Patent Application
20230144557
The present application relates to the preservation and/or stabilization of chemical and/or biological species in sugar glasses, in particular materials comprising pullulan and trehalose. The application includes the materials and methods for their use in stabilizing and/or preserving chemical and/or biological species.
Bacteriophages (phages) are viruses that infect bacteria. From the early days of their discovery in 1917, lytic bacteriophages have been used as potent antimicrobial agents.1,2 An advantage of phage over other antimicrobial agents is its specificity. Whereas most broad-spectrum antimicrobial agents function like a sledgehammer, wiping out any and all bacteria, bacteriophages specifically target only certain species/strains of bacteria within a mixed population. This specificity has made bacteriophage antimicrobials very attractive for food processing/packaging applications, amongst others.3177 The odor, taste, and texture of most food products, particularly fresh produce, is negatively affected by commercial antimicrobial agents. Bacteriophages exist naturally on fruits and vegetables and adding phage antimicrobials will not affect the appearance, taste, odor, or texture of produce.12 Bacteriophage antimicrobials are also particularly useful for decontaminating products such as cheese for which the quality of the product strongly depends on the presence of beneficial bacteria, or honey which is consumed raw and without additives or antibiotics. In addition to their specificity, phage antimicrobials have garnered significant attention in the past 20 years as a result of the ever-growing crisis of antibiotic resistance.13-16 Presence of multidrug resistant bacteria in animal products poses a serious threat to public health, especially in countries where antibiotics are used liberally and without constraint as an integral part of animal husbandry.17,18
Using phage-impregnated coatings on food preparation surfaces, surfaces in food processing plants, and for food packaging may be a promising way to ensure food safety.5,8,14,9 However, to effectively incorporate phage as part of a functional antimicrobial coating, certain challenges must be addressed, including the issue of phage stability.7 Bacteriophages are generally resilient to most environmental conditions such as temperature, pH, and salt concentration, although their sensitivity varies significantly amongst strains.20 However, desiccation cannot be endured by many phages and thus a challenge in developing phage-functionalized coatings is finding methods to protect phage against the effects of desiccation.
Amongst methods proposed to date for long-term stabilization of bacteriophage preparations, freeze-drying21,22 (also known as lyophilization) and freezing in liquid nitrogen are the most prevalent. Neither method can preserve phage stability unless a protectant, such as glycerol, alginate,8,23-28 pectin,24,29 chitosan,25,26 whey protein,16,30 liposome,31,32 poly(ethylene oxide)/cellulose diacetate,33 and sucrose/trehalose34 is present. Also, the lyophilized samples must be maintained in vacuum ampoules for effective phage stabilization. Both methods require access to specialized equipment for sample preparation (freeze dryer, vacuum pumps) and sample storage (liquid nitrogen storage).
Vaccines are an essential part of global health. Every year, millions of lives are saved through vaccination. Unfortunately, almost all available vaccines are thermally labile and must be kept between 2-8° C. at all times to retain their efficacy.35 Failure to maintain an uninterrupted refrigerated supply chain from production to dispensation, called the “cold chain,” leads to vaccine wastage and administration of ineffective vaccines.36 The need for refrigeration is one of the major causes of under-vaccination globally as the cold chain presents economical and logistical problems for vaccination programs. The problem is especially serious for developing countries and remote areas where there are often a lack of dependable cold chain infrastructure and access to reliable electricity is limited.37-39
The development of thermally-stable vaccines that can remain active outside of the cold chain can greatly increase the accessibility of vaccination programs and significantly decrease the cost. Therefore, significant efforts have been made in creating thermally-stable vaccines and/or vaccine carriers. One approach has been to engineer vaccines that are thermally stable without preservative adjuvants. The engineering of protein-based vaccine had shown some promise.40-43 Other reports modified viral vectors to create thermally stable viral vaccines.44-45 Although designing thermally stable vaccines hold some promise, many of the engineered vaccine still have short shelf-life (˜7 days) at elevated temperature (>37° C.). Moreover, engineering new vaccines is labor intensive and the new vaccines must obtain governmental approval before deployment.
Another common approach to thermally stabilize vaccine is the addition of stabilizing adjuvants46 In addition to stabilizing adjuvants, vaccines are often dried to further increase thermal stability. Prausntz's group encapsulated inactivated influenza vaccine in microneedle patches with different stabilizing adjuvant formulations and the vaccine maintained immunogenicity after 4 months at 60° C.47-49 Lyophilized anthrax vaccine was found to have preserved immunogenicity after 16 weeks at 40° C.50 and lyophilized recombinant ricin toxin A vaccine was stable after 4 weeks at 40° C.51 Recombinant hepatitis B vaccine and a protein-polysacharide conjugate vaccine for meningitis A was shown to be stable for 24 months at 37° C. after spray drying.52 Foam drying of attenuated Salmonella enterica vaccine using trehalose methionine and gelatin were able to maintain vaccine potency for 12 weeks at 37° C.53 Spray drying formulations using sugars and proteins with live attenuated measles vaccine were shown to be stable for up to 8 weeks at 37° C.54 Lovalenti et al. stabilized live-attenuated influenza vaccines in a sucrose containing excipient using three drying methods, freeze drying, spray drying, and foam drying. It was found that foam drying with the right excipient composition produced the most thermally stable vaccine that had a shelf life of 4.5 months at 37° C.55 Different lyophilized formulations of rotavirus vaccines were able to retain potency for 20 months at 37° C.56 for up to 20 months.57. Alcock et al. used sucrose and trehalose to dry adenovirus and modified vaccinia virus Ankara onto polypropylene or glass fiber membranes, the viruses retained titer for up to 6 months at 45° C.58 Many of the reports use freeze drying, spray drying, or foam drying which all require specialized equipment for sample preparation (freeze dryer, vacuum pumps) and expose vaccines to extreme temperatures or pressure conditions.53 Moreover, some formulations require a large number of adjuvants which can increase the cost and complexity of the vaccine product.
