by weight) of the total Hib (i.e., conjugated to the free TT +) present in the sample containing particles 700-65 (pellet + supernatant) detected by HPAEC-PAD during storage at 4 ° C.
Figure 3 - Proportion of total Hib (unconjugated and TT conjugated) in the supernatant fraction as a percentage (% by weight) of all Hib present (supernatant + pellet) in the sample containing the particles 841-57-1 during storage at 4 ° C, detected by HPAEC-PAD.
Figure 4 - Proportion of "free" (unconjugated) Hib as a percentage (% by weight) of the total Hib (i.e., conjugated to the free TT +) present in the sample containing 841-57-1 particles (pellet + supernatant) detected by HPAEC-PAD during storage at 4 ° C.
Figure 5 - Total Hib (unconjugated and TT conjugated) detectable by HPAEC-PAD (in μg / ml) in samples containing 841-57-2 and 841-57-25 particles (pellet + supernatant) during storage at 4 ° C.
Figure 6 - Proportion of total Hib (unconjugated and TT conjugated) in the supernatant fraction as a percentage (% by weight) of all Hib present (supernatant + pellet) in aliquots of the sample containing the 855-118-A particles stored respectively for approximately four hours at pH 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.2, and 7.4, detected by HPAEC-PAD.
Figure 7 - Proportion of Total Hib (Unconjugated and TT conjugated) in supernatant fraction as a percentage (% by weight) of all Hib present (supernatant + pellet) at various concentrations of particles (2) mg / ml, 1 mg / ml and 0.5 mg / ml) of the sample containing the 855-14 particles stored respectively for approximately four hours at pH 6.8, 7.4 and 8.0, detected by HPAEC- PAD.
Figure 8 - (A) Optical microscopy showing the dissolution of the particles of the 700-65 sample aged approximately one year, the particles of the sample 817-83-1 aged approximately one week, when the pH is increased in increments around the threshold pH. (B) Optical microscopy showing the dissolution of the 841-12-1 sample and 841-12-3 sample particles over 10 minutes as a result of a sudden increase in sample pH.
Figure 9 - Proportion of "free" (unconjugated) Hib as a percentage (% by weight) of the total Hib (i.e., conjugated to the free TT +) present in the samples containing the 855-72-2 and 855-72-4 particles (pellet + supernatant) during storage at 25, 37 and 45 ° C (with value at T = 0 subtracted in each case), detected by HPAEC-PAD on day 14 The control sample 855-72-5 contained Hib-TT but no particles.
Figure 10 - Mean Hib Antibodies at day 21 ("PII") and ("PIII") after the first immunization of adult rats. Groups of animals received, from left to right in Figure 10: Group 1 (control) - Infanrix Hexa (Infanrix Penta used to extemporaneously reconstitute freeze-dried Hib-TT); group 2 (control) - Infanrix Penta and Hiberix (freeze-dried Hib-TT reconstituted in saline) coadministered at different sites; group 3 - particle sample containing Hib-TT 841-57-1 co-administered at a site other than Infanrix Penta; group 4-particle sample containing Hib-TT 841-57-2 mixed with Infanrix Penta and administered after storage at 4 ° C for 4 weeks; Group 5 - Particle sample containing Hib-TT 700-66 mixed with Infanrix Penta and administered after storage at 4 ° C for 16 months.
Abbreviations
Hib-CRM: Haemophilus influenzae type b polysaccharide conjugated to CRM197 diphtheria protein; Hib-TT: Haemophilus influenzae type b polysaccharide conjugated to tetanus toxoid; HPAEC-PAD: high pressure anion exchange chromatography with pulsed amperometric detection; kDa: kilodaltons;
NaCl: sodium chloride; NaOH: sodium hydroxide; PBS: phosphate buffer solution; PEG: polyethylene glycol; PET: polyethylene terephthalate; PLGA: poly (lactide-co-glycolide) polymer; PMAA: poly (methacrylic acid); PMMA: poly (methyl methacrylate); PMMA-co-PMAA: polymer poly (methyl methacrylate) -co-poly (methacrylic acid); PRP: polyribosyl-ribitol; PVOH: polyvinyl alcohol; PVP: poly (vinylpyrrolidone); T: time; THF: tetrahydrofuran; TT: tetanus toxoid; PPI: water for injection.
Detailed description of the invention
The present invention relates to the stable storage and administration of drug compositions which contain, in a one-part aqueous liquid composition, substances (constituents) which are incompatible such as mutually physically or biochemically reactive or, if the composition is an immunogenic composition or a vaccine, prone to interfere immunologically. This is achieved by sequestering at least one of the incompatible substances within micro- or nanoparticles so that it is not exposed to the immediate environment (i.e., the aqueous composition) containing the substance with which it is incompatible. The substance sequestered within such particles is here called the "biologically active cargo". The particles are created to be pH sensitive, which, as referred to herein, means that they are sensitive to a "trigger" pH such that below a predetermined "threshold pH", the cargo remains sequestered within the particles while above the threshold pH, the cargo is no longer sequestered and is accessible to the immediate environment. By regulating the particle matrix for an appropriate threshold pH relative to the target site target site's local pH, the "delivery" (accessibility of cargo following "release" from particles) occurs only after administration.
particles
Accordingly, in one aspect, the invention provides a plurality of pH sensitive drug delivery particles comprising a biologically active cargo within a matrix, wherein said particles are triggered to release said cargo when they are released. are present in an aqueous environment having a pH higher than the pH of an aqueous environment in which said particles are present before being thereby triggered. In other words, the particles are induced to "give up" their cargo by increasing, above a certain threshold pH, the pH of the local environment. For example, particles present as a component of a final formally parenteral composition that retain a sub-physiological pH during storage are triggered to release their cargo by the high pH level encountered when the composition is injected into the muscle tissue. a human subject.
In some embodiments, the matrix of the particle, within which the biologically active cargo is included, is insoluble in an aqueous environment at a sub-physiological pH, but soluble in an aqueous environment at a "triggering" physiological pH ( that is, at a pH above the threshold pH). As such, in some embodiments, the particles are intact at a sub-physiological pH, while upon subjecting said particles to a triggering physiological pH, said particles are substantially or completely degraded / dissolved in 24 hours or less, by example are degraded / dissolved to at least 80, 85, 90, 95, 99 or 100%. The degree to which the particles are intact or degraded / dissolved can be determined in vitro by light microscopy.
In another aspect, the invention more particularly provides a plurality of pH sensitive drug delivery particles comprising a biologically active cargo within a matrix, wherein the amount of cargo released from said plurality of particles when present in an aqueous environment for at least 6 months at a sub-physiological pH is not more than 30% by weight of the total quantity of the cargo, and when the said particles are subjected to a physiological pH trigger (at or above a threshold pH) the amount of cargo released in 24 hours or less is not less than 50% by weight of the total quantity of the cargo.
By "released" in the context of the above aspect, it is meant that the cargo is detectable in the supernatant, rather than the pellet, of a sample containing the particles after separation of particles for drug delivery, for example, by centrifugation. (Therefore, the aqueous portion of a composition containing the particles that remains when the particles are separated therefrom is referred to herein as the "aqueous environment" or the "storage buffer." This may or may not contain one or more components biologically active). It is expressed as the proportion of cargo measured as present in the supernatant relative to the "total" amount detected at the same time point, i.e., the amount of supernatant plus the amount detected as being associated with the fraction containing the particles, for example, the pellet of the centrifugation. The amount of cargo released can be determined in vitro, as by HPAEC-PAD. However, it should be noted that, more generally, the concept of particles "releasing" the cargo as used herein means that the cargo is no longer "sequestered" by the particle, in the sense that the cargo is exposed or accessible to the cargo. aqueous environment. Thus, except as noted above in the specific context of quantifying the association of cargo with particles, "released" is not meant to necessarily involve physical dissociation or separation of cargo from the matrix. the particle; rather, it means that the structure or integrity of the particle has been altered by the change in pH so that the cargo becomes accessible to the local aqueous environment.
The reference to particles "comprising a biologically active cargo within a matrix" as used herein is intended to encompass various ways in which such cargo and such matrix material may together form a particle. When it is "included in a matrix" in this sense, it can be said that the cargo is sequestered, which means that it is largely or completely inaccessible to the external environment, for example, the aqueous environment of the composition. In some embodiments, the cargo is encapsulated within the matrix. In preferred embodiments, the cargo is substantially homogeneously dispersed throughout the matrix or entangled with the matrix of the particle.
In some embodiments, the aqueous environment at sub-physiological pH comprises a buffer, such as saline, or a phosphate buffer, Tris, borate, succinate, histidine, citrate or maleate.
As used herein, the meaning of "subphysiological pH" is relative to the local physiology of the intended recipient subject of the particles (formulated in an administrable composition), i.e., the pH of the site tissue. injection of the subject. In preferred embodiments, "sub-physiological pH" and "physiological pH" mean respectively subphysiologically and physiologically relative to the pH of human tissue, particularly human muscle and / or infant tissue. In some embodiments, the sub-physiological pH differs from the physiological pH by at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 , 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 pH units, i.e. the physiological pH is at least 0.1, , 2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 units of pH above the sub-physiological pH. These values may represent the lower limit of a range that is bound to the upper end by a value selected from 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0, 8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0 or 3.0. In some embodiments, the sub-physiological pH is at or below 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1 or 6.0, or includes a range with these respective values as the upper limit and a lower limit selected from 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6 , 0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1 or 5.0. In some embodiments, the physiological pH is at or above 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7, 6 or 7.7, or includes a range with these respective values as the lower limit and an upper limit selected from 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 , 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4 or 8.5.
In some embodiments, the amount of cargo released from said plurality of particles when present in an aqueous environment for at least 6 months at a sub-physiological pH is less than or equal to 30% by weight of the amount. total cargo, as not more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 , 9, 8, 7, 6, 5, 4, 3, 2 or 1% by weight. These values may represent the upper limit of a range which is related to the lower end by a value selected from among 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1. Such a rate of release of the cargo (conversely, such a sequestration rate within particles) when present in an aqueous environment at a subphysiological pH may be, in some embodiments, obtained over a longer period, such as at least 7, 8, 9, 10, 11, 12, 14, 16 , 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 months. These values may represent the lower limit of a range which is related to the upper end by a value selected from among 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 48 or 60 months.
The amount of the cargo released from said particles in 24 hours or less of submitting said particles to a physiological triggering pH is, in some embodiments, greater than or equal to 50% by weight of the total amount of the cargo, such as not less than 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% by weight. These values may represent the lower limit of a range that is bound to the higher end by a value selected from 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or Such a release rate of the cargo in response to a physiological triggering pH may be, in some embodiments, obtained over a shorter period of time, such as at 20, 16, 12, 10, 8, 6, 4, 2 or 1 hour (s) or 45, 30, 15, 10 or 5 minutes.
These values may represent the upper limit of a range which is bound to the lower end by a value selected from among 16, 12, 10, 8, 6, 4, 2 or 1 hour (s) or 45, 30, 15, 10, 5 or 1 minute (s).
In some embodiments, the aqueous sub-physiological pH environment in which the particles are present for at least 6 months is maintained at a temperature of between 2 and 8 ° C, for example, at about 4 ° C. However, in certain embodiments, the particles of the invention are heat-stable. This means that while present in the aqueous environment of sub-physiological pH at a temperature in the range of 2 to 8 ° C (i.e., not exceeding 2, 3, 5, 6, 7, 8 or, preferably, 4 ° C), the particles may be subjected to a temperature excursion (i.e., a temperature exceeding 2, 3, 4, 5, 6, 7 or 8 ° C ) not exceeding about 25 ° C or 37 ° C for up to 12 weeks, such as for a period of between 1 day and 2, 4, 6, 8, 10 or 12 weeks.
In some embodiments, the subjecting of the particles to a triggering physiological pH occurs at a temperature around the body temperature of the recipient, particularly around the tissue temperature of the recipient's injection site, such as at or about 37 ° C. in the case of a human being.
In some embodiments, the particles are highly uniform in shape, size, and / or composition, for example as a result of molding. One way in which such particles can be made is to use PRINTTM Technology (Liquidia Technologies,
Inc.), which is a process capable of forming (micro- and / or nano-) particles that: (i) are monodisperse in uniform size and shape; (ii) may be molded in any form; (iii) may be composed essentially of any matrix material; (iv) can be formed under mild conditions (compatible with delicate cargoes); (v) may be subject to post-functionalization chemistry (eg bioconjugation of active agents and / or targeting components); and (vi) which initially produces particles in an addressable two-dimensional lattice (which opens combinatorial approaches since the particles can be provided with a "barcode"). The methods and materials for making the particles of the present invention are further described and disclosed in the issued patents and copending patent applications of the co-applicants, each of which is hereby incorporated by reference in its entirety: US Patent No. 8 518,316; 8,444,907; 8,420,124; 8,268,446; 8,263,129; 8,158,728; 8,128,393; 7,976,759; U.S. Patent Application Publication Nos. 2013-209564; 2013-0249138, 20130241107, 2013-0228950, 2013-0202729, 2013-0011618, 2013-0256354, 2012-0189728, 2011-151015, 2010-0003291, 2009-0165320, 2008-0131692; PCT Publication No. WO2015 / 073831; and pending applications 13/852 683 filed March 28, 2013 and 13/950 447 filed July 25, 2013.
Particles produced using PRINT ™ Technology are manufactured by casting materials intended to make the particles in mold cavities. PRINT ™ Technology generally uses low surface energy molds made from materials such as silicones, perfluoro-polyether-based elastomers (PFPE) or other hydrocarbon-based materials to replicate structures of the micron or nanometer size on a reference model. The polymers used in the molds are often liquid at room temperature and can be crosslinked by photochemistry to elastomeric solids that allow high resolution replication of micron or nanometer sized structures. The liquid polymer "solidifies" when in contact with a reference model, thereby forming a replica image of the structures on the reference model. The solidification of the mold in contact with the reference model can take place by hardening (thermally or photochemically), by cooling by vitrification, and / or by crystallization. After removal of the polymeric mold from the reference model, the polymer forms a patterned model that includes cavities or replicas of recesses of the micron or nanometer size characteristics of the reference model. The micron or nanometer sized cavities in the pattern can be used for high resolution particle fabrication. PRINT ™ Technology enables the manufacture of monodisperse organic and inorganic particles with simultaneous control over the structure (eg shape, size and composition) and function (eg surface structure). The monodispersed nature of the particles in terms of physical constitution and composition provides highly uniform and predictable particle properties such as particle degradation / dissolution rates and thereby cargo release rates and dosage ranges.
Technical aspects to consider when designing a particle support system using PRINT ™ Technology include, but are not limited to: (i) compatibility of the cargo of particles or matrix materials with the materials of the PRINT ™ Technology PRINT ™ polymer mold; (ii) desired particle degradation / dissolution profile for cargo release, (iii) surface functionalization for particle targeting or particle compatibility, (iv) particle modulus; and (v) combining the points (i) to (iv) in forming a precursor solution of the particles that can be subjected to the molding process. Cargo compatibility within a particle matrix can be addressed, for example, by adjusting the hydrophilicity of the matrix to match that of the cargo by the judicious choice of matrix materials. Particle degradation / dissolution is discussed here. The particle modulus can be adjusted by changing, for example, the constituents of the particle. Finally, the precursor of the particle can be optimized for the manufacture of particles, where appropriate, by the addition of comonomers or cosolvents to modify the physical properties of the precursor solution of the particles.
In embodiments of the invention in which the particles are molded, the particles produced in this way will have a size and shape that substantially mimic the shape and size of the mold cavity in which each particle has been formed. Depending on the size of a mold cavity, the particles may be microparticles or nanoparticles. By choosing a mold of appropriate size, the size and shape of the particles can be adjusted to meet specific administration needs such as, for example, cargo loading, degradation / dissolution rate, etc. In certain embodiments, the microparticles according to the present invention may have the largest dimension less than about 1000 microns, less than about 900 microns, less than about 800 microns, less than about 700 microns, less than about 600 micrometers, less than about about 500 μm, less than about 400 μm, less than about 300 μm, less than about 200 μm, less than about 100 μm, less than about 50 μm, less than about 10 μm, less than about 5 μm, or about 1 μm . In other embodiments, the particles are nanoparticles and may have the largest dimension less than about 1000 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm. A person of average skill in the field will understand that the mold cavities and the corresponding particles produced from these molds may have a size between the sizes explicitly mentioned above. In addition, the dimension may be a length, a width, or a diameter of the particle.
In some embodiments, the longest axis of the particles is between about 1 and 10 μm, especially between 5 and 7 μm, such as 6 μm. In some embodiments, the particles have at least one axis that is less than 200 nm, and may optionally be sterilized by filtration.
A person of average skill in the field will also understand that the particles can be sized to have the selected aspect ratios. As defined herein, "aspect ratio" describes the ratio of the longest axis to the shortest axis of a particle. In some embodiments, the aspect ratio is at least 1/1, at least 2/1, at least 5/1, at least 10/1, at least 50 / 1, or at least 100/1. In particular embodiments, the aspect ratio is from about 1: 1 to about 5: 1, from about 5: 1 to about 10: 1, or from about 10: 1 to 100: 1. The particles can be molded into any desired shape. In some embodiments, the particles are ring-shaped or rod-shaped. In some embodiments, the particles are for parenteral administration, such as intradermal or subcutaneous or, preferably, intramuscular administration.
Particle Matrix
The particles of the present invention comprise a biologically active cargo within a matrix. The matrix provides a structural substrate for forming the particles and influences the stability of the particles and the kinetics of their degradation / dissolution in response to a triggering pH. Therefore, the particles of the invention have adjustable cargo release profiles, in part through the choice of matrix materials and their relative proportions, etc. The particle matrix can be made using a variety of materials including synthetic proteins, natural proteins, recombinant proteins, peptides, synthetic polymers, bioabsorbable polymers, polysaccharides, nucleic acids, small molecules, or any of their combinations. Suitable bioabsorbable polymers include polyvinyl alcohol (PVOH), polyethylene glycol (PEG), polyacrylic acid, polyacrylamide, polyvinylpyrrolidone (PVP), synthetic or natural polyamino acids, and PMMA-co -PMAA. Suitable polysaccharides include dextran, dextran derivatives, chitosan, chitosan derivatives, hyaluronic acid, alginic acid, agarose, pectin, cellulose, cellulose derivatives, cellulose ethers, xanthan gum, carrageenan, guar gum, starch, and inulin. Gelatin is also suitable for the matrix of the particle.
Thus, in some embodiments, the matrix is polymeric, i.e., it is composed of one or more polymers. The polymer may be a homopolymer, or a hetero- or co-polymer, such as an alternating or sequenced polymer. Preferably, such a polymer matrix is biocompatible, biodegradable, bioabsorbable and / or excretable in and from the human body. In some embodiments, the polymer of the polymer matrix, when in the aqueous environment at sub-physiological pH, is in an at least partially protonated state and / or may have an approximately or exactly neutral charge and be insoluble .
In some embodiments, the polymer matrix comprises a polymer having a pKa below the threshold physiological pH.
In some embodiments, the polymer matrix comprises a copolymer (poly (methyl methacrylate) -co-poly (methacrylic acid) (PMMA-co-PMAA copolymer), poly (glutamic acid) -co-poly (lysine); zwitterionic hetero or homo-poly (amino acids), carboxymethyl chitosan, hypromellose phthalate, hypromellose acetosuccinate, or an acrylate copolymer represented by the general formula (1): wherein: R 1 represents a hydrogen atom or a methyl group, R2 represents a hydrogen atom or a methyl group, and R3 represents a methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl or sec-butyl group; The PMMA-co-PMAA copolymer can have a weight average molecular weight (Mw) in the range of 1 to 200 kDa, as in the range of 50 to 60 kDa or 35 to 45 kDa or 22 to 40 kDa. at 28 kDa or 8-12 kDa, such as a weight average molecular weight (Mw) of 10, 25, 4 0, 55 or 125 kDa.
In some embodiments, the polymer matrix comprises a PMMA-co-PMAA copolymer, wherein the molar ratio of the methyl methacrylate monomer (MMA) to the methacrylic acid monomer (MAA) in the copolymer is in the range of from 1/1 to 4/1, as in the range of 1.5 to 2/1. In other embodiments, the polymer matrix comprises poly (glutamic acid) -co-poly (lysine), wherein the molar ratio of glutamic acid monomer to lysine monomer is approximately or exactly 1/1 .
cargoes
The pH sensitive drug delivery particles of the present invention comprise, in addition to the matrix discussed above, a biologically active cargo. Such cargo is sequestered within the particles, allowing storage within an aqueous environment while preventing any undesirable interactions between the cargo and the components of the aqueous environment. By "biologically active" as used herein in relation to the cargo, it is meant that the cargo is not inert with respect to the biological (eg, physiological, immunological, etc.) functioning of the recipient's body to which the particles are administered. In other words, such a biologically active cargo is able to interact with the recipient's body to somehow mediate the biological effect. The biological effect may be a therapeutic or prophylactic effect and, therefore, the cargo may be a "medicine", which in relation to the particles of the invention is used here in its broadest sense. Therefore, the cargo may be an active agent, a pharmaceutical agent, a therapeutic agent, or a vaccine agent. In some embodiments, the particles may each comprise more than one biologically active cargo, such as one, two, three or four different cargoes or more. In some embodiments, the particles may each comprise more than one biologically active cargo of the same type, such as two, three or four or more drugs or two, three or four therapeutic agents or more. In some embodiments, the particles may each comprise more than one biologically active cargo that are of different types, such as one, two, three, or four or more drugs, and one, two, three, or four or more vaccine agents.
The biologically active cargo may more particularly comprise an antigen, an antibody, a small molecule drug compound, an immunoglobulin, a protein, a polysaccharide, a protein-polysaccharide conjugate, a nucleic acid or an adjuvant (non-specific immunomodulatory agent). The biologically active cargo may, in some embodiments, be hydrolytically sensitive, meaning that, subject to prevailing parameters such as pH, temperature, ionic strength, etc., the cargo is susceptible to a material degree of degradation. by hydrolysis when in contact with an aqueous environment. For example, a "material" degree of degradation by hydrolysis could be, in the context of a cargo of antigen, a degree of degradation that causes a detectable reduction in immunogenicity or antigenicity. In some embodiments, the biologically active cargo has a low isoelectric point (pi), such as a pI of 4 or less, especially 3 or 2 or less. In some embodiments, the biologically active cargo includes phosphate groups, as in phosphodiester bonds.
In particular embodiments, the biologically active cargo comprises an antigen. The term "antigen" is well understood by those skilled in the art as meaning an agent capable of eliciting an immune response in a human or animal body. Therefore, antigens are the "active ingredients" in immunogenic compositions / vaccines. An antigen may comprise or consist of, for example, a protein or polypeptide, a saccharide such as an oligo- or polysaccharide, a conjugate of a protein and a saccharide or an acid nucleic. The antigens can be in various forms, such as purified or recombinant proteins, polysaccharides, conjugates of these proteins and polysaccharides, nucleic acid vectors for in vivo antigen production, bacteria or inactivated whole viruses. , viral fragments, pseudoviral particles, live attenuated bacteria, replicating attenuated viruses or bacterial outer membrane complexes. The antigens, being the cargo according to the invention, can be of any type of antigen as described above, and can be antigens derived from or associated with a pathogen (such as a bacterium, a virus or another pathogen) , a cancer / tumor, an allergic or autoimmune condition, a non-infectious pathological condition, a dependency condition, or any other physiological condition justifiable by a prophylactic or therapeutic intervention via immunization.
In some embodiments in which the biologically active cargo is an antigen, said cargo comprises a saccharide such as an oligo- or polysaccharide. The term "oligo / polysaccharide" will be used herein to mean an oligosaccharide or polysaccharide that has been isolated from a pathogen. In some of these embodiments, the oligo / polysaccharide has a low isoelectric point (pI), such as a pI of 4 or lower, particularly of 3 or 2 or less. The oligo / polysaccharide can be used in its native form isolated from the pathogen, or it can be treated. Such a treatment may be, for example, a reduction of native saccharides, for example by microfluidization (other techniques are described in EP 0497524).
In some embodiments, the oligo / polysaccharide is derived from a bacterial pathogen and in particular can be derived from a bacterial capsular saccharide or a lipooligosaccharide (LOS) or a lipopolysaccharide (LPS). For example, the oligo / polysaccharide may be derived from a bacterial pathogen selected from the group consisting of: Haemophilus influenzae type b ("Hib"); Neisseria meningitidis (in particular serotypes A, C, W and / or Y); Streptococcus pneumoniae (in particular serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20 , 22F, 23F and / or 33F); Staphylococcus aureus; Bordetella pertussis; and
Salmonella typhi.
In a particular embodiment, the saccharide in said cargo of antigen comprising a saccharide is an oligo / polysaccharide conjugated to a carrier protein, i.e. the cargo is an oligo / polysaccharide conjugate antigen. protein. Such conjugates are well known in the art as a means of conferring on the oligo / polysaccharide antigen the T-cell dependent character of the immune response triggered by the carrier protein. Therefore, the carrier proteins are selected for their ability to provide a source of helper T cell epitopes. In a given oligo / polysaccharide-protein conjugate, the carrier protein may be derived from the same pathogen as the oligo / polysaccharide, or a different pathogen. Suitable carrier proteins for use in the conjugate oligo / polysaccharide-protein conjugate cargoes of the invention are well known in the art, and include: tetanus toxoid, tetanus toxoid fragment C , diphtheria toxoid, CRM197 or other nontoxic mutant of diphtheria toxin, nontypeable Haemophilus influenzae protein D, Neisseria meningitidis outer membrane protein complex (OMPC), pneumococcal PhtD, pneumococcal pneumolysin , Pseudomonas aeruginosa exotoxin A (EPA), Staphylococcus aureus detoxified hemolysin, Bordetella sp. detoxified adenylate cyclase, Escherichia coli detoxified heat labile enterotoxin, or cholera toxin subunit B (CTB) or detoxified cholera toxin.
As discussed above, the biologically active cargo of the particles of the invention may be sensitive to hydrolysis. In the case of an oligo / polysaccharide-protein conjugate antigen, sensitivity to hydrolysis may manifest as hydrolytic cleavage within the saccharide chain or between saccharide and carrier protein, in a case such as in the other resulting in the production of "free" (non-conjugated) saccharide, that is, a saccharide that is not conjugated to the protein, which is undesirable. Since the sequestration of the conjugated oligo / polysaccharide-protein antigen within the particles of the invention serves to protect the conjugated antigen against possible hydrolytic interactions with an aqueous environment (sub-physiological pH) in which the particles may be present, the loss of conjugate integrity during particle storage in an aqueous environment is minimized. Thus, in certain embodiments in which the biologically active cargo is an oligo / polysaccharide-protein conjugate antigen, the amount of free (non-conjugated) saccharide, derived from said oligo / polysaccharide conjugate, present collectively in the particles to the administration of drugs and the aqueous environment is not greater than 30 or 25 or 20 or 15 or 10% by weight of the total amount of conjugated and free saccharide present collectively in the particles and the aqueous environment for at least 6 months in an aqueous environment at sub-physiological pH. These values may respectively represent the upper end of a range which is linked to the lower end by a value chosen from 25, 20, 15, 10 or 5% by weight.
In some of these embodiments, such a maximum rate of increase of the free saccharide applies for a period greater than 6 months, such as at least 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 months. These values may represent the lower limit of a range which is related to the upper end by a value selected from among 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 48 or 60 months. In some of these embodiments, the aqueous environment of sub-physiological pH in which the particles are present for at least 6 months is maintained at a temperature of between 2 and 8 ° C. In particular embodiments, the particles may be subjected to a single excursion at a temperature in excess of this range, however not exceeding about 37 ° C for longer than about 2 weeks. Preferably, the excursion does not exceed about 25 ° C.
In preferred embodiments, the biologically active cargo comprises an oligo / polysaccharide derived from the capsular saccharide of Haemophilus influenzae type b ("Hib", polyribosylribitol phosphate or "PRP"), optionally in its native full-length conjugated form. CRM 197 or, more preferably, tetanus toxoid. In other preferred embodiments, the oligo / polysaccharide is derived from the capsular saccharide of Neisseria meningitidis, in particular serotype A. In these preferred embodiments, the conjugated antigens are preferably substantially homogeneously dispersed throughout the region. matrix of the particle.
compositions
The pH sensitive drug delivery particles of the invention can be stored and administered to a subject, which is an animal, particularly a mammal, more particularly a human, in a composition acceptable for use. parenteral. Accordingly, in one aspect of the invention there is provided a composition comprising a plurality of pH sensitive drug delivery particles of the invention in an aqueous environment, preferably sterile. Such a composition of the invention may be an immunogenic composition, i.e., a composition capable of eliciting in a subject an immune response directed specifically against one or more antigenic components present in the composition. Such an immunogenic composition may be a vaccine. In other words, the invention provides a vaccine comprising an (immunogenic) composition of the invention as described herein.
Preferably, such compositions retain a pH below the threshold physiological pH of the particles or, in other words, are of sub-physiological pH. Such a sub-physiological pH of the composition or its aqueous environment is determined with respect to the local physiological pH of the particular tissue type / anatomic region (e.g., intramuscular, intravenous) of the particular subject type (e.g. animal, mammal, human, adult, infant) to which the composition is intended to be administered directly. In some embodiments, the aqueous composition / environment is adjusted to have a pH at or below 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2 , 6.1 or 6.0. The values can respectively define the upper end of a range which is defined at the lower end by a value chosen from among 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1 or 5.0. The aqueous environment may contain one or more physiologically acceptable excipients and / or a buffer such as saline, or a phosphate buffer, Tris, borate, succinate, histidine, citrate or maleate.
The composition may comprise more than one population of pH-sensitive particles, each population containing different cargoes and comprising the same or different particle matrices. Thus, in some embodiments, the composition comprises a first plurality and a second plurality of particles, wherein said second plurality of particles comprises cargo other than the cargo of the first plurality of particles. Alternatively, the composition may contain a plurality of particle populations differing in physical characteristics such as matrix polymer, size, and shape; such populations may include respectively the same cargoes or different cargoes.
In some embodiments, for example, when the polymer matrix of the particles comprises a PMMA-co-PMAA copolymer, the particles are present in the composition at a concentration of 0.1 to 15, 0.5 to 12.5, 1 at 10 or 2 to 5 mg / ml, such as 0.5 to 3 mg / ml, in particular 1.0 to 2.5 mg / ml. In such embodiments, the PMMA-co-PMAA copolymer may have, for example, a molar ratio of the methyl methacrylate monomer (MMA) to the methacrylic acid monomer (MAA) in the range of 1: 1. at 4/1 (as in the range of 1.5 to 2/1) and may have a weight average molecular weight (Mw) in the range of 1 to 200 kDa, such as in the range of 50 to 60 kDa or 35 to 45 kDa or 22 to 28 kDa or 8 to 12 kDa, such as a weight average molecular weight (Mw) of 8.5, 10, 23.9, 25, 37, 40, 51, 55 or 125 kDa.
As a consequence of the sequestered cargo within the particle matrix, the accessibility of the cargo to the aqueous environment is impaired or substantially prevented. Therefore, the cargo is substantially prevented by the matrix from interacting with the components of the aqueous environment, or such interaction is at least reduced with respect to the situation in the absence of the particle matrix. In some embodiments, said interaction is prevented or reduced for at least 6 months, such as 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 , 34 or 36 months (these values may represent the lower limit of a range which is bound to the upper end by a value selected from 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 48 or 60 months); in some of these embodiments, the composition is stored at about 4 ° C.
In some embodiments, wherein the composition is an immunogenic composition, the aqueous environment (i.e., not including the particles present therein) comprises one or more antigens, and optionally associated components. such as one or more adjuvants. Such an adjuvant may have a high pI, such as a pI of 8 or higher, such as 9 or 10 or higher, especially 11 or higher. In a preferred embodiment, the adjuvant is aluminum hydroxide.