Pullulan is a polysaccharide that has excellent film forming properties and has been used in the food industry as an oxygen barrier to prolong the shelf life of foods.59-65 Previous studies have shown that pullulan is able to provide outstanding thermal stability and protection against oxidation of various labile biomolecules.66-67 Trehalose is a disaccharide sugar that has been used extensively as a cryoprotectant and stabilizing agent during lyophilization.68-79
U.S. Pat. No. 7,749,538 describes a shaped product such as a film, sheet or film capsule shape having a high pullulan content in combination with trehalose wherein the content of the pullulan provides a shaped product that has stability to changes in humidity.
US20190111006 describes a solid material comprising pullulan and trehalose (optionally called a “PT material”) in the form of a film, coating or shaped object, that possesses a synergistic effect that leads to long-term stability of chemical and/or biological species. The contents of this application are specifically incorporated herein by reference in their entirety.
The Applicant has found that the combination of pullulan and trehalose in a solid material (optionally called a “PT material”), has beneficial properties when in the form of a powder. Further, the Applicant has found that PT material dried to, and maintained at, a water/moisture content of 10 wt % or less, has beneficial properties for storage and transportation, in particular for degradation-sensitive medicaments.
Accordingly, the present application includes a polymer matrix comprising pullulan and trehalose and one or more chemical and/or biological species, wherein the one or more chemical and/or biological species are incorporated within the polymer matrix and the polymer matrix preserves and/or stabilizes the one or more chemical and/or biological species and the polymer matrix is in powder form.
The present application includes a polymer matrix comprising pullulan and trehalose and one or more chemical and/or biological species, wherein the one or more chemical and/or biological species are incorporated within the polymer matrix and the polymer matrix preserves and/or stabilizes the one or more chemical and/or biological species and the polymer matrix has a water content of 10 wt % or less.
The present application also includes a method of preserving and/or stabilizing one or more chemical and/or biological species comprising:
The present application also includes a method of preserving and/or stabilizing one or more chemical and/or biological species comprising:
The present application also includes a method of creating a material for preserving chemical and/or biological species comprising:
The present application also includes a method of creating a material for preserving chemical and/or biological species comprising:
In some embodiments, at least one of the species is a biomolecule. In some embodiments, the biomolecule is selected from one or more of protein, enzyme, antibody, peptide, nucleic acid, phage, antidote, antigen and vaccine.
In some embodiments, at least one of the species a microorganism. In some embodiments, wherein the microorganism is selected from one or more of anaerobic bacteria, aerobic bacteria, mammalian cells, bacterial cells and viruses.
The present application also includes a pullulan/trehalose powder comprising one or more chemical and/or biological species wherein the wt % pullulan in the dried pullulan/trehalose powder is less than 50 wt %.
The present application also includes a dried pullulan/trehalose powder comprising one or more chemical and/or biological species wherein the wt % pullulan in the dried pullulan/trehalose powder is in the range of 30-45 wt %.
The present application also includes a PT material comprising one or more chemical and/or biological species in the form of a powder, and a method of making a PT material comprising one or more chemical and/or biological species in the form of a powder. In some embodiments, a dried PT material comprising one or more chemical and/or biological species is formed and then mechanically converted into a powder. In some embodiments, the initial form (i.e. the form converted into a powder) is a film. In some embodiments, a mixture comprising a pullulan/trehalose solution and one or more chemical and/or biological species is dried by vacuum drying. In some embodiments, the dried PT material comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6% or less than 5% water by weight.
The present application also includes methods of making, storing and/or transporting a PT material comprising one or more chemical and/or biological species. In some embodiments, a PT material comprising one or more chemical and/or biological species is dried and/or sealed into a package under a vacuum or in an environment with less than 50%, less than 45, less than 40, less than 35, or less than 33%, relative humidity. In some embodiments, the packaged PT material comprising the one or more chemical and/or biological species has a moisture (i.e. water) content of 10 wt % or less. In some embodiments, the packaged PT material comprising the one or more chemical and/or biological species is stored and/or transported at a temperature in the range of −20° C. to 40° C. In some embodiments, the PT material comprising one or more chemical and/or biological species is in powder form.
The present application also includes methods of reconstituting and/or administering a medicament comprising pullulan/trehalose and one or more chemical and/or biological species, for example an antigen. In some embodiments, a PT material comprising one or more chemical and/or biological species is provided in the form of a powder in a sealed container delivered to the administration site or place of treatment. In some embodiments, the PT material has a water content of 10 wt % or less. In some embodiments, a diluent is mixed with the PT material at the administration site/place of treatment to dissolve the PT material and produce an aqueous medicament, for example an aqueous vaccine. In some embodiments, the diluent is added to a container containing the PT material, the PT material is added to a container of the diluent, the PT material and the diluent are transferred to another container, or a barrier between the PT material and the diluent in a container is broken. In some embodiments, the diluent contains water, for example sterile water, and optionally other compounds such as an adjuvant, a salt, a buffer, etc. The aqueous medicament is then administered to a patient, for example by way of injection (for example intramuscular or subcutaneous injection), ingestion (oral administration) or nasal spray (intranasal administration).
The present application also includes a PT material comprising one or more chemical and/or biological species for use in a medicament, for example an immunogenic composition, and/or for use in providing a therapy, for example an immune response, in a patient. The present application also describes a method of providing a therapy, such as an immune response, in a patient comprising administering an effective dose of a medicament comprising one or more chemical and/or biological species, for example an antigen, to the patient, wherein the one or more chemical and/or biological species was previously combined with the PT material.