The one or more antigens included in the aqueous environment in certain embodiments of the immunogenic composition may, in some embodiments, be selected from: diphtheria toxoid, tetanus toxoid, acellular pertussis antigens (such as pertussis toxoid, filamentous haemagglutinin, pertactin), hepatitis B surface antigen (HBsAg) and inactivated polio vaccine (IPV), the Haemophilus influenzae oligo / polysaccharide conjugate antigen. type b, the N. meningitidis oligo / polysaccharide conjugated antigen of serotype C, the N. meningitidis oligo / polysaccharide conjugated antigen of serotype A, the N. meningitidis oligo / polysaccharide conjugated antigen serotype W, the N. meningitidis oligo / polysaccharide conjugated antigen of serotype Y and N. meningitidis serotype B antigen. In particular, the following combinations of The genes may be included within the aqueous environment of the immunogenic compositions of the invention: diphtheria toxoid and tetanus toxoid; ii. diphtheria toxoid, tetanus toxoid, acellular pertussis antigens; iii. diphtheria toxoid, tetanus toxoid, acellular pertussis antigens and HBsAg; iv. diphtheria toxoid, tetanus toxoid, acellular pertussis antigens and IPV; v. diphtheria toxoid, tetanus toxoid, acellular pertussis antigens, HBsAg and IPV; vi. Neisseria meningitidis capsular oligo / polysaccharide serotype C (MenC) conjugated to Neisseria meningitidis capsular oligo / polysaccharide carrier protein of serotype W (MenW) conjugated to Neisseria meningitidis capsular oligo / polysaccharide carrier protein of serotype Y (MenY) conjugated to a carrier protein; vii. Neisseria meningitidis capsular oligo / polysaccharide serotype C (MenC) conjugated to Neisseria meningitidis capsular oligo / polysaccharide carrier protein serotype W (MenW) conjugated to Neisseria meningitidis capsular oligo / polysaccharide carrier protein serotype Y (MenY) conjugated to a carrier protein and antigen derived from Neisseria meningitidis serotype B (MenB).
Preferably, when the aqueous environment of the immunogenic composition comprises one of the combinations (i) to (v) above, the cargo is an oligo / Hib polysaccharide conjugate antigen. Also preferably, when the aqueous environment of the immunogenic composition comprises one of combinations (vi) and (vii) above, the cargo is a MenA oligo / polysaccharide conjugated antigen. In some of these embodiments in which diphtheria toxoid, tetanus toxoid, acellular pertussis antigens and / or HBsAg are present in the aqueous environment, diphtheria toxoid, tetanus toxoid, antigens Acellular pertussis is adsorbed on aluminum hydroxide and the HBsAg is adsorbed on aluminum phosphate. In some of these embodiments, diphtheria toxoid is present at the dosage level of 1 to 10 international units (IU) (e.g., exactly or approximately 2 IU) or 10 to 40 IU (for example, exactly or approximately 20 or 30 IU) or 1 to 10 units of flocculation limit (Lf) (e.g., exactly or approximately 2 or 2.5 or 9 Lf) or 10 to 30 Lf (e.g., exactly or approximately 15 or Lf), and the tetanus toxoid is present at the dose level of 10 to 30 IU (e.g., exactly or approximately 20 IU) or 30 to 50 IU (e.g., exactly or approximately 40 IU) or 1 to 15 Lf (for example, exactly or approximately 5 or 10 Lf).
In some embodiments, in addition to the particle-associated oligon / Hib polysaccharide conjugate antigen, the immunogenic composition comprises, in its aqueous environment, diphtheria toxoid and tetanus toxoid in exact or approximate amounts. respective per dose of: 30/40 IU; 25/10 Lf; 20/40 IU; 15/5 Lf; 2/20 UI; 2.5 / 5 Lf; 2/5 Lf; 25/10 Lf; 9/5 Lf. Cell-free pertussis antigens (Pa) including pertussis toxoid (PT), filamentous haemagglutinin (FHA) and pertactin (PRN) may also be present, such that the aqueous environment comprises DTPa antigens in the amounts following: 20 to 30 μg, for example, exactly or approximately 25 μg of PT; 25 to 30 μg, for example, exactly or approximately 25 μg of FHA; 1 to 10 μg, for example, exactly or approximately 3 or 8 μg of PRN; 10 to 30 Lf, for example, exactly or approximately 15 or 25 Lf of D; and 1 to 15 Lf, for example, exactly or approximately 5 or 10 Lf of T; or 2 to 10 μg, for example, exactly or approximately 2.5 or 8 μg PT; 2 to 10 μg, for example, exactly or approximately 5 or 8 μg of FHA; 0.5 to 4 μg, for example, 2 to 3 μg as exactly or approximately 2.5 or 3 μg of PRN; 1 to 10 Lf, for example, exactly or approximately 2 or 2.5 or 9 Lf of D; and 1 to 15 Lf, for example, exactly or approximately 5 or 10 Lf of T.
As mentioned above, the interaction between the cargo and the components of the aqueous environment is reduced or substantially prevented by the particle matrix. In this way, in embodiments of the immunogenic compositions of the invention, the particle matrix prevents or reduces aggregation or flocculation and / or prevents or reduces immunological interference and / or prevents degradation by hydrolysis of the cargo, compared to an equivalent composition in which the cargo is not sequestered within the particles and is accessible to the aqueous environment. In some embodiments, said interaction, and in particular said aggregation / flocculation and / or immunological interference and / or hydrolytic degradation, is prevented or reduced for at least 6 months, such as 7, 8, 9, 10, 11, 12 , 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 months (these values may represent the lower limit of a range that is related to the upper end by a value selected from 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 48 or 60 months), optionally wherein said composition is maintained at about 4 ° C.
The phenomenon of aggregation or flocculation, which can be observed visually or by optical microscopy, occurs when certain cargoes interact with certain components of the aqueous environment leading to the formation of a network of particles. In the case of an immunogenic composition containing a cargo of antigen, such a network of particles can mask epitopes and "interfere" negatively with the triggered immune response. In some cases, the aggregation / flocculation and the resulting interference may be the result of the cargo and component of the aqueous environment having respectively low and high isoelectric points (pi) (or vice versa), so that they are attracted to interact with each other. This is thought to be the reason for the aggregation / flocculation / interference observed between the PRP saccharide of the Hib conjugate vaccine (low pI) and the aluminum hydroxide adjuvant (high pI) used to adsorb other antigens in some combined vaccines containing the Hib conjugate. Thus, in some embodiments, said cargo has a low pI such that a pI of 4 or less, especially 3 or 2 or less, and / or the aqueous environment comprises a component having a high pI, such as a pI of 8. or higher, such as 9 or 10 or 11. Such low pH cargo may include phosphate groups, for example, in the context of phosphodiester linkages. However, immunological interference reduced or prevented by the immunogenic compositions provided herein is not necessarily associated with flocculation or aggregation. In embodiments in which the aqueous environment comprises one or more antigens, preferably the particle matrix does not interfere with the immunogenicity of said one or more antigens.
In particular embodiments of the immunogenic compositions of the invention, the particle matrix prevents or reduces the aggregation or flocculation of Hib-TT or Hib-CRM197 or MenA-CRM197 and / or prevents or reduces immunological interference with Hib-TT or Hib-CRM197 or MenA-CRM197.
Hydrolytic degradation, such as cleavage, of the hydrolysis-sensitive cargo is discussed above, and may occur by the interaction of said cargo with the water molecules in the aqueous composition. Therefore, preventing or minimizing the exposure of the cargo to the water molecules can reduce or prevent the occurrence of hydrolysis. Therefore, the present immunogenic compositions achieve this through the sequestration of the cargo of antigen within the particle. Certain saccharide antigens, such as Hib and MenA, may be particularly prone to hydrolytic degradation, such as depolymerization. In particular embodiments of the immunogenic compositions of the invention, the particle matrix prevents or reduces hydrolytic degradation of Hib-TT or Hib-CRM197 or MenA-CRM197.
The immunogenic compositions of the invention, in some embodiments, are suitable for parenteral administration. One skilled in the art knows how to formulate therapeutic compositions for compatibility with a given parenteral route of administration, for example, intramuscularly. In particular, one skilled in the art knows how to formulate such compositions to have a particular pH, this being a key feature of the immunogenic compositions of at least some embodiments of the invention in which the aqueous environment in which which particles are administered (and possibly stored) is of sub-physiological pH. The invention further provides a plurality of particles or a composition of the invention packaged in a suitable container for therapeutic use. The particles or composition may be in a vial from which the contents can be extracted when necessary, for example, using a needle and a syringe. Alternatively, the particles or composition may be poured in advance into a parenteral delivery device such as a syringe. Such a syringe may be a conventional single chamber syringe, or it may be a dual chamber syringe. The dual chamber syringe may be configured to deliver the respective contents of the chambers sequentially, or simultaneously after extemporaneous mixing within the syringe.
Use and administration of particles for the administration of drugs
In one aspect of the invention, there is provided a method of preventing or reducing the interaction between a biologically active cargo and the components of an aqueous environment in which said cargo is present, comprising (i) the formation of a plurality of pH responsive drug delivery particles as defined herein comprising said cargo, and (ii) formulating said plurality of particles in said aqueous environment, including any adjustments necessary to render said pH environment sub- physiological. (In step (ii) above, said adjustment may not be necessary if the aqueous environment is already at sub-physiological pH). Thus, in one embodiment of this aspect of the invention, there is provided a method of preventing or reducing the interaction between a biologically active cargo and the components of an aqueous environment of subphysiological pH wherein said cargo is present, comprising (i) forming a plurality of particles for administering pH-sensitive drugs as herein defined comprising said cargo, and (ii) formulating said plurality of particles in said aqueous environment. In some embodiments, the result of the formulation according to steps (ii) above is the production of a composition, an immunogenic composition, or a vaccine as defined herein.
As discussed above, such prevention or reduction of said interaction is advantageous in a variety of circumstances. Therefore, in some embodiments, the above methods are methods for storing a biologically active cargo in an aqueous environment (wherein storage is effected by preventing or reducing interactions between the biologically active cargo and components of an aqueous environment). Similarly, in some of these embodiments, as well as in other embodiments, the above methods are for the prevention or reduction of the interaction between said biologically active cargo and the molecules of the invention. water in said aqueous environment, or between said biologically active cargo and a component of the aqueous environment other than water. Such a component may be, for example, an adjuvant or an antigen or a biological or pharmaceutical active ingredient, or a formulation excipient. In particular, said methods may be for the prevention or reduction of degradation, such as degradation by hydrolysis (i.e. by interaction with water molecules), of said cargo in said aqueous environment.
When the above methods are for storing a biologically active cargo in an aqueous environment, said storage may be for at least 6 months, for example 7, 8, 9, 10, 11, 12, 14, 16, 18 , 20, 22, 24, 26, 28, 30, 32, 34 or 36 months. These values may represent the lower limit of a range which is related to the upper end by a value selected from among 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 48 or 60 months.
When the above methods are for the prevention or reduction of the interaction between said biologically active cargo and an adjuvant component of the aqueous environment, in some embodiments, said adjuvant is aluminum hydroxide. In some embodiments, the cargo contains phosphate groups such as in phosphodiester and / or low acid radicals. In some embodiments, the cargo is Hib-TT or Hib-CRM197 or MenA-CRM197.
The above methods may alternatively be intended for preventing or reducing the interaction between said biologically active cargo and a second cargo. Such embodiments include, in addition to steps (i) and (ii) cited above, a step (iii): forming a second plurality of particles comprising said second cargo within a matrix, in a which said second plurality of particles is as defined herein for the plurality of particles but provided that the cargo is not identical to said biologically active cargo, i.e. the second plurality of particles is as defined herein for the plurality of particles with the exception of the cargo, in the sense that in such embodiments comprising two pluralities (populations) of particles, the two pluralities comprise different cargoes. In some alternative embodiments, the two pluralities may comprise the same cargo in a different polymer matrix.
The plurality of particles and the compositions (immunogens) and vaccines of the invention can be used in medicine, as prophylactically or therapeutically. They can be administered to a subject in need. Generally, the subject is an animal, such as a mammal, and is preferably a human subject. In some embodiments, the subject is an infant or a child or adolescent or an adult or an older adult. The subject may be a pregnant woman / woman, possibly in which the pregnant infant is the subject in need. The subject may be an immunocompromised individual. In a particular embodiment, the plurality of particles and immunogenic compositions and vaccines are for use in immunization, such as pediatric immunization.
In one aspect, the invention provides a plurality of particles or an immunogenic composition / composition / vaccine as disclosed herein for use in medicine, particularly in human medicine. More particularly, the invention provides a plurality of particles as defined herein, or an immunogenic composition / composition / vaccine as defined herein, for use in the treatment or prevention, in particular in a human being, of i) an infection or pathology caused directly or indirectly by a pathogen or allergen, or (ii) a pathology associated with immunologically distinct host cells, such as cancer. The invention further provides the use of a plurality of particles as defined herein, or an immunogenic composition / composition / vaccine as defined herein, in the manufacture of a medicament for use in the treatment or preventing, in an animal, particularly a human being, (i) an infection or pathology caused directly or indirectly by a pathogen or allergen, or (ii) a pathology associated with immunologically distinct host cells, such as the cancer.
In another aspect, there is provided a method of treatment or prophylaxis against (i) an infection or pathology caused directly or indirectly by a pathogen or allergen, or (ii) a pathology associated with immunologically distinct host cells, such as cancer, comprising the step of administering to a subject, in particular a human, an effective amount of a plurality of particles or an immunogenic composition / composition / vaccine as disclosed herein. A method, including the same step of administration, is also provided to elicit an immune response against such a pathogen or allergen or such immunologically distinct host cells.
The plurality of particles or the immunogenic composition / composition / vaccine of the invention may be administered in liquid form, i.e. in the form of a suspension containing the particles. Prior to administration, the particles may be stored in the formally-formable liquid composition, such as the composition / immunogenic composition / vaccine of the invention wherein the particles are in the aqueous environment as defined herein. However, the particles can be stored in lyophilized form, as freeze-dried, or in powder form, to be reconstituted in liquid form via the mixture with the aqueous environment (as defined herein) extemporaneously with the administration to a subject. Alternatively, the particles may be stored in a liquid medium other than said aqueous environment, such that mixing with said aqueous environment, to provide the immunogenic composition / composition / vaccine of the invention, takes place extemporaneously with the administration. The particles of the composition / immunogenic composition / vaccine of the invention may be in unit dose or multiple dose sealed containers such as vials, or they may be dispensed in advance in delivery devices such as syringes.
The plurality of particles or the immunogenic composition / composition / vaccine of the invention may be administered to a subject by various routes, such as intramuscular, intravenous, intraperitoneal, intra- or trans-dermal, subcutaneous, intrapulmonary. (eg, inhalation), trans- or sub-mucosal. Thus, in some embodiments, the administration is parenterally.
An appropriate effective dose of the biologically active cargo, for example, an antigen (and hence particles and the composition in which they are present), can be readily determined by those skilled in the art. Such a dose can be calculated based on the administration of a specified mass of particles. In this case, a specific mass of the composition containing the particles is measured by weight or by volume. Alternatively, the dose may be based on the administration of a specified mass of cargo, in which case loading of the cargo into the particles may be taken into account.
In a particular embodiment of the plurality of particles, compositions / immunogenic compositions / vaccines above; their use; or a method of treating or triggering an immune response, the pathogen is selected from the list consisting of: Haemophilus influenzae type b (Hib); Neisseria meningitidis (in particular serotypes A, C, W and / or Y);
Streptococcus pneumoniae; Staphylococcus aureus; Bordetella sp. ; and Salmonella typhi. In preferred embodiments, the pathogen is Haemophilus influenzae type b (Hib) or Neisseria meningitidis serotype A (MenA).
Particle manufacturing and formulation
The particles of the present invention comprise a biologically active cargo within a matrix. Cargo can associate with the particle matrix in different ways such that it is "included within the matrix". For example, the cargo may be encapsulated within the matrix, or the cargo may be dispersed within or physically mixed with the matrix. The combination of the cargo and the matrix can be described as a physical association, such as a non-covalent association.
In particular embodiments, the cargo is dispersed substantially homogeneously throughout the matrix of the particle. This can be achieved by making particles from a homogeneous mixture (a solution) comprising the cargo and the polymer (s) in the matrix.
Thus, in one aspect, there is provided a method of manufacturing a plurality of drug delivery particles comprising a biologically active cargo within a matrix, comprising the step of making a solution (e.g. that is, a homogeneous mixture) comprising said cargo and a polymer of the matrix, optionally wherein said polymer is as defined herein for the polymer matrix. Also provided is a method of manufacturing a plurality of drug delivery particles comprising a biologically active cargo within a matrix, the method comprising using a solution comprising a polymer as defined herein for the polymer matrix, and a cargo, wherein said cargo is optionally as defined herein.
In some embodiments of the above methods, said solution comprises said cargo at an amount not exceeding 30% by weight and a complement of PMMA-co-PMAA copolymer. For example, the solution may comprise the cargo at a level of 0.1 to 5% by weight, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 , 0.8, 0.9, 1, 1.5, 2, 2.5 or 5% by weight. In some embodiments, the solution further comprises a plasticizer. In some embodiments, the plasticizer is polyvinylpyrrolidone (PVP). In a particular embodiment, the polymer of the matrix is PMMA-co-PMAA and the solution further comprises PVP, in which the weight ratio of PVP / PMMA-co-PMAA does not exceed 1 / 1, for example, the weight ratio of PVP / PMMA-co-PMAA is in the range of 0.1 to 1/1, such as 0.15 / 1, 0.2 / 1, 0.25 / 1, 0.3 / 1, 0.35 / 1, 0.4 / 1, 0.45 / 1, 0.5 / 1, 0.55 / 1, 0.6 / 1, 0.65 / 1,
0.7 / 1, 0.75 / 1, 0.8 / 1, 0.85 / 1, 0.9 / 1, or 0.95 / 1. PVP may have, for example, a molecular weight (Mw) of about 2.5 kDa and a polydispersity of approximately 1.9. In other embodiments of these methods, the solution comprises a poly (glutamic acid) -co-poly (lysine) copolymer in place of the PMMA-co-PMAA copolymer and / or glycerol in place of the PVP.
The cargo can be chosen, for example, from Hib-TT, Hib-CRM197 and MenA-CRM197. In particular embodiments, said solution comprises 0.2 to 1.2, more particularly 0.4 to 1% by weight of Hib-TT or Hib-CRM197 or MenA-CRM197 and a complement of PVP / PMMA-co- PMAA or poly (glutamic acid) -co-poly (lysine) / glycerol, optionally in a ratio in% by weight of 1/1.
The particles of the invention can be made from these solutions by known techniques for the production of micro- or nanoparticles, for example, homogenization (emulsification), extrusion and drying such as spray. In particular, the particles are formed by molding. Therefore, in some embodiments, the above methods include another step of molding said solution to form the plurality of particles. The solution may comprise a plasticizer other than PVP (optionally in addition to PVP); such a plasticizer may be a blowing agent. In some embodiments, the methods include removing substantially all of said PVP or other plasticizer and / or blowing agent from said particle. By "removal from said particle" in this sense, it is meant that the plasticizer and / or blowing agent may be substantially absent from particles formed from said solution (containing a plasticizer and / or blowing agent) as a result of process, i.e. the resultant particle may be substantially free of any plasticizer and / or pore-forming agent. This does not necessarily mean that the particles formed will temporarily contain such a plasticizer and / or pore-forming agent (the plasticizer and / or pore-forming agent may be lost as a consequence of the particle manufacturing process), as may be the case for particles which are collected in a dry state, in which the plasticizer and / or pore-forming agent is lost from the particle during a subsequent step of "transitioning" the particles to a sub-physiological pH acceptable for parenteral administration. Inescapably, the composition of the particles, after the loss or removal of any plasticizer and / or pore-forming agent, will differ from the composition of the solution used in their manufacture.
In another aspect, the present invention provides a method of manufacturing a plurality of drug delivery particles comprising a biologically active cargo within a matrix, comprising the steps of: i. at least partial deprotonation of a polymer, which is insoluble in its protonated state, in an aqueous environment such that the polymer has a net negative charge and is soluble in said aqueous environment; ii. combining said polymer with said cargo to produce a solution; iii. forming the particles by molding said solution and removing the aqueous environment.
For the sake of clarity, it should be noted that "aqueous environment" as used in the above paragraph should not be confused with the use of this term elsewhere in this document in connection with the compositions of the invention or the properties of the particles of the invention when present in an aqueous environment below the triggering pH.
In step (i) above, a polymer is solubilized through deprotonation (at least partial), for example by adding the polymer to an aqueous environment of alkaline or basic pH. The dissolved polymer is then combined with the cargo to produce a solution, often called a "stock solution." The solution is then molded as described above in connection with the PRINT ™ Technology of the Liquidia Technologies co-applicant and as illustrated in the examples herein, one result of which is the removal of the aqueous environment. This step (iii) of the above process causes the non-covalent association or physical entanglement of the polymer chains with the cargo. In alternative embodiments, the particles may be formed not by molding but by other known techniques for making micro- or nanoparticles, for example spray drying.
After particle formation, they can be collected in a dry state, or in an organic or aqueous liquid collection environment. Such a collection environment may be acidic, for example, with a pH of less than 5. In some embodiments, the above method may further include protonating the polymer of the matrix of collected particles such that the polymer returns to an insoluble state. The particles can then be stored at a sub-physiological pH that is acceptable for parenteral administration, for example, in an aqueous environment.
In another aspect, the invention provides a plurality of particles for drug delivery that can be obtained or obtained by the foregoing methods.
In another aspect, there is provided a method of making a composition, comprising making a plurality of particles according to a method as defined herein and formulating said particles in an aqueous environment. In some embodiments, wherein the composition is an immunogenic composition, said environment comprises an antigen and / or an adjuvant. Said antigen and / or said adjuvant may, for example, be as defined herein in relation to the immunogenic compositions of the invention. The invention further provides, in another aspect, a method of making a composition, such as an immunogenic composition, comprising a plurality of particles for administering drugs comprising a biologically active cargo within a matrix, in a which matrix comprises a polymer, comprising the steps of: i. introducing a plurality of particles, fabricated according to a method as defined herein, into an acidic aqueous environment, such that the acidic environment protonates the polymer of the matrix rendering the polymer insoluble in said environment; ii. elevating the pH of the aqueous acidic environment to a sub-physiological pH that is acceptable for parenteral administration while maintaining the insoluble state of the matrix polymer in said environment; and possibly iii. formulating said particles in an aqueous environment of sub-physiological pH.
In step (i) of this process, referred to herein as "stabilization", the particles are contacted with a "stabilizing solution" at acidic pH, producing protonation of the polymer of the matrix rendering the polymer insoluble. For example, for particles comprising a matrix polymer initially containing COOH groups predominantly in the negatively charged ionized form (i.e., COO-), such protonation alters the balance in favor of the COOH form of neutral charge. Protonation of the polymer in the matrix causes the insolubility of the particles in the stabilizing solution. The pH of the stabilization solution should be such that when the particles are brought into contact with the stabilizing solution, the protonation rate exceeds the dissolution rate. In some embodiments, the stabilizing solution has a pH in the range of 1 to 5, such as about or exactly pH 3.5 or 4.5. Such "contacting" may be, for example, by sprinkling the dry particles into the stabilizing solution while continuing the stirring of the latter. The duration of such contact (i.e., before step (ii) begins) may be up to 60, 50, 40, 30, 20, 10 or 5 minutes, or these values may be delimit respectively the upper end of a time range that is linked to the lower end by 1 minute.
In step (ii), known herein as "neutralization", the environment containing the insoluble particles is adjusted to a pH that is compatible with parenteral administration. Such a pH should clearly be below the threshold pH above which the particles will release their cargo, i.e. the pH must be sub-physiological. Both "sub-physiological" and "acceptable for parenteral administration" in this sense must be relative to the particular target site of administration of / to the pathway in the particular intended subject. This pH adjustment is carried out, for example, by continuous or stepwise addition of a higher pH solution (for example, by addition of a buffer which is less acidic than the stabilizing solution), to be performed at a pH value of appropriate rate that maintains the insoluble state of the polymer matrix. Thus, in some embodiments, step (ii) comprises increasing the pH of the aqueous environment to a sub-physiological pH in a stepwise manner. In some embodiments, the pH of the aqueous environment is increased from 0.1 to 10 pH units per minute, such as 0.5, 1, 2 or 5 pH units per minute, particularly 0.5 units. pH per minute. For clarification purposes, this means that every minute between the first adjustment and reaching the sub-physiological and acceptable pH for finally adjusted parenteral use, the pH increase of the solution is within range from 0.1 to 10 pH units. The combined steps of stabilization and neutralization are referred to here as "transition".
In the optional step (iii), the particles are formulated in an aqueous environment of subphysiological pH, i.e. the solution containing the particles at sub-physiological pH is combined with the other components to produce a composition, which composition must also be at subphysiological pH so that the particles retain the cargo during storage. These "other components" present within the aqueous composition of step (iii) may comprise formulation excipients such as buffers, tonicity modifiers, preservatives, adjuvants, etc., as well as active ingredients. such as drug compounds or antigens. In particular, the aqueous environment of step (iii) may be as defined herein for the composition of the invention. In another aspect, the invention provides a composition, such as an immunogenic composition, obtainable or obtained by the foregoing methods.
The following examples are provided to illustrate particular features and / or particular embodiments. These examples should not be construed as limiting the invention to the particular features or embodiments described.
Examples
Example 1 - Preparation of Particles and Formulations Containing Particles
Particle Manufacturing: The particles for drug delivery were manufactured. First, a series of stock solutions has been prepared. A homogeneous aqueous solution of approximately 10% by weight of PMMA-co-PMAA (Mm ~ 125 kDa, molar ratio of MMA / MAA = 2/1, Eudragit® S100, Evonik Industries) was manufactured by dissolving the polymer at a pH of approximately 8. A homogeneous aqueous solution of approximately 15% by weight of PVP (2.5 kDa, Polysciences, Inc.) was prepared. The concentration of the tetanus toxoid-type Haemophilus influenzae polysaccharide (Hib-TT) conjugate solution was 0.0946% by weight in water.
The following volumes were combined to produce the mother stock solution: 19,000 ml of PMMA-co-PMAA, 12.667 ml of PVP, 26.540 ml of Hib-TT, and 2.11 ml of PPI water. Based on the solid components, the stock solution of the particles had the following percentage weight ratio: 49.67% by weight of PMMA-co-PMAA, 49.67% by weight of PVP, and 0.66% by weight. weight of Hib-TT. The resulting stock solution was cast at room temperature onto a 0.005 inch (0.127 mm) thick PET film using a No. 10 Mayer bar. To form the particles for drug delivery, the film was was pre-rolled against a PRINT® 6 μm ring-shaped mold (Liquidia Technologies, Inc., Morrisville, NC). The mold was then filled by passing through a rolling instrument at 290 ° F (143 ° C) at 2 feet (60 cm) per minute. Particles for drug delivery were removed from the film by mechanical scraping under dry conditions (i.e., less than 30% relative humidity) using a scraper.
Particle Transition: Under dry conditions (10 to 30% relative humidity), approximately 1.000 g of particles for the administration of drugs was sprayed onto 40 ml of 0.2 M sodium succinate pH 3.5 / PEG400, 50/50 by volume with rapid stirring. The particles were stirred for about 10 minutes. After 10 minutes, aliquots of 4x20 ml of 200 mM sodium maleate pH 6.1 were added to the suspension, with 1 to 2 minutes of agitation between each aliquot addition.
The suspension was divided into four polycarbonate tubes, approximately 30 ml per tube. The suspension was pelleted by centrifugation at 18,000 x g for approximately 10 minutes at 4 ° C. The supernatant was removed and discarded. Each pellet was resuspended in 15 ml of 200 mM sodium maleate pH 6.1 and two tubes were combined into one tube. The suspension was pelleted by centrifugation at 18,000 x g for approximately 10 minutes at 4 ° C. The supernatant was removed and discarded. The pellet was resuspended in 30 ml of 200 mM sodium maleate pH 6.1. The suspension was pelleted by centrifugation at 18,000 x g for approximately 10 minutes at 4 ° C. The supernatant was removed and discarded. The pellet was resuspended in 30 ml of 200 mM sodium maleate pH 6.1. The suspension of the particles was diluted by combining 15 ml of the suspension with 30 ml of 10 mM sodium maleate pH 6.1.
The suspension of the particles was filtered through a nylon net of 41 μη in 10 mM sodium maleate pH 6.1. The filtrate was concentrated by centrifugation of the suspension at 19,000 x g for approximately 20 minutes at 4 ° C. The concentrated filtrate was pelleted by centrifugation at 19,000x for approximately 20 minutes at 4 ° C. The supernatant was removed and discarded. The pellet was resuspended in 40 ml of 10 mM sodium maleate pH 6.1 for washing. The particles were washed twice more for a total of three washes. The particles were again agglomerated and resuspended in a small volume (~ 3 ml per tube) of 10 mM sodium maleate pH 6.1.
Particle content was determined gravimetrically as 25.925 mg / ml. The total Hib (conjugate + free) content was determined by HPAEC-PAD to be 37,750 μg / ml.
Particle Formulation - The suspension of the above particles was formulated as follows.
To produce a sample to be coadministered with Infanrix ™ Penta (GSK Vaccines), 700-65, 8 ml of 10 mM sodium maleate / 300 mM NaCl / 0.02% thimerosal pH 6.1 was dispensed into a container. . 8 ml of the suspension of the particles described above were added. 62.297 ml of 10 mM sodium maleate / 150 mM NaCl / 0.01% thimerosal, pH 6.1 was added producing a particle concentration of approximately 2.649 mg / ml.
To produce a sample containing Infanrix ™ Penta, 700-66, 6 ml of the particle suspension described above was agglomerated. The pellet was resuspended in 1.021 ml of 10 mM sodium maleate pH 6.1 and 2.669 ml of 210 mM sodium maleate / 0.22% thimerosal pH 6.1. 5 ml of the resuspended particles were removed and 50 ml of Infanrix ™ Penta was added to the 5 ml.
To produce a second sample containing Infanrix ™ Penta, [700-83-02], 5.0 ml of the particle suspension described above was agglomerated. The pellet was resuspended in 0.787 ml of 10 mM sodium maleate pH 6.1 and 2.224 ml of 210 mM sodium maleate / 0.22% thimerosal pH 6.1. 3 ml of the resuspended particles were removed and 30 ml of Infanrix ™ Penta was added to the 3 ml.
The formulations were aliquoted into flasks and stored at 4 ° C until use. As used in these examples, the liquid medium in which the particles of the respective samples were resuspended will be referred to as the "storage buffer".
EXAMPLE 2 Stability of Formulated Particles of HPAEC-PAD Example 1: Quantification with HPAEC-PAD was carried out on samples containing the particles for the administration of drugs formulated in storage buffer according to Example 1. Samples were analyzed for total oligo / polysaccharide, which encompassed oligo / polysaccharide ("Hib") both conjugated (Hib-TT or Hib-CRM) and unconjugated ("free").
Some samples were also analyzed for free Hib.
In the analysis of samples containing the particles, particularly those containing no aluminum adjuvant, both the drug delivery particles and the storage buffer were analyzed for total Hib (conjugate + free). . In these cases, the particles for drug delivery were recovered from the storage buffer using centrifugation. The storage buffer (supernatant) was removed and stored for analysis. Particles for drug delivery (pellet) were resuspended in the same volume of storage buffer that was recovered in the supernatant. Particles for the administration of resuspended drugs were then triggered to "release" the cargo by the addition of base to produce a suspension of triggered drug particles. Before the analysis, with the naked eye, the sample was clear. These samples were analyzed for both total and free Hib for both pellet and supernatant.