The present application also describes a method of making a stabilized composition, for example an immunogenic composition, comprising one or more chemical and/or biological species, for example an antigen. In some embodiments, the method includes mixing one or more chemical and/or biological species with a pullulan/trehalose solution, drying the mixture, and converting the dried mixture to a powder. In addition, or alternatively, the method includes mixing one or more chemical and/or biological species with a pullulan/trehalose solution, and drying the mixture to a water content of less than 10 wt %.
Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the application, are given by way of illustration only and the scope of the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the description as a whole.
The embodiments of the application will now be described in greater detail with reference to the attached drawings in which:
Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present application herein described for which they are suitable as would be understood by a person skilled in the art.
In understanding the scope of the present application, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and/or steps.
Terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.
As used in this application, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. For example, an embodiment including “a bacteriophage” or “a vaccine” should be understood to present certain aspects with one bacteriophage or vaccine, or two or more additional (and different) bacteriophage or vaccines.
The term “and/or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of” or “one or more” of the listed items is used or present.
The term “biomolecule” as used herein means an organic macromolecule (such as a protein or nucleic acid) in living organisms.
The term “microorganism” as used herein means a microscopic organism that may exist as a single cell or as a colony of cells
The term “preserving” or “preservation” as used herein with respect to the chemical and/or biological species means to maintain at least a measurable or detectable level of function or activity for the chemical and/or biological species for a desired period of time under specified conditions.
The term “stabilizing” or “stabilization” as used herein with respect to the chemical and/or biological species refers to any reduction in the degradation or loss of activity of the chemical and/or biological species compared to a control.
The term “drying” as used herein refers to a process of allowing a solution of a polymer to cure or set, by removal of water/moisture, until a solid, movable material is obtained.
The term “pullulan” as used herein refers to a natural polysaccharide which is produced extracellularly by Aurebasidium pullulans when cultivated with starch hydrolyzates as a carbon source.
The term “trehalose” as used herein refers to (D)-(+)-trehalose which is a disaccharide composed of two glucose molecules bound together via the α,α-1,1-glucosidic linkage.
The expression “incorporated within” as used herein means that the one or more biological and/or chemical species are interspersed throughout the pullulan/trehelose polymer matrix.
The term “polymer matrix” as used herein means a material which is made of at least one polymer and which forms a surrounding medium or structure.
The term “food grade” as used herein means that the specified material is compatible for ingestion by humans and/or animals.
The term “medical grade” as used herein means that the specified material is compatible for administration to humans and/or animals including, for example, by way of injection.
The term “vaccine” as used herein may mean, where appropriate given the context, an antigen of a vaccine, but does not necessarily exclude the presence of other parts of a vaccine, such as an adjuvant or diluent.
The term “immunogenic” as used herein relates to or denotes substances able to produce an immune response.
The term “essentially free from” as used herein means that the presence of the stated features, elements, or components, is in an amount that does not materially affect the characteristics of the composition or material being referenced.
As used herein, the term “effective amount” or “therapeutically effective amount” means an amount that is effective, at dosages and for periods of time necessary to achieve a desired result.
The term “medicament” as used herein refers to any substance used for medical treatment or therapy.
The Applicant has found that a material made from pullulan and trehalose acts synergistically to provide a stabilized matrix that is capable of preserving and/or stabilizing chemical and/or biological species, such as bacteriophages and viruses, that are incorporated within the matrix and further certain beneficial properties are provided when the matrix is in powder form. For example, the powdered polymer matrixes of the present application have faster dissolution rates. The Applicant has also found that PT material dried to, and maintained at, a water content of 10 wt % or less, has beneficial properties for storage and transportation, in particular for degradation-sensitive medicaments.
Accordingly, the present application includes a polymer matrix comprising pullulan and trehalose and one or more chemical and/or biological species, wherein the one or more chemical and/or biological species are incorporated within the polymer matrix and the polymer matrix preserves and/or stabilizes the one or more chemical and/or biological species, wherein the polymer matrix is in the form of a powder.
The present application includes a polymer matrix comprising pullulan and trehalose and one or more chemical and/or biological species, wherein the one or more chemical and/or biological species are incorporated within the polymer matrix and the polymer matrix preserves and/or stabilizes the one or more chemical and/or biological species and the polymer matrix has a water content of 10 wt % or less.
In some embodiments the polymer matrix comprises about 10 wt % to about 50 wt % of pullulan and about 50 wt % to about 90 wt % of trehalose, based on the dry weight of the matrix. In some embodiments the polymer matrix comprises about 20 wt % to about 40 wt % of pullulan and about 60 wt % to about 80 wt % of trehalose, based on the dry weight of the matrix. In some embodiments the polymer matrix comprises about 30 wt % to about 40 wt % of pullulan and about 60 wt % to about 70 wt % of trehalose, based on the dry weight of the matrix. In some embodiments the polymer matrix comprises about 37 wt % of pullulan and about 63 wt % of trehalose, based on the dry weight of the matrix.
In some embodiments the pullulan has a molecular weight in the range of about 100,000 to about 200,000. Pullulan having such molecular weights is commercially available, for example from Hayashibara Co, Ltd., Okayama, Japan. Trehalose is available from a variety of commercial sources, including, for example, Hayashibara Co, Ltd., Okayama, Japan. In some embodiments, for use in food products, the pullulan and trehalose are both food grade materials. In some embodiments, for use in medical products, the pullulan and trehalose are both medical grade, or pharmaceutically acceptable, materials.
In some embodiments, the polymer matrix is in the form of a powder. In some embodiments, the polymer matrix is formed into a powder by milling, crushing and/or grinding. In some embodiments, the powder comprises particles having a median surface area:volume ratio of 10:1 mm−1 to about 120:1 mm−1. In some embodiments, the powder comprises particles having a median surface area:volume ratio of 10:1 mm−1 to about 100:1 mm−1. In some embodiments, the powder comprises particles having a median surface area:volume ratio of 12:1 mm−1 to about 60:1 mm−1. In some embodiments, the powder comprises particles having a median surface area:volume ratio of 12:1 mm−1 to about 40:1 mm−1.
In some embodiments, the polymer matrix is formed first into a dried film or a shaped object prior to forming into a powder. In some embodiments, the dried film or shaped object is amorphous. In some embodiments, the shaped object is a pill or capsule.
In some embodiments, the polymer matrix has a water content of less than 10 wt %. In some embodiments, the polymer matrix has a water content of less than 9 wt %. In some embodiments, the polymer matrix has a water content of less than 8 wt %. In some embodiments, the polymer matrix has a water content of less than 7 wt %. In some embodiments, the polymer matrix has a water content of less than 6 wt %. In some embodiments, the polymer matrix has a water content of less than 5 wt %. In some embodiments, the polymer matrix has a water content of about 5 wt % to about 10 wt %. In some embodiment, the polymer matrix has a water content of about 6 wt % to about 10 wt %. In some embodiment, the polymer matrix has a water content of about 7 wt % to about 10 wt %. In some embodiment, the polymer matrix has a water content of about 8 wt % to about 10 wt %.
In some embodiments, the polymer matrix powder comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6% or less 5%, by weight, of water. In some embodiments, the polymer matrix powder comprises less than 10%, less than 9% or less than 8%, by weight, of water.
In some embodiments, the one or more chemical and/or biological species are preserved and/or stabilized without requiring refrigeration. In some embodiments, the one or more chemical and/or or biological species are preserved at a temperature of from about 2° C. to about 40° C., about 10° C. to about 30° C. or about 20° C. to about 25° C. In some embodiments, the one or more chemical and/or or biological species are preserved at a temperature of from about −20° C. to about 40° C., about 10° C. to about 30° C. or about 20° C. to about 25° C. In some embodiments, the one or more chemical and/or biological species are preserved and/or stabilized for at least 3 months at the above temperatures. In some embodiments, the one or more chemical and/or biological species are preserved and/or stabilized for at least 4 days, or from 4 to 10 days at temperatures below freezing.
In some embodiments, the one or more chemical species is a biomolecule. In some embodiments, the biomolecule is chosen from one or more of a protein, an enzyme, an antibody, a peptide, a nucleic acid, an antidote and a vaccine. In some embodiments, the biomolecule is a vaccine. In some embodiments, the biomolecule is an immunogenic protein. In some embodiments, the biomolecule is an antigen. In some embodiments, the one or more biological species is a microorganism. In some embodiments, the microorganism is chosen from one or more of anaerobic bacteria, aerobic bacteria, mammalian cells, bacterial cells and viruses. In some embodiments, the microorganism is a virus. In some embodiments, the virus is a bacteriophage. In some embodiments, the virus is an enveloped virus. In some embodiments, the virus is a DNA virus. In some embodiments, the virus is Herpes Simplex Virus (HSV-2). In some embodiments, the virus is HSV-2 In some embodiments, the virus is a RNA virus. In some embodiments, the virus is influenza virus. In some embodiments, the virus is PR8. In some embodiments, the virus is formulated for administration in a biological preparation. In some embodiments, the virus is formulated for administration as a live-attenuated vaccine. In some embodiments, the virus is formulated for administration as an inactivated vaccine.
In some embodiments the one or more chemical species are biomolecules that act as immunogens or that are used to generate an immune response, including, DNA, RNA, peptides and/or proteins. In some embodiments, these biomolecules are incorporated within the matrix along with other agents that are used in vaccine formulations, such as adjuvants. In some embodiments, the polymer matrix further comprises one or more additional substances or additives such as, but not limited to, seasonings, spices, colorings, flavors, emulsifiers and plasticizers. In some embodiments, the physical properties of the polymer matrix, such as solubility, transparency, tactile impression, texture, plasticity, etc. are changed using additives.
In some embodiments, the polymer matrix is without or essentially free from any additives that are non-GRAS or not acceptable for injection in a person. In some embodiments, the polymer matrix is without or essentially free from PMAL-C16. In some embodiments, the polymer matrix is free from or without, or contains less than 0.1% to 10%, of a zwitterionic surfactant having a lipid group with a chain length of 13-30 carbon atoms. In some embodiments, the polymer matrix is free from or without, or contains less than 0.1 mg/ml to 50 mg/ml, of a zwitterionic surfactant having a lipid group with a chain length of 13-30 carbon atoms.
The present application also includes an immunogenic composition comprising an antigen, the antigen formulated in a polymer matrix of the application.
The Applicants have found that chemical and/or biological species can be preserved and/or stabilized by incorporating the species within in a polymer matrix comprising certain amounts of pullulan and trehalose, wherein the polymer matrix is in powder form and/or wherein the polymer matrix has a water content of less than 10 wt %.
Accordingly, the present application includes a method of preserving and/or stabilizing one or more chemical and/or biological species comprising:
In some embodiments, the trehalose is added to an aqueous solution of pullulan and the one or more chemical and/or biological species. In some embodiments, the trehalose is added at a concentration of about 0.1 to 1 M, or about 0.5 M.
In some embodiments, the one or more chemical and/or biological species, an aqueous solution comprising pullulan and an aqueous solution comprising trehalose are mixed thoroughly to ensure uniform distribution of all of the ingredients.