For example, 500 μ! of a sample having a particle concentration of approximately 2 mg / ml were centrifuged to separate the particles for drug delivery from the storage buffer. The supernatant was measured, collected, and stored for analysis. A volume of 10 mM sodium maleate pH 6.1 equal to the volume of supernatant removed was added to the pellet to resuspend the particles for drug delivery. To trigger / dissolve the particles for drug delivery, 4-7 μΐ of 0.5 N sodium hydroxide was added to the resuspended particles to raise the pH to approximately 7.0-7.5, targeting approximately 7.2. The added base volume was adjusted as needed to maintain the ratio of the base to the particles as provided in this example. For some samples, particularly those containing aluminum adjuvant, total Hib was determined. In these cases, the particles for drug delivery were not recovered from the storage buffer using centrifugation. The sample was triggered by the addition of base to produce a suspension of triggered drug particles. Before the analysis, with the naked eye, the sample was clear. These samples were analyzed for total Hib. This measure of total Hib reflected the total Hib contained in the sample (both particles for drug delivery and storage buffer).
For example, 500 μ! of a sample containing a particle concentration of approximately 2 mg / ml were triggered by the addition of base. To the sample, 4-7 μl of 0.5 N sodium hydroxide was added to raise the pH of the sample to approximately 7.0-7.5, targeting approximately 7.2. The added base volume was adjusted as needed to maintain the ratio of the base to the particles as provided in this example.
Stallion / control preparation - Standards: Hib standards were prepared in the concentration range of interest. For example, standards at 0.625 μg / ml, 1.25 μg / ml, 2.50 μg / ml, 5.00 μg / ml, 10.0 μg / ml, 15.0 μg / ml, 20.0 μg / ml, and 25.0 μg / ml were prepared by dilution of a known concentration of Hib using 10 mM sodium maleate pH 6.1. Witness: possibly, control samples may be included in the analysis. For example, HIBERIX ™ [conjugate vaccine for Haemophilus b (tetanus toxoid conjugate)] having a known concentration can be included as a control sample. If HIBERIX ™ is used, a first control sample can be produced by reconstituting the lyophilized vaccine using 0.9% NaCl as described in the Prescribing Information. To produce a second control sample, a portion of the reconstituted vaccine is then diluted tenth using more than 0.9% NaCl. Both control samples can be analyzed.
Analysis of Free Polysaccharide: To analyze the free oligo / polysaccharide, deoxycholate was used to precipitate and remove the conjugated polysaccharide from the sample of interest.
A 1% (w / v) deoxycholate (DOC) solution in deionized water was prepared in advance and stored at -20 ° C in aliquots for no more than six months. Approximately 1 g of deoxycholate was added to 90 ml of deionized water with stirring. After complete dissolution of the deoxycholate, the pH of the solution was slowly increased to 6.8 by the dropwise addition of 1 M sodium hydroxide. The precipitate formed after the addition of each drop of base was allowed to dissolve before the addition of another drop of base. After adjusting the pH, deionized water was added to bring the final volume to 100 ml. To prepare a sample for analysis, 100 μ! supernatant or suspension of the triggered drug particles (from the pellet that was resuspended) were placed in a 1.5 ml LoBind microcentrifuge tube (Eppendorf) and 100 μl of the 1% DOC solution. were added and the tube was vortexed to ensure uniform mixing. The sample was cooled on ice for approximately 30 minutes. After 30 minutes, 10 μl of 1 N HCl was added and the sample was vortexed. The conjugated oligo / polysaccharide, Hib-TT or Hib-CRM, bound to DOC creating a precipitate. The precipitate was removed using centrifugation at 19,500 x g for 15 minutes at approximately 4 ° C and room temperature. The pellet was discarded and the supernatant was stored for free Hib analysis.
Sample Preparation - Ribitol ribose 5-phosphate hydrolysis: The samples were hydrolysed and produced ribitol ribose 5-phosphate as an analyte of interest.
Standards / control: To 100 μl of standard or control, 150 μl of 1 N sodium hydroxide were added. The sample was vortexed to uniformly mix the base in the sample. The sample was hydrolysed at room temperature for approximately 12 hours.
Samples of interest: at 100 μl of sample (total oligo / polysaccharide of the sample, total polysaccharide of the pellet, total polysaccharide of the supernatant, free polysaccharide of the pellet, or free polysaccharide of the supernatant), 150 μ! 1N sodium hydroxide were added. The sample was vortexed to uniformly mix the base in the sample. The sample was hydrolysed at room temperature for approximately 12 hours.
Instrument parameters
Sample concentrations were calculated by comparing the peak area of ribitol ribose 5-phosphate in the sample of interest to a fit of the standards to linear regression using the least squares method. The concentration has been reported in μg / ml. The vials containing sample 700-65 were stored at 4 ° C and the distribution (i.e., particle associated or not) of Hib-TT conjugate and unconjugated ("free") Hib was was evaluated over time (T = 0, 1, 8, 15, 18, 28, 49, 64, 84, 242, 593, 649, and 663 days) by HPAEC-PAD. Table 1 shows the proportion (in% by weight) of the total Hib (i.e., conjugate + free) measured at each time point, which is in the pellet fraction (i.e. ie, associated with the particles) and in the fraction of the supernatant, that is to say, not associated with the particles. FIG. 1 represents the part of the total Hib (conjugate + free) found in the fraction of the supernatant, that is to say not associated with the particles.
Table 1
Table 2 and Figure 2 show the proportion of free (unconjugated) Hib (in% by weight) as a percentage of the total Hib (i.e., conjugate + free) contained in the sample (collectively in the pellet and supernatant), measured at each time point. At each time point, both total Hib (conjugated and unconjugated) and free Hib (unconjugated) were determined for pellet (particles) and supernatant.
pH: the pH of samples 700-65 and 700-66 were also monitored over time. An aliquot of 200 to 300 μ! of the test sample was dispensed into a 1.5 ml tube and the pH was determined using a pH meter (Hach Corporation, Model IQ 150) with an ISFET probe (Hach Corporation, pH 17.SS) . Prior to pH determination, the pH meter was calibrated using standards of pH 4.01 (Orion 910104) and pH 7.00 (Orion 910107). Table 3 shows the pH values collected at several time intervals (NA = sample not analyzed).
Example 3 - Preparation of Other Particles and Formulations Containing Particles
Particle Making: Drug delivery particles were made as in Example 1 except that the concentration of the Hib-TT stock solution was 0.0957 wt.% In water.
Particle Transition: Under dry conditions (20 to 30% relative humidity), approximately 800 mg of particles for the administration of drugs were sprayed onto 40 ml of 0.1 M sodium succinate buffer pH 4, 5 / PEG400, 50/50 by volume with rapid stirring.
The particles were stirred for about 5 to 10 minutes. After 5 to 10 minutes, aliquots of 4 x 20 ml of 0.2 M sodium maleate pH 6.1 were added to the suspension, with 1 to 2 minutes of agitation between each aliquot addition.
The transition was continued as in Example 1, with minor modifications: after dividing the suspension into four polycarbonate tubes, it was agglomerated by centrifugation at 18,000 xg for at least 15 minutes at 4 ° C, and the following centrifugation was performed at 18,000 xg for 5 minutes.
Particle content was determined gravimetrically as 14.12 mg / ml. The total Hib content (conjugate + free) was determined to be 39,595 μg / ml by HPAEC-PAD.
Particle Formulation: The suspension of the above particles was formulated as follows. Sample 841-57-1 was produced for coadministration with Infanrix ™ Penta. To produce sample 841-57-1, a volume of 7 ml of the particle suspension described above was diluted using 7 ml of 10 mM sodium maleate / 300 mM sodium chloride / 0.01 % thimerosal, pH 6.1 and 25.136 ml of 10 mM sodium maleate pH 6.1 producing a particle concentration of 2.526 mg / ml. Sample 841-57-2 was produced. To produce sample 841-57-2, 11 ml of the particle suspension described above was agglomerated. The pellet was resuspended in 0.878 ml of 10 mM sodium maleate pH 6.1 and 2.795 ml of 210 mM sodium maleate / 0.21% thimerosal pH 6.1. 5 ml of the resuspended particles were removed and 50 ml of Infanrix ™ Penta was added to the 5 ml.
Samples containing the particles were aliquoted in flasks for various in vivo studies and stability studies. Sample 841-57-2S was produced by aliquoting the 841-57-2 sample in syringes for a stability study. The formulations were stored at 4 ° C until use.
EXAMPLE 4 Stability of Formulated Particles of Example 3 HPAEC-PAD: the vials containing the samples of Example 3 were stored at 4 ° C. and the distribution (that is, associated with the particles, or no) Hib-TT conjugate and unconjugated Hib ("free") was evaluated in time (T = 0, 14, 34, 67, 94, 124 and 199 days) by HPAEC-PAD, as described for example 2. For sample 841-57-1 (Hib-TT-containing particles stored in buffer at pH 6.1). Table 4 shows the proportion (in% by weight) of the total Hib (i.e., conjugate + free), measured at each time point, which is found in the pellet fraction (i.e. ie associated with the particles) and in the fraction of the supernatant, that is to say, not associated with the particles. Figure 3 shows the portion of the total Hib (conjugate + free) found in the supernatant fraction, i.e., not associated with the particles.
Table 4
Table 5 and Figure 4 show the proportion of free (unconjugated) Hib (in% by weight) as a percentage of the total Hib (i.e., conjugate + free) contained in the sample (collectively in the pellet and supernatant), measured at each time point. At each time point, both total Hib (conjugated and unconjugated) and free Hib (unconjugated) were determined for pellet (particles) and supernatant.
Table 5
For samples 841-57-2 and 841-57-2S (Hib-TT-containing particles stored in InfanrixTM Penta, the latter in a syringe), and 841-57-1 discussed above, Table 6 and Figure Show the concentration (g / ml) of total Hib (i.e., conjugate + free) measured by HPAEC-PAD at the same time points. The reported concentration reflects the total Hib in the sample (particles as well as aqueous environment).
Table 6
Example 5 - Sensitivity of the particles to the triggering pH Release of the cargo around the threshold pH Manufacture of the particles: particles for the administration of medicaments were manufactured. First, a series of stock solutions has been prepared. A homogeneous aqueous solution of 10% by weight of PMMA-co-PMAA (Mm ~ 125 kDa, molar ratio of MMA / MAA = 2/1, Eudragit® S100, Evonik Industries) was manufactured by dissolving the polymer at a pH of approximately 8.0. A homogeneous aqueous solution of 15% by weight of PVP (2.5 kDa, Polysciences, Inc.) was prepared. The concentration of the Hib-TT solution was 0.0902% by weight in water.
The following volumes were combined to produce the mother stock solution: 17.325 ml of PMMA-co-PMAA, 11.550 ml of PVP, 25.575 ml of Hib-TT, and 0.550 ml of PPI water. Based on the solid components, the stock solution of the particles had the following percentage weight ratio: 49.67% by weight of PMMA-co-PMAA, 49.67% by weight of PVP, and 0.66% by weight. weight of Hib-TT. The resulting stock solution was cast at room temperature onto a 0.004 inch (0.102 mm) thick PET film using a No. 10 Mayer bar. To form the particles for drug delivery, the film was laminated against a PRINT® 6 μm ring-shaped mold (Liquidia Technologies, Inc., Morrisville, NC). The film / mold was passed through a rolling instrument at 290 ° F (143 ° C) at 2 feet (60 cm) / min. The laminate was cooled slowly in air. The particles, 855-51, were collected under dry conditions (20% relative humidity) using a scraper. Particles for the administration of 855-51 drugs have been transitioned into various vehicles to produce a series of formulations using the following procedures.
Particle Transition: Sample 855-61-1 was produced. Approximately 0.95 g of 855-51 was added to a tube containing 40 ml of 0.2 M sodium succinate pH 3.5 / PEG400, 50/50 by volume with stirring. After stirring for approximately 10 minutes, aliquots of 4 x 20 ml of 200 mM sodium maleate pH 6.1 were added with approximately one minute of agitation between the additions. The suspension was divided into 4 tubes (~ 30 ml per tube) and the particles were pelleted by centrifugation at 18,000 x g at 4 ° C for approximately 20 minutes. The supernatant was discarded and the particles resuspended using 15 ml of 200 mM sodium maleate pH 6.1 per tube. Four tubes were combined into two tubes and the particles were agglomerated. The supernatant was removed and discarded. Each pellet was resuspended in 30 ml of 200 mM sodium maleate pH 6.1. The suspension was pelleted by centrifugation at 19,000 x g at 4 ° C for approximately 10 minutes. The supernatant was removed and discarded. Each pellet was resuspended in 15 ml of 10 mM sodium maleate pH 6.1 and the suspension was diluted 1/3 with the addition of 30 ml of 10 mM sodium maleate pH 6.1 per tube.
The suspension of the particles was filtered through a nylon net of 41 μm in 10 mM sodium maleate pH 6.1. The filtrate was concentrated by centrifugation of the suspension at 19,000 x g at 4 ° C for approximately 15 minutes. The concentrated filtrate was pelleted by centrifugation at 20,000 x g at 4 ° C for approximately 20 minutes. The supernatant was removed and discarded. The pellet was resuspended in 40 ml of 10 mM sodium maleate pH 6.1 for washing. The particles were washed three more times for a total of four washes. The particles were again agglomerated and resuspended in a small volume (~ 3 ml per tube) of 10 mM sodium maleate pH 6.1. The suspension was analyzed for particle concentration and polysaccharide content by HPAEC-PAD. The particle concentration of 855-61-1 was 28.26 mg / ml, and contained 16.802 μg / ml of total Hib (conjugate + free).
Variable pH Incubation: An aliquot of Sample 855-61-1, particle concentration of 28.26 mg / ml in 10 mM sodium maleate pH 6.1, was diluted to approximately 2 mg / ml using 10 mM sodium maleate pH 6.1 to produce 855-118-A.
Fourteen 0.5 ml aliquots were dispensed into 1.5 ml tubes which were then centrifuged at 17,000 x g for approximately 20 minutes at 4 ° C to agglomerate the particles for drug delivery. The supernatant was removed and discarded. The pellets were resuspended in a series of increasing pH buffers. The buffers were produced using 1X PBS at the following pH values: 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.2, and 7.4. The particles were held in the respective buffers at room temperature without stirring for about four hours. After four hours, the particle samples were agglomerated at 17,000 x for approximately 20 minutes at 4 ° C. The supernatant was collected for analysis and the pellet resuspended in 1X PBS pH 7.4 giving a clear suspension. Samples (supernatant and pellet) were analyzed for total Hib (conjugate + free) by HPAEC-PAD. Table 7 and Figure 6 show the results, showing a marked increase in cargo release (total Hib in the supernatant) as the pH increases from 6.5 to 7.4.
Table 7
Release of the cargo triggered by the pH at various concentrations of the particles Synthesis of the polymer
A series of PMMA-co-PMAA polymers was synthesized according to the reaction scheme above. A target molar ratio of 2/1 (MMA / MAA) was maintained. Table 8 details the raw materials used.
Raw materials
Synthesis Process: The inhibitors were removed from the reactive methyl methacrylate monomer (Aldrich M55909) by passing the monomer through a column filled with beads to remove the inhibitors (Aldrich 306312). The procedure was repeated with methyl acrylic acid (Aldrich 155721) using a column with fresh beads. The monomers with the inhibitors removed were stored in the dark before use.
Volumes of the various reagents and solvents were calculated and are listed in Table 9. The materials were added to a 25 or 50 ml round-bottomed flask. After adding the reagents, the vial was sealed and swept a second time with dry nitrogen for at least five minutes. A reflux condenser was added and the system purged with nitrogen. The flask was heated to 80 ° C. The reaction was carried out overnight at 80 ° C with nitrogen under stirring reflux conditions.
The next morning, the reaction was removed from the heat and allowed to cool. The gelatinous product was dissolved in 15 ml of ACS grade THF. The polymer was drip precipitated in 300 ml of ice-cold ethyl ether (anhydrous, ACS grade). The precipitated polymer was recovered using centrifugation (3000 x 3 minutes). The precipitate was transferred to a 50 ml Erlenmeyer flask and dissolved in 20 ml of ACS grade THF. The polymer was precipitated a second time in ice-cold ethyl ether with stirring (anhydrous, ACS grade). The precipitated polymer was recovered using centrifugation (3000 g x 3 minutes) and washed with 4 x 50 ml of ethyl ether (anhydrous, ACS grade) at room temperature. The polymer pellet was then transferred to an observation glass and allowed to dry under vacuum for twenty minutes.