In some embodiments, one or more additional substances or additives such as, but not limited to, seasonings, spices, colorings, flavors, emulsifiers, salts, adjuvants, buffers and plasticizers are added to the mixture, and/or to the one or more chemical and/or biological species, the aqueous solution comprising pullulan or the aqueous solution comprising trehalose prior to drying. In some embodiments, the physical properties of the polymer matrix, such as solubility, transparency, tactile impression, texture, plasticity, etc. is changed using additives.
In some embodiments, the mixture is drop cast into a specific shape prior to drying. In some embodiments, the shape is a pill or a capsule.
In some embodiments, the mixture is formed into a film prior to drying. In some embodiments, the mixture is formed into a thin film of any of a variety of shapes, for example a strip or patch. In some embodiments, the shaped object is a pill or capsule.
In some embodiments, the polymer matrix is formed into a powder by milling, crushing and/or grinding. In some embodiments, the powder comprises particles having a median surface area:volume ratio of 10:1 mm−1 to about 120:1 mm−1. In some embodiments, the powder comprises particles having a median surface area:volume ratio of 10:1 mm−1 to about 100:1 mm−1. In some embodiments, the powder comprises particles having a median surface area:volume ratio of 12:1 mm−1 to about 60:1 mm−1. In some embodiments, the powder comprises particles having a median surface area:volume ratio of 12:1 mm−1 to about 40:1 mm−1.
In some embodiments the polymer matrix comprises about 10 wt % to about 50 wt % of pullulan and about 50 wt % to about 90 wt % of trehalose, based on the dry weight of the matrix. In some embodiments the polymer matrix comprises about 20 wt % to about 40 wt % of pullulan and about 60 wt % to about 80 wt % of trehalose, based on the dry weight of the matrix. In some embodiments the polymer matrix comprises about 30 wt % to about 40 wt % of pullulan and about 60 wt % to about 70 wt % of trehalose, based on the dry weight of the matrix. In some embodiments the polymer matrix comprises about 37 wt % of pullulan and about 63 wt % of trehalose, based on the dry weight of the matrix.
In some embodiments the pullulan has a molecular weight in the range of about 100,000 to about 200,000. Pullulan having such molecular weights is commercially available, for example from Hayashibara Co, Ltd., Okayama, Japan. Trehalose is available from a variety of commercial sources, including, for example, Hayashibara Co, Ltd., Okayama, Japan. In some embodiments, for use in food products, the pullulan and trehalose are both food grade materials. In some embodiments, for use in medical products, the pullulan and trehalose are both medical grade, or pharmaceutically acceptable, materials.
In some embodiments, the one or more chemical and/or biological species are preserved and/or stabilized without requiring refrigeration. In some embodiments, one or more chemical or biological species are preserved at a temperature of from about 2° C. to about 40° C., about 10° C. to about 30° C. or about 20° C. to about 25° C. In some embodiments, the one or more chemical and/or or biological species are preserved at a temperature of from about −20° C. to about 40° C., about 10° C. to about 30° C. or about 20° C. to about 25° C. In some embodiments, the one or more chemical and/or biological species are preserved and/or stabilized for at least 3 months at the above temperatures. In some embodiments, the one or more chemical and/or biological species are preserved and/or stabilized for at least 4 days, or from 4 to 10 days at temperatures below freezing.
In some embodiments, the one or more chemical species is a biomolecule. In some embodiments, the biomolecule is chosen from one or more of a protein, an enzyme, an antibody, a peptide, a nucleic acid, an antidote and a vaccine. In some embodiments, the biomolecule is an immunogenic protein. In some embodiments, the biomolecule is an antigen. In some embodiments, the biomolecule is a vaccine. In some embodiments, the one or more biological species is a microorganism. In some embodiments, the microorganism is chosen from one or more of anaerobic bacteria, aerobic bacteria, mammalian cells, bacterial cells and viruses. In some embodiments, the microorganism is a virus. In some embodiments, the virus is a bacteriophage. In some embodiments, the virus is an enveloped virus. In some embodiments, the virus is a DNA virus. In some embodiments, the virus is Herpes Simplex Virus (HSV-2). In some embodiments, the virus is HSV-2 TK−. In some embodiments, the virus is a RNA virus. In some embodiments, the virus is influenza virus. In some embodiments, the virus is PR8. In some embodiments, the virus is formulated for administration in a biological preparation. In some embodiments, the virus is formulated for administration as a live-attenuated vaccine. In some embodiments, the virus is formulated for administration as an inactivated vaccine.
In some embodiments the one or more chemical species are biomolecules that act as immunogens or that are used to generate an immune response, including, DNA, RNA, peptides and/or proteins. In some embodiments, these biomolecules are incorporated within the matrix along with other agents that are used in vaccine formulations, such as adjuvants.
In some embodiments, the polymer matrix is without or essentially free from any additives that are non-GRAS or not acceptable for injection in a person. In some embodiments, the polymer matrix is without or essentially free from PMAL-C16. In some embodiments, the polymer matrix is free from or without, or contains less than 0.1% to 10%, of a zwitterionic surfactant having a lipid group with a chain length of 13-30 carbon atoms. In some embodiments, the polymer matrix is free from or without, or contains less than 0.1 mg/ml to 50 mg/ml, of a zwitterionic surfactant having a lipid group with a chain length of 13-30 carbon atoms.
In some embodiments, the present application also includes a method of preserving and/or stabilizing one or more chemical and/or biological species comprising:
In some embodiments an aqueous mixture of pullulan, trehalose and one or more chemical and/or biological species is dried in a partial vacuum, dried under a nitrogen-enhanced atmosphere, and/or dried in a desiccated environment. In some embodiments, the mixture is not freeze dried. Freeze drying can produce PT materials with a cloudy or opaque appearance, which is believed to be caused by the formation of crystals in the PT material. The cloudy or opaque appearance produced during freeze drying is associated with a loss of activity of at least some chemical or biological species intended to be preserved and/or stabilized by the PT material.
In some embodiments, the polymer matrix is dried to a water content of less than 10 wt %. In some embodiments, the polymer matrix is dried to a water content of less than 9 wt %. In some embodiments, the polymer matrix is dried to a water content of less than 8 wt %. In some embodiments, the polymer matrix is dried to a water content of less than 7 wt %. In some embodiments, the polymer matrix is dried to a water content of less than 6 wt %. In some embodiments, the polymer matrix is dried to has a water content of less than 5 wt %. In some embodiments, the polymer matrix is dried to a water content of about 5 wt % to about 10 wt %. In some embodiment, the polymer matrix is dried to a water content of about 6 wt % to about 10 wt %. In some embodiment, the polymer matrix is dried to a water content of about 7 wt % to about 10 wt %. In some embodiment, the polymer matrix is dried to a water content of about 8 wt % to about 10 wt %.
In some embodiments, the PT materials of the present application comprising one or more chemical and/or biological species and dried to a water content of less than 10 wt % are formed in, or has the form of, small particles, for example a powder. In some embodiments, a brittle solid material is formed, which is mechanically broken into a powder. In some embodiments, the powder comprises particles having a median surface area:volume ratio of 10:1 mm−1 to about 120:1 mm−1. In some embodiments, the powder comprises particles having a median surface area:volume ratio of 10:1 mm−1 to about 100:1 mm−1. In some embodiments, the powder comprises particles having a median surface area:volume ratio of 12:1 mm−1 to about 60:1 mm−1. In some embodiments, the powder comprises particles having a median surface area:volume ratio of 12:1 mm−1 to about 40:1 mm−1.
Once solidified, the PT material with one or more chemical and/or biological species may be subjected to freezing. The PT material may be used to preserve and/or stabilize one or more chemical or biological species over a temperature range of at least −20° C. to 40° C.
The PT material with one or more chemical and/or biological species may be stored in a sealed container. Optionally, the sealed container may also contain a desiccant and/or be sealed while under vacuum. However, acceptable performance can be achieved if the PT material with one or more chemical and/or biological species is filled and sealed into containers while inside of a room or other vessel (for example a biosafety or other controlled environment cabinet) having 50% relative humidity or less, or 45%, 40%, 35% or 33% relative humidity or less. The permanent gasses in the room or other vessel may be air or a more nearly inert gas, for example nitrogen enriched air or nitrogen. The gasses in the room or other vessels may be treated, for example filtered or passed through a sorbent, to remove contaminants.
The present application also includes methods of reconstituting and/or administering a medicament comprising pullulan/trehalose and one or more chemical and/or biological species, for example an antigen. In some embodiments, a PT material comprising one or more chemical and/or biological species is provided in the form of a powder in a sealed container delivered to the administration site or place of treatment. In some embodiments, the PT material has a water content of 10 wt % or less. In some embodiments, a diluent is mixed with the PT material at the administration site/place of treatment to dissolve the PT material and produce an aqueous medicament, for example an aqueous vaccine. In some embodiments, the diluent is added to a container containing the PT material, the PT material is added to a container of the diluent, the PT material and the diluent are transferred to another container, or a barrier between the PT material and the diluent in a container is broken. In some embodiments, the diluent contains water, for example sterile water, and optionally other compounds such as an adjuvant, a salt, a buffer, etc. The aqueous medicament is then administered to a patient, for example by way of injection (for example intramuscular or subcutaneous injection), ingestion (oral administration) or nasal spray (intranasal administration).
In some embodiments, the PT material with one or more chemical and/or biological species are stored in a container. The container may be sealed, for example by a septum. In some embodiments, multiple PT materials, each with one or more chemical and/or biological species, are stored in the same container. For example, one PT material may contain one antigen while another PT material contains another antigen, one PT material may contain an antigen while one or more other PT materials contain one or more of a buffer, a salt or an adjuvant. In some embodiments. an aqueous mixture including pullulan, trehalose and one or more chemical and/or biological species is reconstituted by adding a diluent, for example by drawing the diluent into a syringe and then injecting the diluent into the container through the septum. In some embodiments, the container is agitated, for example shaken, to help increase the rate of dissolution of the PT material or to help ensure than no un-dissolved PT material remains. A portion of the reconstituted mixture can then be drawn out of the container by way of a needle inserted through the septum. The reconstituted mixture can then be administered to a patient (i.e. a human or non-human mammal or other animal), for example by being injected into the patient. Alternatively, in some embodiments, the reconstituted mixture is administered to the patient by an intranasal route by way of an atomizer or sprayer, or the patient may swallow some of the reconstituted mixture.
The present application also includes a polymer matrix comprising pullulan and trehalose and one or more chemical and/or biological species, wherein the one or more chemical and/or biological species are incorporated within the polymer matrix and the polymer matrix preserves and/or stabilizes the one or more chemical and/or biological species, and the polymer matrix is in the form of a powder for use as a medicament, for example an immunogenic composition. In some embodiments, the medicament is reconstituted from the powder and injected into a mammal. In some embodiments, the one or more chemical and/or biological species are immunogenic and the polymer matrix is for use in a method of providing an immune response in a mammal.
The present application also includes a method of providing an immune response in a mammal comprising administering, to a mammal in need thereof, an effective amount of a polymer matrix comprising pullulan and trehalose and one or more chemical and/or biological species, wherein the one or more chemical and/or biological species are immunogenic and are incorporated within the polymer matrix and the polymer matrix preserves and/or stabilizes the one or more chemical and/or biological species, and the polymer matrix is in the form of a powder. In some embodiments, a diluent is added to the powder to provide an aqueous mixture and administering the aqueous mixture to the mammal, for example by injecting the aqueous mixture into the mammal. The present application also includes a method of making and/or storing and/or transporting a stabilized medicament or component thereof comprising a) producing a mixture of pullulan, trehalose and one or more chemical and/or biological species, b) drying the mixture to 10 wt % water content or below to provide a dried material and c) packaging the dried material in a sealed container. In some embodiments, the dried material is in the form of a powder. In some embodiments, the container comprises a septum. In some embodiments, the mixture is reconstituted by injecting a diluent through the septum. In some embodiments, the container is stored and/or transported at a temperature between −20° C. and 40° C. In some embodiments, the the container is stored for a period of time above 8° C.