After drying under vacuum, the polymer was manually ground to a fine powder using a mortar and pestle. The polymer was transferred to a pre-weighed flask and placed in a vacuum oven at 60 ° C to dry overnight. The next morning, the oven was allowed to cool to room temperature while maintaining a vacuum. The resulting polymer was weighed for yield, analyzed by GPC for molecular weight, and analyzed by NMR for MMA content.
Table 9
Volumes / masses of raw materials
Polymer Analysis: GPC and NMR: The resulting polymers were analyzed for GPC molecular weight and PMMA content by NMR. For NMR analysis, the percentage of PMMA was determined by comparing the integration of the proton signal from the methyl group on the methacrylate with the proton signal from the proton of the acid onto the methacrylic acid. Table 10 summarizes the results of the analysis. The overall yield is also included in Table 10. The use of a chain transfer agent reduced the molecular weight of the resulting polymer. Increasing the amount of the chain transfer agent further reduced the molecular weight of the resulting polymer.
Results of molecular weights and PMMA by NMR
Particle manufacturing: Particles for drug delivery were made. First, a series of stock solutions has been prepared. A homogeneous aqueous solution of 6% by weight PMMA-co-PMAA (Lots 852-2-2, 852-4-3, 852-4-4, 852-9-6, 852-13-7, and 852-13 -8) was made by dissolving the polymer at a pH of approximately 8 to 12. A homogeneous aqueous solution of 15% by weight of PVP (2.5 kDa, Polysciences, Inc.) was prepared. The concentration of the Hib-TT solution was 0.0902% by weight in water.
The following volumes were combined to produce the stock solution of the particles: 52.560 ml of PMMA-co-PMAA, 21.024 ml of PVP, 46.200 ml of Hib-TT, and 0.216 ml of PPI water. Based on the solid components, the stock solution of the particles had the following percentage weight ratio: 49.67% by weight of PMMA-co-PMAA, 49.67% by weight of PVP, and 0.66% by weight. weight of Hib-TT. The resulting stock solution was cast at room temperature onto a 0.005 inch (0.127 mm) thick PET film using a Mayer bar No. 12. To form the particles for drug delivery, the film was laminated against a PRINT® 6 μm ring-shaped mold (Liquidia Technologies, Inc., Morrisville, NC). The film / mold was passed through a rolling instrument at 290 ° F (143 ° C) and a line speed of 2 feet (60 cm) / min. The laminate was cooled slowly in air. The particles were collected under dry conditions (5 to 15% relative humidity) using a scraper. The batch number of the particles was 855-12.
Particle Transition: Harvested particles were divided into four samples. Under dry conditions (10 to 20% relative humidity, approximately 900 mg of drug delivery particles were sprayed onto 40 ml of stabilizing solution (0.2 M sodium succinate buffer pH 3.5 / PEG400, 50/50 by volume) with stirring The particles were stirred for about 10 minutes After 10 minutes of stirring, the sample was moved under a sterile hood and 8 x 10 ml aliquots of 400 mM Sodium maleate pH 6.1 was added to the suspension, with stirring for 1 minute between each aliquot addition.The suspension was divided into four polycarbonate tubes, approximately 30 ml per tube.The suspension was agglomerated by centrifugation at 18,000 xg for approximately 25 minutes at 4 ° C. The supernatant was removed and discarded, each pellet was resuspended in 15 ml of 400 mM sodium maleate pH 6.1 and four tubes were combined in The suspension was pelleted by centrifugation at 18,000 x g for approximately 25 minutes at 4 ° C. The supernatant was removed and discarded. Each pellet was resuspended in 30 ml of 200 mM sodium maleate pH 6.1. The suspension was pelleted by centrifugation at 19,000 x g for approximately 10 minutes at 4 ° C. The supernatant was removed and discarded. Each pellet was resuspended in 15 ml of 10 mM sodium maleate pH 6.1 and diluted with another 30 ml of 10 mM sodium maleate pH 6.1. The suspension of the particles was filtered through a nylon net of 41 μm in 10 mM sodium maleate pH 6.1. The filtrate was concentrated by dividing the material into two tubes and centrifuging the suspension at 19,000 x g for approximately 10 minutes at 4 ° C. The concentrated filtrate was pelleted by centrifugation at 19,000 x g for approximately 10 minutes at 4 ° C. The supernatant was removed and discarded. Each pellet was resuspended in 40 ml of 10 mM sodium maleate pH 6.1 for washing. The particles were washed twice more for a total of three washes. The particles were resuspended in approximately 3 ml of 10 mM sodium maleate pH 6.1 per tube. The particle concentration of the suspension was determined to be 12.5 mg / ml. An aliquot of the suspension was tested for total Hib concentration by HPAEC-PAD. The result was 17.52 μg / ml. The particle concentration of the suspension was 12.50 mg / ml. The batch number for the particle suspension was 855-14. The sample was stored at 4 ° C until further use.
Incubation at Variable pH and Concentration of Particles: The formulation of 855-14 particles, with a particle concentration of 12.5 mg / ml in 10 mM sodium maleate pH 6.1, was dispensed into vials. For a sample with a particle concentration of 2 mg / ml, 80 μ! have been distributed in the bottle; for 1 mg / ml, 40 μl were dispensed; and for 0.5 mg / ml, 20 μl were dispensed. The dispensed aliquot was agglomerated at 17,000 x g for approximately 20 minutes at 4 ° C to reconstitute the particles for drug delivery at each concentration of particles of interest. 1X PBS pH 6.8 and pH 8.0 was prepared. 1X PBS was also used as is with a pH of 7.4. Samples agglomerated at 2 mg / ml were resuspended in 1X PBS at pH 6.8, 7.4, and 8.0. The procedure was repeated for the 1 mg / ml and 0.5 mg / ml samples as well. After incubation for four hours with agitation at ambient conditions, the samples were agglomerated at 17,000 x g for approximately 20 minutes at 4 ° C. The supernatant was collected for analysis and the pellet was resuspended in 1X PBS pH 7.4. Samples (supernatant and pellet) were analyzed for total Hib by HPAEC-PAD. Table 11 and Figure 7 present the results obtained for the samples at various particle concentrations and pH values.
Table 11
The samples demonstrated total Hib release dependent on both particle concentration and pH; for a given particle concentration, higher pH levels led to a higher percentage of Hib in the supernatant. For a given pH value, a lower particle concentration resulted in a higher percentage of Hib in the supernatant.
Dissolution of the particles at the threshold pH by electron microscopy: after approximately one year of storage at 4 ° C., 700 μ! from sample 700-65 were distributed in a vial in dram. 0.01 N NaOH was added in increments using a 250 μl Hamilton syringe equipped with a 27 gauge needle. The pH was monitored using a pH meter. A small aliquot was taken periodically and analyzed using an optical microscope. Table 12 details the data collected in the study. Figure 8A illustrates the optical images.
Table 12
In Figure 8A, the optical images show that at pH 6.40, the particles for drug delivery remained insoluble. At pH 6.64, the drug delivery particles began to swell and dissolve. At pH 6.72, optical imaging showed that most of the particles were dissolved and a small portion of the swollen particles remained. At pH 6.80, optical imaging showed mostly dissolved particles and some large aggregates consisting of swollen and shaped particles. The aggregates appeared to dissolve over time, after approximately 20 minutes at pH 6.80. The studies were repeated using particles for the administration of drugs that had recently been manufactured. After approximately one week of storage at 4 ° C, 700 μ! of sample 817-83-1 were distributed in a vial in dram. 0.02 N NaOH was added in increments using a 250 μl Hamilton syringe equipped with a 27 gauge needle. The NaOH additions were at least 2 minutes apart. 0.02 N NaOH was used in this study to minimize any potential dilution effects. Table 13 details the data collected in the study. Figure 8A shows the optical images.
Table 13
In Figure 8A, the optical images show that at pH 6.41, the particles for drug delivery remained insoluble. At pH 6.56, the particles for administration of drugs swell. At pH 6.74, optical imaging showed that most of the particles were dissolved. At pH 6.85, optical imaging showed mostly dissolved particles and a small amount of particulate debris. Based on the experimental data, there were small differences between the particles for the administration of drugs manufactured and stored for approximately one week and the particles for administration of drugs manufactured and stored for approximately one year. Particles for drug delivery began to swell when exposed to pH 6.50 to 6.55. The majority of the drug delivery particles were dissolved when exposed to pH 6.65 to 6.74. Particles for drug delivery were almost completely dissolved when exposed to pH 6.85 or higher.
Kinetics of particle dissolution by optical microscopy
Particle manufacturing: Particles for drug delivery were made. First, a series of stock solutions has been prepared. A homogeneous aqueous solution of 5% by weight of PMMA-co-PMAA (~ 125 kDa, molar ratio of MMA / MAA = 2/1, Eudragit® S100, Evonik Industries) was manufactured by dissolving the polymer at a pH of approximately 8. A second homogeneous aqueous solution of 5% by weight of PMMA-co-PMAA (Lot 831-30, Example 5) was manufactured in the same way. A homogeneous aqueous solution of 15% by weight of PVP (2.5 kDa, Polysciences, Inc.) was prepared. The concentration of the Hib-TT solution was 897 μg / ml. The following volumes were combined to produce a stock solution of particles: 1,932 ml of PMMA-co-PMAA, 0,644 ml of PVP, and 1,424 ml of Hib-TT. Based on the solid components, the stock solution of the particles had the following percentage weight ratio: 49.67% by weight of PMMA-co-PMAA, 49.67% by weight of PVP, and 0.66% by weight. weight of Hib-TT. A stock solution of the particles was produced with each of Eudragit® S100 and [831-30]. The resulting stock solutions were poured at room temperature onto a crude PET film using a Mayer No. 13 bar. To form the particles for drug delivery, the film was laminated against a ring-shaped mold. of 6 μm PRINT® (Liquidia Technologies, Inc., Morrisville, NC) using a bench-top rolling instrument. Particles for drug delivery were removed from the film by mechanical scraping under dry conditions (i.e., less than 30% relative humidity). The process was repeated with both stock solutions.
Particle Transition: For each set of particles, under dry conditions (10 to 30% relative humidity), approximately 90 to 100 mg of the drug delivery particles were sprayed onto 4 ml of 0.2 M sodium succinate pH 3.5 / PEG400, 50/50 by volume with rapid stirring.
The particles were stirred for about five minutes. After about five minutes, aliquots of 4 x 2 ml of 200 mM sodium maleate pH were added to the suspension, with about one minute of agitation between each aliquot addition. The suspension was divided into six tubes, approximately 2 ml per tube. The suspension was pelleted by centrifugation at 17,000 x g for at least 5 minutes at 4 ° C. The supernatant was removed and discarded. Each pellet was resuspended in 1 ml of 200 mM sodium maleate pH 6.1 and two tubes were combined into one tube. The suspension was pelleted by centrifugation at 17,000 x g for at least 5 minutes at 4 ° C. The supernatant was removed and discarded. The pellet was resuspended in 2 ml of 200 mM sodium maleate pH 6.1. The suspension was pelleted by centrifugation at 12,000 x g for approximately 5 minutes at 4 ° C. The supernatant was removed and discarded. The pellet was resuspended in 2 ml of 10 mM sodium maleate pH 6.1. The suspension of the particles was diluted by combining 1 ml of the suspension with 1 ml of sodium maleate pH 6.1.
The suspension of the particles was filtered through a nylon net of 41 μm in 10 mM sodium maleate pH 6.1. The filtrate was concentrated by centrifugation of the suspension at 12,000 x g for at least 3 minutes at 4 ° C. The concentrated filtrate was pelleted by centrifugation at 12,000 x g for approximately 3 minutes at 4 ° C. The supernatant was removed and discarded. The pellet was resuspended in 2 ml of 10 mM sodium maleate pH 6.1 for washing. The particles were washed twice more for a total of three washes. The particles were agglomerated at 12,000 x g for approximately 3 minutes at 4 ° C. The supernatant was removed and discarded. The pellet was resuspended in 2 ml of 10 mM sodium maleate / 150 mM NaCl pH 6.1. The particles were agglomerated at 12,000 x g for approximately 3 minutes at 4 ° C. The supernatant was removed and discarded. The pellet was resuspended in 1 ml of 10 mM sodium maleate / 150 mM NaCl pH 6.1.
The suspensions were gravimetrically analyzed for particle concentration. The concentration for sample 841-12-1 (Eudragit® S100 containing the particles) was 7.75 mg / ml. The concentration for sample 841-12-3 (Lot [831-30] containing the particles) was 1.725 mg / ml. The suspensions were diluted or concentrated to a particle concentration of approximately 2 mg / ml. 50 μ! of sample 841-12-1 and sample 841-12-3 were placed in individual vials and shaken at approximately 300 rpm at room temperature. 0.02 N NaOH was added to each sample, 10 μl to 841-12-1 and 18 μl to 841-12-3, and the time was started. At T = 2, 4, 6, 8, and 10 minutes, 2 μl of the sample was taken and evaluated using light microscopy. Figure 8B shows the images. Table 14 summarizes particle observations using microscopy.
Table 14
Example 6 - Thermostability of pH-sensitive particles
A study was conducted to evaluate the thermal stability, measured by free Hib (by HPAEC-PAD) over time (T = 0, 14, 68, and 86 days), for particles for the administration of drugs stored at various temperatures (25, 37, 45 ° C).
Sample 855-72-2: 1, 1 ml of 855-61-1 was combined with 1.1 ml of 10 mM sodium maleate / 300 mM NaCl / 0.02% thimerosal pH 6.1 and 10 ml. 857 ml of 10 mM sodium maleate / 150 mM NaCl / 0.01% thimerosal pH 6.1 to produce a final particle concentration of approximately 2.381 mg / ml with a total Hib calculated content of 20 μg / ml. The production of 85561-1 has been described in Example 5.
Sample 855-72-4: 2.4 ml of 855-14 were combined with 2.4 ml of 10 mM sodium maleate / 300 mM NaCl / 0.02% thimerosal pH 6.1 and 8.340 ml of 10 mM sodium maleate / 150 mM NaCl / 0.01% thimerosal pH 6.1 to produce a final particle concentration of approximately 2.283 mg / ml with a calculated total Hib content of 20 μg / ml. The production of 855-14 was described in Example 5. Compared to 855-72-2 above, 855-72-4 contains particles made from a PMMA-co-PMAA polymer matrix. a different origin, and which were transitioned in 0.4 M, as opposed to 0.2 M, of sodium maleate pH 6.1. 855-72-5: As a control, a sample containing soluble Hib (i.e., no particles) was produced by combining 0.27 ml of Hib-TT (902 μg / ml) with 11.910 ml of 10 mM sodium maleate / 150 mM NaCl / 0.01% thimerosal pH 6.1.
The samples were dispensed into vials in 0.65 ml aliquots and stored at three temperatures: 25 ° C, 37 ° C, and 45 ° C. They were removed from storage at day 0, 14, 68, and 86 (approximately 0, 2, 10, 12 weeks) and analyzed for total Hib and free Hib in both particles (pellet) and the environment. aqueous (supernatant) by HPAEC-PAD. Tables 15 to 17 show the proportion of Hib in free form (non-conjugated) (pellet + supernatant, for the samples containing the particles) at these time points, after subtraction of the respective amounts of free Hib present at T = 0. Figure 9 graphically presents the information for the time points 14, 68 and 86 days at 25, 37 and 45 ° C. There appears to be a protective effect of the particles on Hib-TT integrity at 37 and 45 ° C.
Table 15 855-72-2
Table 16 855-72-4
Table 17 855-72-5
Example 7 In Vivo Study
An in vivo study was performed to demonstrate the ability of pH-sensitive particles to protect the Hib-TT cargo against deleterious interaction with the Infanrix Penta formulation during storage, while allowing the cargo to trigger an immune response to following the administration. An adult rat model was used.
Material and Methods: Hib-TT-containing particles were added to Infanrix Penta to make a hexavalent DTPa-HepB-IPV-Hib composition. After a minimum of 4 weeks storage at 4 ° C, these compositions were administered to the rats in groups 4 and 5, discussed below. Adult female Sprague-Dawley rats 6 weeks old (Ico: OFA-SD) were divided into 5 groups (see below, n = 20 per group). Immunization with one-tenth of human dose administered intramuscularly occurred on days 0, 14, and 28. The animals were bled for serology on days 21 ("7PII") and 35 ("7PIII").
Group 1: Freeze-dried Hib-TT (adsorbed on aluminum phosphate) was reconstituted with Infanrix Penta (= Infanrix Hexa) in a final volume of approximately 625 μl. 50 μ! (Hib dose of 1 μg / ml) were administered extemporaneously (i.e., within one hour of reconstitution).
Group 2: lyophilized Hib-TT (Hiberix) was reconstituted with 625 μl of 150 mM NaCl (Hib dose of 1 μg / ml in 50 μl) and coadministered (at different sites) with Infanrix Penta (50 μl) .
Group 3: the particles containing Hib-TT 84157-1 were diluted in 150 mM NaCl buffer / 10 mM maleate pH 6.1 to a concentration of Hib-TT of 20 μg / ml. 55 μl of the diluted sample were administered and Infanrix Penta (50 μΐ) was coadministered (at a different site).
Group 4: 55 μl of particles containing Hib-TT in Infanrix Penta 841-57-2 were administered after storage at 4 ° C for 4 weeks.
Group 5: 55 μl of Hib-TT-containing particles in Infanrix Penta 700-66 were administered after storage at 4 ° C for 16 months.
Serologic analysis for Hib antigen (PRP): sera from all rats were collected individually seven days after the second (7PII) or third (7PIII) immunization and tested for the presence of polyribosyl-specific IgG antibodies. -ribitol-phosphate (PRP) of Haemophilus influenzae type b according to the following protocol. 96 well plates were sensitized with tyramine PRP (1 μg / ml) in carbonate-bicarbonate buffer (50 mM) and incubated overnight at 4 ° C. The sera from the rats were diluted 1/10 in 0.05% PBS-Tween and serially diluted in the wells of the plates (12 dilutions, 1/2 step). An anti-rat IgG (H + L) polyclonal antibody coupled to peroxidase was added (1/5000 dilution). A colorimetric reaction was observed after the addition of the peroxidase substrate (OPDA), and quenched with HCl (1 M) before reading by spectrophotometry (wavelengths: 490 to 620 nm). For each serum tested and standard added to each plate, a logistic curve with 4 parameters was adapted to the relationship between OD and dilution (Softmaxpro). This allowed the derivation of the title of each sample expressed in STD titles. The statistical procedure employed was a variance analysis (ANOVA) on log10 values with 2 factors (group and TP) using a heterogeneous variance model, that is, identical variances did not have been assumed for different factor rates. The interaction between the 2 factors was tested; the results are provided by the second factor rate since the interactions appeared to be qualitative in nature. Estimates of the geometric mean of the ratios between the groups and their 95% confidence intervals (CI) were obtained by using a retro-transformation on the log10 values. The adjustment for multiplicity was performed using the Tukey method. The 95% confidence intervals adjusted for multiplicity were provided. Hib serology results: The serology results for Hib are shown in Figure 10. In both 7PII and 7PIII, groups 1 and 2 were statistically significantly different, validating the ability of the experiment to demonstrate the occurrence of interference between Hib-TT and Infanrix Penta (apparent in the form of a reduced anti-Hib immune response). At 7PIII, group 1 (extemporaneous reconstitution of freeze-dried Hib-TT with Infanrix Penta = Infanrix Hexa) was statistically significantly different from groups 2 to 5 in which Hib-TT was coadministered (i.e. level of distinct, unmixed sites, groups 2 and 3) with Infanrix Penta and / or was present within a particle for the administration of drugs (groups 3 to 5). Therefore, by mixing Hib-TT-containing particles with Infanrix Penta, a reduction in interference was observed. Note that the lower anti-Hib immune response in group 1 occurs during extemporaneous mixing, i.e., even without storage of the mixture for any extended duration. At 7PIII, no significant difference was found between the co-administration (no mixing) of Hib-TT-containing particles with Infanrix Penta (group 3) and the administration of particles that had been mixed Infanrix Penta for 4 weeks ( group 4). This demonstrates that the particle prevents or minimizes the deleterious interaction between Hib-TT and Infanrix Penta during storage, while allowing access to the Hib-TT of the immune system once administered.
Statistical equivalence was detected between groups 2 and 3 to 7PIII, showing that the formulation of Hib-TT within a particle does not impair its ability to induce an immune response once administered. Groups 4 and 5 have also been shown to be equivalent to 7PIII, suggesting no loss or minimal loss of Hib-TT immunogenicity, and no minimal loss or loss of Hib-TT from the particles during the period. storage time of 4 weeks to more than one year, that is, the Hib-TT in the particles is stable in an aqueous formulation.

权利要求:
Claims (113)
[1]
A plurality of pH sensitive drug delivery particles comprising a biologically active cargo within a matrix, wherein said particles are triggered to release said cargo by being present in an aqueous environment having a higher pH by in relation to the pH of an aqueous environment in which said particles are formulated and maintained before being so initiated.
[2]
The plurality of particles according to claim 1, wherein said matrix is insoluble in an aqueous environment at a sub-physiological pH, and wherein said matrix is soluble in an aqueous environment at a physiological triggering pH.
[3]
3. A plurality of particles according to claim 1 or claim 2, wherein said particles are intact at sub-physiological pH, and wherein when subjecting said particles to a physiological triggering pH, said particles are substantially or completely dissolved or degraded in 24 hours or less.
[4]
4. Plurality of pH-sensitive drug delivery particles comprising a biologically active cargo within a matrix, wherein the amount of cargo released from said plurality of particles when present in an aqueous environment for at least 6 months at a sub-physiological pH is not more than 30% by weight of the total amount of the cargo, and in which upon submission of said particles at a physiological triggering pH, the amount of cargo released in 24 hours or less is not less than 50% by weight of the total quantity of the cargo.
[5]
The plurality of particles according to claim 4, wherein said particles are further defined in claims 1 or 3 and / or said matrix is further defined in claim 2.
[6]
6. Plurality of particles according to any one of claims 2 to 5, wherein said subphysiological pH and physiological pH are defined with respect to the pH of human muscle tissue, especially infant.
[7]
7. Plurality of particles according to any one of claims 2 to 6, wherein said subphysiological pH and physiological pH differ from each other by at least 0.1, 0.2, 0.3, 0, 4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 pH units.
[8]
The plurality of particles according to any one of claims 2 to 7, wherein said subphysiological pH is at or below 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1 or 6.0.
[9]
The plurality of particles according to any one of claims 2 to 8, wherein said physiological pH is at or above 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 or 7.7.
[10]
10. Plurality of particles according to any one of claims 4 to 9, wherein said steps more than 30% by weight of the total amount of the cargo are not more than 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% by weight.
[11]
11. Plurality of particles according to any one of claims 4 to 10, wherein said steps less than 50% by weight of the total amount of the cargo are not less than 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% by weight.
[12]
The plurality of particles according to any one of claims 4 to 11, wherein said at least 6 months are 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 months.
[13]
13. Plurality of particles according to any one of claims 4 to 12, wherein said in 24 hours or less are in 20, 16, 12, 10, 8, 6, 4, 2 or 1 hour (s) or 45, 30, 15, 10 or 5 minutes.
[14]
14. A plurality of particles according to any one of claims 4 to 13, wherein the release of the cargo is as determined in vitro, such as HPAEC-PAD or ELISA.
[15]
15. The plurality of particles according to any one of claims 4 to 14, wherein during said at least 6 months at a sub-physiological pH, said aqueous environment is maintained at a temperature between 2 and 8 ° C, for example at about 4 ° C.
[16]
16. Plurality of particles according to any one of claims 4 to 14, wherein during said at least 6 months at a sub-physiological pH, said aqueous environment does not exceed 2, 3, 4, 5, 6, 7, 8 ° C, or preferably 4 ° C, for longer than an excursion of a duration between 1 day and 12 weeks, which excursion does not exceed about 37 ° C, preferably about 25 ° C.
[17]
The plurality of particles according to any one of claims 4 to 16, wherein said subphysiological pH aqueous environment comprises a buffer, such as saline, or a phosphate buffer, Tris, borate, succinate, histidine, citrate or maleate.
[18]
18. Plurality of particles according to any one of claims 3 and 5 to 17, wherein the particle is intact or substantially or completely dissolved or degraded is determined in vitro by optical microscopy.
[19]
19. Plurality of particles according to any one of claims 1 to 18, wherein said matrix is polymeric.
[20]
The plurality of particles according to claim 19, wherein said polymeric matrix is biocompatible or biodegradable or bioabsorbable or excretable, in or from the human body.
[21]
21. Plurality of particles according to any one of claims 19 or 20, wherein said polymer matrix comprises a polymer having a pKa lower than said physiological triggering pH.
[22]
A plurality of particles according to any one of claims 19 to 21, wherein said polymer matrix comprises a polymer which is in at least partially protonated state, and / or has an approximately or exactly neutral charge, and is insoluble, when it is in said aqueous environment at a sub-physiological pH.
[23]
23. Plurality of particles according to any one of claims 19 to 22, wherein said polymer matrix comprises a copolymer (poly (methyl methacrylate) -co-poly (methacrylic acid) (copolymer PMMA-co-PMAA); poly ( glutamic acid) -co-poly (lysine); zwitterionic hetero- or homo-poly (amino acids); carboxymethyl-chitosan; hypromellose phthalate; hypromellose acetosuccinate; or an acrylate copolymer represented by the general formula (1 ):

(1) wherein: R1 represents a hydrogen atom or a methyl group, R2 represents a hydrogen atom or a methyl group, and R3 represents a methyl, ethyl, propyl, isopropyl, n-butyl or iso-butyl group; , tert-butyl, sec-butyl, phenyl, or benzyl.
[24]
The plurality of particles according to any one of claims 19 to 23, wherein said polymer matrix comprises the PMMA-co-PMAA copolymer, wherein the molar ratio of the methyl methacrylate monomer (MMA) to the acid monomer methacrylic acid (MAA) in the copolymer is in the range of 1/1 to 4/1, especially 1.5 to 2/1.
[25]
The plurality of particles according to any one of claims 19 to 24, wherein said PMMA-co-PMAA copolymer has a weight average molecular weight (Mw) in the range of 200 kDa, as in the range of 50. at 60 kDa or 35 to 45 kDa or 22 to 28 kDa or 8 to 12 kDa, such as a weight average molecular weight (M w) of 10, 25, 40, 55 or 125 kDa.
[26]
The plurality of particles according to any one of claims 19 to 23, wherein said polymeric matrix comprises poly (glutamic acid) -co-poly (lysine), wherein the molar ratio of the glutamic acid monomer to the monomer lysine is approximately or exactly 1/1.
[27]
27. Plurality of particles according to any one of claims 1 to 26, wherein said cargo is a drug or an antigen.
[28]
28. Plurality of particles according to any one of claims 1 to 27, wherein said cargo comprises an oligo / polysaccharide antigen, optionally conjugated to a carrier protein such as tetanus toxoid, the toxoid fragment C tetanus, diphtheria toxoid, CRM197 or other nontoxic mutant of diphtheria toxin, nontypeable Haemophilus influenzae protein D, an outer membrane protein complex (OMPC) of Neisseria meningitidis, pneumococcal PhtD, pneumolysin pneumococcal, Pseudomonas aeruginosa exotoxin A (EPA), Staphylococcus aureus detoxified hemolysin, Bordetella sp. detoxified adenylate cyclase, Escherichia coli detoxified heat labile enterotoxin, or toxin B subunit cholera (CTB) or detoxified cholera toxin.
[29]
29. Plurality of particles according to any one of claims 1 to 28, wherein said cargo is sensitive to hydrolysis.
[30]
30. Plurality of particles according to any one of claims 1 to 29, wherein said cargo has a low isoelectric point (pI), such as a pI of 4 or less, in particular of 3 or 2 or less.
[31]
31. Plurality of particles according to any one of claims 1 to 29, wherein said cargo is an oligo / polysaccharide-protein conjugated antigen and wherein said oligo / polysaccharide fraction before conjugation has a low isoelectric point (pI), such as a pI of 4 or lower, especially 3 or 2 or less.
[32]
32. Plurality of particles according to any one of claims 27 to 31, wherein said antigen comprises an oligo / polysaccharide derived from a pathogen selected from the group consisting of Haemophilus influenzae type b (Hib); Neisseria meningitidis (in particular serotypes A, C, W and / or Y); Streptococcus pneumoniae; Staphylococcus aureus; Bordetella pertussis; and Salmonella typhi.
[33]
33. Plurality of particles according to any one of claims 27 to 32, wherein said antigen comprises an oligo / polysaccharide derived from a bacterial capsular saccharide or a lipooligosaccharide (LOS) or a lipopolysaccharide (LPS).
[34]
34. Plurality of particles according to any one of claims 27 to 33, wherein said antigen is the Hib / capsular oligo / polysaccharide antigen (PRP) of Hib conjugated to tetanus toxoid (TT) or CRM197.
[35]
35. Plurality of particles according to any one of claims 27 to 33, wherein said antigen is the capsular oligo / polysaccharide of Neisseria meningitidis serotype A (MenA) conjugated to CRM197.
[36]
36. Plurality of particles according to any one of claims 1 to 35, wherein said cargo comprises phosphate groups or phosphodiester bonds.
[37]
37. Plurality of particles according to any one of claims 1 to 36, wherein said cargo is substantially homogeneously dispersed throughout the matrix.
[38]
38. Plurality of particles according to any one of claims 1 to 36, wherein said cargo is encapsulated within the matrix.
[39]
39. Plurality of particles according to any one of claims 27 to 38, wherein said antigen is an oligo / polysaccharide conjugated to a carrier protein, and wherein during said at least 6 months in an aqueous environment at sub-physiological pH the amount of free (non-conjugated) saccharide derived from said oligo / polysaccharide conjugate, present collectively in the particles and the aqueous environment is not more than 30 or 25 or 20 or 15 or 10% by weight of the total amount of conjugated and free saccharide present collectively in the particles and the aqueous environment.
[40]
40. Plurality of particles according to any one of claims 1 to 39, wherein said particles are molded resulting in a precisely predetermined size and shape, in particular a ring shape.
[41]
41. Plurality of particles according to any one of claims 1 to 40, wherein the longest axis of said particles is between 1 and 10 microns, in particular between 5 and 7 microns such as 6 microns.
[42]
42. Plurality of particles according to any one of claims 1 to 41, wherein at least one axis is less than 200 nm, as less than 100 nm, and said particle can be sterilized by filtration.
[43]
43. Plurality of particles according to any one of claims 1 to 42, wherein said particles are for parenteral administration, such as intradermal or subcutaneous administration, in particular intramuscular administration.
[44]
44. A composition comprising a plurality of particles as defined in any one of claims 1 to 43 in an aqueous environment, optionally wherein said environment is sterile.
[45]
45. The composition of claim 44, said composition having a pH below the threshold physiological pH of the particles and / or said composition being of sub-physiological pH.
[46]
46. A composition according to claim 44 or claim 45, said composition having a pH at or below 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6 , 1 or 6.0.
[47]
47. A composition according to any one of claims 44 to 46, wherein said particles are present in said composition at a concentration of 0.1 to 15, 0.5 to 12.5, there or 2 to 5 mg / ml as from 0.5 to 3 mg / ml, optionally wherein said particles are as defined in claim 25.
[48]
A composition comprising a plurality of particles as defined in any one of claims 1 to 43, wherein said plurality is a first plurality with respect to another second plurality of particles, wherein said second plurality of particles comprises a cargo other than the cargo of the first plurality of particles.
[49]
49. The composition according to any one of claims 44 to 48, wherein said aqueous environment comprises one or more physiologically acceptable excipients.
[50]
The composition of any one of claims 44 to 49, wherein said aqueous environment comprises a buffer, such as physiological saline, or a phosphate buffer, Tris, borate, succinate, histidine, citrate or maleate.