The present application also includes a method of making and/or storing and/or transporting a stabilized medicament or component thereof comprising a) producing a mixture of pullulan, trehalose and one or more chemical and/or biological species, b) drying the mixture to form a polymer matrix, c) forming the polymer matrix into a powder, and d) packaging the dried powder in a sealed container. In some embodiments, the mixture is dried to a water content of less than 10 wt %. In some embodiments, the container comprises a septum. In some embodiments, the mixture is reconstituted by injecting a diluent through the septum. In some embodiments, the container is stored and/or transported at a temperature between −20° C. and 40° C. In some embodiments, the the container is stored for a period of time above 8° C.
In some embodiments, a syringe is pre-loaded with a PT material comprising one or more chemical and/or biological species in the form of a powder and/or having a water content of less than 10 wt %. The PT material dissolves when a diluent is drawn into the syringe. The resulting aqueous mixture can be injected into a patient through a needle attached to the same syringe. In some embodiments, the reconstituted mixture is a whole vaccine (i.e. a combination of antigen and any required adjuvants or other additives).
In an alternative method, syringe 128 is produced containing the PT material 104 containing one or more chemical and/or biological species in the form of a dry powder. The needle 132 of syringe 128 is inserted into the vial 108 and plunger 130 pulled back to draw diluent 112 into the syringe 128. The incoming diluent 112 may dissolve the PT material. Optionally, the syringe 128 is shaken until the PT material 104 is essentially dissolved and the one or more chemical and/or biological species is thereby released. The reconstituted medicament (including PT material with one or more chemical and/or biological species) is then injected in step 134 into a patient.
In an alternative method, after the PT material 104 is dissolved, the vial 102 or its contents may be transferred to a nasal sprayer. For example, the cap 106 may be removed and a nasal sprayer head installed over the vial 102. For example, the nasal sprayer head may have a threaded or snap fit feature that engages a corresponding feature of the vial 102. The reconstituted medicament (i.e. an aqueous mixture of pullulan, trehalose and PT solution and one or more chemical and/or biological species, for example an antigen) may then be administered to a patient intranasally.
In some embodiments, diluent 112 is sterile water.
In some embodiments, a composition includes pullulan, trehalose, and one or more chemical and/or biological species, wherein the one or more chemical and/or biological species are incorporated within the polymer matrix and the polymer matrix preserves and/or stabilizes the chemical or biological species. In some embodiments, the composition is an immunogenic composition having a viral vector or an antigen. In some embodiments, the composition is essentially without or free from any other components, or at least without or free from any other components that are not generally regarded as safe (GRAS) and are suitable for administration to a person, for example by way of injection. In some embodiments, the composition is substantially without or free from surfactants. In some embodiments, the composition is without or free from PMAL-C16; without any, or without from about 0.1% to 10%, of a zwitterionic surfactant having a lipid group with a chain length of 13-30 carbon atoms; and/or, without any, or without from about 0.1 to 50 mg/ml, of a zwitterionic surfactant having a lipid group with a chain length of 13-30 carbon atoms. In some embodiments, the composition excludes any composition disclosed, for example by way of enabling disclosure and/or by way of specific example, in U.S. Pat. No. 9,974,850.
The following non-limiting examples are illustrative of the present application:
Pullulan (PI20 food grade, 200 kDa) was obtained from Hayashibara Co, Ltd., Okayama, Japan. D-(+)-trehalose dehydrate, D-(+)-maltose monohydrate, sucrose, calcium chloride (CaCl2)), magnesium sulfate (MgSO4.7H2O), Tris, gelatin, Tryptic Soy Broth (TSB), and Listeria enrichment broth (LEB) were purchased from Sigma-Aldrich. Agar and agarose were purchased from Becton, Dickinson and Company (BD). Phosphate buffered saline (PBS) was purchased from BioShop Canada. Listeria monocytogenes serotype ½a, E. coli O157:H7, and Salmonella Newport, were routinely cultured and maintained in our lab. Two Myoviridae phages, E. coli O157:H7 phage, EcoM-AG10 (AG10), and Salmonella phage, SnpM-CG4-1 (CG4-1), were obtained from Canadian Research Institute for Food Safety, University of Guelph. LISTEX™ P100 was purchased from Micreos Food Safety (Wageningen, The Netherlands). Distilled deionized water was obtained from a Milli-Q Advantage A10 water purification system (EMD Millipore) and was autoclaved.
The infectivity of phages encapsulated in dried films was quantified using the overlay technique.80 The phage-containing film was dissolved in 1 mL of CM buffer (prepared by mixing 2.5 g MgSO4. 7H2O, 0.735 g of CaCl2, 0.05 g gelatin, and 6 mL 1 M Tris-HCl at pH 7.5, with water for a final volume of 1 L) through repeated pipetting. The reconstituted film solution was then serially diluted in CM buffer, each dilution was mixed in equal volumes with 100 μL of the bacterial host (109 CFU/mL) and then incubated at 30° C. for 10 minutes to allow for phage adsorption. The phage-host mixture was then added to 4 mL of soft Tryptic Soy Agar (TSA, prepared by adding 0.5% agarose to TSB) and overlayed onto a TSA plate (1.5% agar to TSB). The plates were incubated at 30° C. overnight. Plaque formation was observed the following day, and the plaques were counted to determine the phage titer of each film. For each dilution, triplicate experiments were conducted. The total number of plaques were averaged and considered as the number of viable phages.
An aqueous mixture, for example an injectable vaccine, may be reconstituted from a PT material including one or more chemical and/or biological species. In some cases, it is desirable to increase the rate of dissolution of the PT material. In some examples, this is achieved by preparing the PT material as a very thin film or in a powder form. For example, after drying a PT material can be converted to a powder through a mechanical process, such as milling, crushing or grinding a larger PT material.
An experiment was conducted to compare the rate of dissolution of an intact film to that of a film with a similar mass but crushed into smaller particles. The films were prepared as follows: from 10 mL solution containing 10% pullulan (0.1 g/mL=100 g/L) and 0.5 M trehalose, 90 uL was taken and mixed with 10 uL of a blue dye. Then, from this 100 uL mixture of pullulan/trehalose and dye, 60 uL was taken and dried for making PT films. The films were in the form of discs, about 5 mm in diameter and about 0.5 mm to 1 mm in thickness. Some samples of the PT film were left intact. Other samples of the PT film were manually crushed with a pipette tip. The PT films were brittle and crushing the PT film mostly produced a powder, but with several larger particles remaining that were not completely crushed to a powder form due to incomplete crushing. The larger particles were generally rectangular prisms with dimensions of about 0.5 to 1.5 mm on each side. Each of the larger particles had a volume of roughly 2-4% of the volume of the original film. The larger particles can be filtered out, but were left in the powder for the further procedures described below.
200 uL of water was added to various samples of intact film and crushed PT material.
To determine if powdering a PT material might have an impact on a biological species that is entrapped in the PT material, two PT films were created containing the phage P11 (useful against Pseudomonas aeruginosa). One PT film was maintained intact and the second PT film was physically disintegrated as described above. These two samples were then stored in a closed container for 2 days at room temperature. The number of viable phage was determined after dissolving each sample separately. The intact PT film yielded 5.67±0.04 log(phage/ml) of viable phage and the disintegrated PT material yielded 5.48±0.01 log(phage/ml) of viable phage. These results indicate that PT material can be physically powdered with very little loss of activity of a biological species preserved and/or stabilized by the PT material.
During transport or storage, it possible that PT films may be exposed to a wide range of temperatures. Experiments described above indicate that PT materials are effective at high temperatures, for example up to 40° C. However, shipping or storing the PT material during winter in or between cold countries may expose the PT material to low temperatures.
PT films were prepared containing the P11 phage and dried to about 7% moisture by weight. Samples of the films were stored for 4 days at room temperature, 4° C., −20° C. and −80° C. After this period of storage, the samples were dissolved and the number of viable phage counted.
Previous work regarding the preparation and storage of PT films was described in Leung, V., L. Groves, A. Szewczyk, Z. Hosseinidoust, and C. D. Filipe, Long-Term Antimicrobial Activity of Phage-Sugar Glasses is Closely Tied to the Processing Conditions. ACS Omega, 2018. 3(12): p. 18295-18303, which is incorporated herein by reference.
As mentioned above, the residual water content of the PT material may be kept at 10 wt % or less. Vacuum drying is more effective than drying with ambient air (RH typically of 50%) in reaching this amount of residual water.
When samples of these volumes (50 uL and 100 uL) are placed in a vessel large enough for the solution to form a droplet the sample dries to a thin (1 mm or less) film covering approximately the footprint of the original droplet. For example, a 50 uL droplet produces a 5-7 mm diameter film. A 100 uL droplet produces a film of about 10 mm in diameter.
Drying should be done such that the formation of crystals is avoided. Small particles in the solution, or formation of crystals through the presence of certain salts (such as phosphate), may lead to loss of the protective ability of the PT material.
No negative effects have been noticed with rapid drying, even to the extent of drying in 2 minutes produced by applying a nitrogen jet to a thin liquid film of PT material. On the contrary, faster drying can reduce degradation of chemical or biological species that degrade in aqueous environments. In some cases, rapid drying under vacuum can cause bubbling in the PT material as it dries. This may modify the shape or form of the dried PT material, but does not appear to reduce the protective ability of the PT material.
Methods that create high shear conditions during the preparation of PT materials can reduce the stability of at least some biological species. In one experiment, an aqueous mixture of pullulan, trehalose and P11 phage was split in two aliquots. The first aliquot was used to create PT films without using vortexing to mix the phage with the pullulan/trehalose solution and the second aliquot was subjected to 60 seconds of vortexing. These two films were reconstituted after 4 days of storage at room temperature. The sample that was not vortexed yielded 5.67±0.04 log(phage/ml) and the sample that was subjected to vortexing yielded 4.2±0.05 log(phage/ml). In this case, vortexing resulted in a substantial loss of phage counts.
Pullulan/trehalose films using three different drying methods: (1) through vacuum drying; (2) through freeze drying (prior art method) and (3) through spray drying. For all the cases, the initial solution contained 10% pullulan and 0.5 M trehalose. The resulting solid films obtained using the three different methods of drying, were observed through optical polarized light microscopy (transmission and/or reflective mode). This microscopic technique is a widely used method to detect the presence of crystals in samples. The crystals can be identified by the presence of structures that have a rainbow-like glow associated with them. The solids obtained using vacuum drying, did not contain any crystals, whereas the solids obtained either through freeze-drying or through spray drying, contained crystals.
While the present application has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.
The present application claims the benefit of priority from U.S. provisional patent application Ser. No. 63/009,041, filed on Apr. 13, 2021, the contents of which are incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2021/050494 | 4/13/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63009041 | Apr 2020 | US |