[51]
51. The composition of any one of claims 44 to 50, wherein the interaction of said cargo with the components of the aqueous environment is prevented or reduced by the particle matrix.
[52]
52. The composition according to claim 51, wherein said interaction is prevented or reduced for at least 6 months, such as 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28. , 30, 32, 34 or 36 months, said composition being optionally maintained at about 4 ° C.
[53]
53. Composition according to any one of claims 44 to 52, said composition being an immunogenic composition.
[54]
54. An immunogenic composition according to any one of claims 44 to 53, wherein said aqueous environment comprises an adjuvant.
[55]
55. The immunogenic composition of claim 54, wherein said adjuvant has a high pI, such as pI of 8 or greater, such as 9 or 10 or higher, especially 11 or greater.
[56]
56. Immunogenic composition according to claim 54 or 55, wherein said adjuvant is aluminum hydroxide.
[57]
57. An immunogenic composition according to any one of claims 44 to 56, wherein said aqueous environment comprises one or more antigens selected from the group consisting of diphtheria toxoid, tetanus toxoid, acellular pertussis antigens (such as pertussis toxoid, filamentous haemagglutinin, pertactin), hepatitis B surface antigen (HBsAg), inactivated polio vaccine (IPV), capsular oligo / polysaccharide Neisseria meningitidis serotype A (MenA) conjugated to a carrier protein, capsular oligomer / polysaccharide Neisseria meningitidis serotype C (MenC) conjugated to a carrier protein, oligomer / capsular polysaccharide of Neisseria meningitidis serotype W (MenW) conjugated to a carrier protein , capsular oligo / polysaccharide of Neisseria meningitidis serotype Y (MenY) conjugated to a carrier protein and antigen derived from Neisseria menin gitidis of serotype B (MenB), in particular in one of the following combinations: i. diphtheria toxoid and tetanus toxoid; ii. diphtheria toxoid, tetanus toxoid, acellular pertussis antigens; iii. diphtheria toxoid, tetanus toxoid, acellular pertussis antigens and HBsAg; iv. diphtheria toxoid, tetanus toxoid, acellular pertussis antigens and IPV; v. diphtheria toxoid, tetanus toxoid, acellular pertussis antigens, HBsAg and IPV; vi. Neisseria meningitidis capsular oligo / polysaccharide serotype C (MenC) conjugated to Neisseria meningitidis capsular oligo / polysaccharide carrier protein of serotype W (MenW) conjugated to Neisseria meningitidis capsular oligo / polysaccharide carrier protein of serotype Y (MenY) conjugated to a carrier protein; vii. Neisseria meningitidis capsular oligo / polysaccharide serotype C (MenC) conjugated to Neisseria meningitidis capsular oligo / polysaccharide carrier protein of serotype W (MenW) conjugated to Neisseria meningitidis capsular oligo / polysaccharide carrier protein of serotype Y (MenY) conjugated to a carrier protein and antigen derived from Neisseria meningitidis serotype B (MenB).
[58]
58. An immunogenic composition according to claim 57, wherein said diphtheria toxoid, tetanus toxoid and cell-free pertussis antigens are adsorbed on aluminum hydroxide, and optionally said HBsAg, if present, is adsorbed on aluminum phosphate.
[59]
59. An immunogenic composition comprising a plurality of particles as defined in any one of claims 1 to 43 in an aqueous environment, wherein: i. said particles comprise a cargo of Hib-TT or Hib-CRM197 dispersed homogeneously within a matrix comprising PMMA-co-PMAA; and ii. said aqueous environment comprises diphtheria toxoid, tetanus toxoid and acellular pertussis antigens adsorbed on aluminum hydroxide, and optionally HBsAg and IPV.
[60]
60. An immunogenic composition comprising a plurality of particles as defined in any one of claims 1 to 43 in an aqueous environment, wherein: i. said particles comprise a cargo of MenA-CRM homogeneously dispersed within a matrix comprising PMMA-co-PMAA; and ii. said aqueous environment comprises the capsular oligo / polysaccharide of Neisseria meningitidis serotype C (MenC) conjugated to a carrier protein, the capsular oligo / polysaccharide Neisseria meningitidis serotype W (MenW) conjugated to a carrier protein, the oligomer / capsular polysaccharide of Neisseria meningitidis serotype Y (MenY) conjugated to a carrier protein, and possibly the antigen derived from Neisseria meningitidis serotype B (MenB).
[61]
61. An immunogenic composition according to claim 59 or claim 60, wherein the particle matrix prevents or reduces the aggregation or flocculation of Hib-TT or Hib-CRM197 or MenA-CRM197 and / or prevents or reduces the immunological interference with Hib-TT or Hib-CRM197 or MenA-CRM197.
[62]
62. An immunogenic composition according to claim 61, wherein said aggregation or flocculation and / or immunological interference is prevented or reduced for at least 6 months, such as 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 months, the composition being optionally kept at about 4 ° C.
[63]
63. An immunogenic composition according to any one of claims 53 to 61, wherein the particle matrix does not interfere with the immunogenicity of said one or more antigens included within the aqueous environment.
[64]
64. A vial containing a plurality of particles according to any one of claims 1 to 43 or a composition according to any one of claims 44 to 63.
[65]
65. Device for parenteral administration, such as a syringe, containing a plurality of particles according to any one of claims 1 to 43 or a composition according to any one of claims 44 to 63.
[66]
66. Device according to claim 65, said device being a double chamber syringe containing in a chamber a plurality of particles according to any one of the claims 43 and in the other chamber an aqueous environment as defined in any one Claims 45, 46, 49, 50 and 54 to 58.
[67]
67. Plurality of particles according to any one of claims 1 to 43 or composition according to any one of claims 44 to 63 for use in medicine, in particular in human medicine.
[68]
68. Plurality of particles according to any one of claims 1 to 43 or composition according to any one of claims 44 to 63 for use in the treatment or prevention, in particular in a human being, of (i) an infection or pathology caused directly or indirectly by a pathogen, or (ii) a pathology associated with immunologically distinct host cells, such as cancer.
[69]
69. Use of the plurality of particles according to any one of claims 1 to 43 or the composition according to any one of claims 44 to 63 in the manufacture of a medicament for use in the treatment or prevention, in particularly in humans, of (i) an infection or pathology caused directly or indirectly by a pathogen, or (ii) a pathology associated with immunologically distinct host cells, such as cancer.
[70]
70. Plurality of particles or composition according to claim 68 or use according to claim 69, said plurality of particles or said composition being for parenteral administration.
[71]
A method of triggering an immune response against (i) a pathogen or allergen causing an infection or pathology, or (ii) immunologically distinct host cells responsible for a condition, such as cancer, comprising the step of administering to a subject, in particular a human, an effective amount of the plurality of particles according to any of claims 1 to 43 or the composition of any one of claims 44 to 63.
[72]
72. A method of treatment or prophylaxis against (i) an infection or pathology caused directly or indirectly by a pathogen, or (ii) a pathology associated with immunologically distinct host cells, such as cancer, comprising the step of administering to a subject, in particular a human, an effective amount of the plurality of particles according to any of claims 1 to 43 or the composition of any one of claims 44 to 63.
[73]
73. The method of claim 71 or claim 72, wherein said administering step is performed by a parenteral route.
[74]
74. Plurality of particles or composition according to claims 68 or 70, or use according to claims 69 or 70, or method according to any one of claims 71 to 73, wherein said pathogen is selected from the list consisting of: Haemophilus influenzae type b (Hib); Neisseria meningitidis (in particular serotypes A, C, W and / or Y); Streptococcus pneumoniae; Staphylococcus aureus; Bordetella sp. ; and Salmonella typhi.
[75]
75. An immunogenic composition or composition according to any one of claims 44 to 63, wherein said immunogenic composition or composition is a vaccine.
[76]
76. A vaccine comprising the immunogenic composition or composition according to any one of claims 44 to 63.
[77]
A method of preventing or reducing the interaction between a biologically active cargo and the components of an aqueous environment in which said cargo is present, comprising: i. forming a plurality of pH-sensitive particles as defined in any one of claims 1 to 43, comprising said cargo; and ii. formulating said plurality of particles in said aqueous environment, including any adjustment necessary to render said sub-physiological pH environment.
[78]
78. A method of preventing or reducing the interaction between a biologically active cargo and the components of an aqueous environment of subphysiological pH in which said cargo is present, comprising: i. forming a plurality of pH-sensitive particles as defined in any one of claims 1 to 43, comprising said cargo; and ii. formulating said plurality of particles in said aqueous environment.
[79]
79. The method of any one of claims 77 and 78, wherein said plurality of particles formulated in said aqueous environment is the composition as defined in any one of claims 44 to 63.
[80]
The method of claim 77 or claim 78, wherein said preventing or reducing the interaction is for: i. storing said cargo in said aqueous environment; or ii. preventing or reducing degradation, such as hydrolytic degradation, of said cargo in said aqueous environment.
[81]
81. The method of claim 77 or claim 78, said method being for the prevention or reduction of the interaction between said biologically active cargo and the water molecules in said aqueous environment, or between said biologically active cargo and a component of the aqueous environment other than water, as an adjuvant.
[82]
The method of claim 81, wherein said adjuvant is aluminum hydroxide, and optionally wherein said cargo is Hib-TT or Hib-CRM197 or MenA-CRM197.
[83]
The method of claim 77 or claim 78, said method for preventing or reducing the interaction between said biologically active cargo and a second cargo present in said aqueous environment, further comprising: iii. forming a second plurality of particles comprising said second cargo within a matrix, said second plurality of particles being as defined in any of the claims thereon provided that said second cargo is not the same as said biologically active cargo.
[84]
84. A method of manufacturing a plurality of particles for drug delivery comprising a biologically active cargo within a matrix, comprising the step of manufacturing a solution of said cargo and a matrix polymer. optionally wherein said polymer is as defined with reference to the polymer matrix or polymer of any one of claims 20 to 26.
[85]
85. A method of manufacturing a plurality of particles for the administration of medicaments comprising a biologically active cargo within a matrix, the method comprising the use of a solution comprising a polymer as defined with reference to the matrix. polymer or polymer according to any one of claims 20 to 26, and a cargo optionally as defined in any one of claims 27 to 36.
[86]
86. The process according to claims 84 or 85, wherein said solution comprises said cargo at a quantity not exceeding 30% by weight and a complement of PMMA-co-PMAA copolymer.
[87]
The method of claim 86, wherein said solution comprises said cargo at a level of 0.1 to 5% by weight, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0. , 6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5 or 5% by weight.
[88]
The method of any one of claims 84 to 87, wherein said cargo is Hib-TT or Hib-CRM197 or MenA-CRM197.
[89]
The method of any one of claims 84 to 88, wherein said solution further comprises PVP, wherein the weight ratio of PVP / PMMA-co-PMAA does not exceed 1/1.
[90]
The process according to claim 89, wherein the weight ratio of PVP / PMMA-co-PMAA is in the range of 0.1 to 1/1, such as 0.2 / 1, 0.3 / 1. , 0.4 / 1, 0.5 / 1, 0.6 / 1, 0.7 / 1, 0.8 / 1 or 0.9 / 1.
[91]
91. The method of any one of claims 89 and 90, wherein said PVP has a molecular weight of about 2.5 kDa and a polydispersity of about 1.9.
[92]
92. A process according to claims 84 or 85, wherein said solution comprises said cargo at a quantity not exceeding 30% by weight and a complement of poly (glutamic acid) -co-poly (lysine) copolymer.
[93]
The method of claim 92, wherein said cargo is Hib-TT or Hib-CRM197 or MenA-CRM197.
[94]
The method of any one of claims 92 and 93, wherein said solution further comprises glycerol, wherein the weight ratio of glycerol / polyglutamic acid-co-poly (lysine) does not exceed 1/1.
[95]
The method according to any one of claims 84 to 91, wherein said solution comprises 0.2 to 1.2, more particularly 0.4 to 1,% by weight of Hib-TT or Hib-CRM197 or MenA. -CRM197 and a complement of PMMA-co-PMAA and PVP, optionally in a weight ratio of 1/1.
[96]
96. A process according to any one of claims 84, 85 and 92 to 94, wherein said solution comprises 0.2 to 1.2, more particularly 0.4 to 1, wt.% Of Hib-TT or Hib- CRM197 or MenA-CRM197 and a complement of poly (glutamic acid) -co-poly (lysine) and glycerol wherein the glycerol is present at 30% by weight.
[97]
The method of any one of claims 84 to 96, further comprising forming said plurality of particles by molding said solution.
[98]
The method of any one of claims 84 to 97, wherein said solution further comprises a plasticizer other than PVP, optionally wherein said plasticizer is a pore-forming agent.
[99]
The method of any one of claims 84 to 98, wherein if said solution comprises PVP, another plasticizer and / or a porogen, said method further comprises removing substantially all of said PVP, of the other plasticizer and / or pore-forming agent from said particle.
[100]
A method of manufacturing a plurality of drug delivery particles comprising a biologically active cargo within a matrix, comprising the steps of: i. at least partial deprotonation of a polymer, which is insoluble in its protonated state, in an aqueous environment such that the polymer has a net negative charge and is soluble in said aqueous environment; ii. combining said polymer with said cargo to produce a solution; iii. forming the particles by molding said solution and removing the aqueous environment.
[101]
The method of claim 100, further comprising collecting the particles in a dry state or in an organic or aqueous liquid collection environment.
[102]
The process of claim 101 wherein the aqueous liquid is acidic.
[103]
The process of claim 101 or claim 102, wherein the aqueous liquid has a pH of less than 5.
[104]
The method of claim 100, further comprising protonating the polymer of the particles such that the polymer of the particles returns to an insoluble state.
[105]
The method of claim 104, further comprising storing the particles at a subphysiological pH acceptable for parenteral administration.
[106]
The method of claim 105 wherein said storage is in an aqueous environment.
[107]
107. Plurality of particles for the administration of medicaments obtainable or obtained by the process according to any of claims 84 to 106.
[108]
A method of making a composition, such as an immunogenic composition, comprising making a plurality of particles according to a method as defined in any one of claims 84 to 106 and formulating said particles in an environment wherein said environment optionally comprises an antigen and / or an adjuvant, wherein said antigen and / or adjuvant is optionally as defined in any one of claims 55 to 58.
[109]
A method of making a composition, such as an immunogenic composition, comprising a plurality of drug delivery particles comprising a biologically active cargo within a matrix, wherein the matrix comprises a polymer, including the following steps: i. introducing a plurality of particles, made according to the method of any one of claims 84 to 101, into an aqueous acidic environment such that the acidic environment protonates the polymer of the matrix rendering the polymer insoluble in said environment; ii. elevating the pH of the aqueous acidic environment to a sub-physiological pH that is acceptable for parenteral administration while maintaining the insoluble state of the matrix polymer in said environment; and possibly iii. formulating said particles in an aqueous environment of sub-physiological pH, optionally wherein said aqueous environment is as defined in any one of claims 45, 46, 49, 50 and 54 to 58.
[110]
The method of claim 109, wherein in step (i) said introducing a plurality of particles into an acidic environment comprises introducing said particles into a stabilizing solution at a pH in the range of from as a pH of 3.5 or 4.5, wherein the particles are introduced into the stabilizing solution up to 60, 50, 40, 30, 20, 10 or 5 minutes.
[111]
The method of claim 109 or claim 110, wherein in step (ii) said raising of the pH of the acidic aqueous environment comprises increasing the pH of said environment to said sub-physiological pH in a progressive manner.
[112]
The method of any one of claims 109 to 111, wherein in step (ii) the pH of the solution is increased from 0.1 to 10 pH units per minute, such as 0.5, 1, 2 or 5 pH units per minute, in particular 0.5 pH units per minute.
[113]
113. A composition, such as an immunogenic composition, obtainable or obtained by the process of any one of claims 108 to 112.

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法律状态:
2018-02-15| FG| Patent granted|Effective date: 20171218 |

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2021-12-16| MM| Lapsed because of non-payment of the annual fee|Effective date: 20210331 |

优先权:

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申请号 | 申请日 | 专利标题

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US201662304399P| true| 2016-03-07|2016-03-07|

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US62/304,399|2016-03-07|

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