Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Disinfection or sterilisation of materials or objects, in general; Accessories therefor
  • A61L2/02—Disinfection or sterilisation of materials or objects, in general; Accessories therefor using physical processes
  • A61L2/08—Radiation
  • A61L2/087—Particle radiation, e.g. electron-beam, alpha or beta radiation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Disinfection or sterilisation of materials or objects, in general; Accessories therefor
  • A61L2/02—Disinfection or sterilisation of materials or objects, in general; Accessories therefor using physical processes
  • A61L2/08—Radiation
  • A61L2/081—Gamma radiation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Disinfection or sterilisation of materials or objects, in general; Accessories therefor
  • A61L2/26—Accessories
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/14—Macromolecular materials
  • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
  • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0068—General culture methods using substrates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2103/00—Materials or objects being the target of disinfection or sterilisation
  • A61L2103/05—Living organisms or biological materials

Definitions

• SAPs relevant for this invention consists of alternating hydrophilic and hydrophobic amino acid residues capable of forming beta- sheets. They autonomously assemble into well-ordered nanostructures in neutral water, while they can temporarily disassemble into individual molecules when high shearing force is applied to them. SAPs can form a hydrogel (also known as SAP gels) depending on their environment such as pH and/or osmolality; for example, they are capable of forming a hydrogel, when they are placed in the body at near neutral pH,.
• SAP gels also known as SAP gels
• SAP gels have been previously described as being used for a variety of medical applications, e.g., improved wound healing, inducement of homeostasis; reduction of adhesion in interior tissues, particularly, in context of surgery; as temporary tissue-void matrix fillers, facilitating ingrowth of natural tissue into such a void.
• Particular SAPs are described in US Patents Nos.5,670,483; 5,955,343; 9,724,448; 10,596, 225 and Int’l Pat. Appln. Pub. WO2014/136081; and foreign equivalents thereof. Sterilization is a very important step in the manufacturing process for most biomaterials, including for self-assembling peptide solutions.
• a solution of SAPs is forced through a porous filter, wherein the sterilizing filter has an average pore size of 0.22 ⁇ m (US Pat. Appln. No.2015/019735).
• some thermally stable self-assembling peptide solutions can be sterilized by autoclaving treatment at about 121 °C for about 25 minutes (US Pat. Appln. No.10/369,237). Furthermore, an additional ethylene oxide sterilization step is required for the outer part of PuraStat ® products. And, it was discovered that autoclaving cannot be used for some SAPs, such as RADA16 in solution, because of its complete thermal degradation (US Pat. Appln. Pub. No.2017/0202986). Thus, another new sterilization method has been needed to reduce losses, particularly, for RADA16 and other SAPs. Gamma irradiation sterilization of self-assembling peptides including RADA16 was described in passing in a prior publication (US Pat.
• peptide structure can be changed by the reactive radical species generated by irradiation such as gamma ray, X-ray, and e-beam.
• reactive radical species generated by irradiation
• Such a structural change can be governed by multiple factors including the amino acid composition, reactive residue position and possibly the conformation acquired by each macromolecule (Vieira R et al, Biol. Pharm. Bull.2013, 36(4) 664-675).
• SAPs RADA16 (Ac-RADARADARADARADA-NH 2 (SEQ ID NO:1)), KLD12 (Ac- KLDLKLDLKLDL-NH 2 (SEQ ID NO:2)), and IEIK13 (Ac-IEIKIEIKIEIKI-NH 2 (SEQ ID NO:3)), do not include aromatic amino acids or sulfur-containing amino acids.
• irradiation sterilization can also affect the secondary structure of self-assembling peptides and their fibrous structure.
• RADA16, KLD12 and IEIK13 have beta-sheet conformation, and these molecules self-assemble to form an ordered nanofibrous structure. Irradiation sterilization process may potentially change the secondary structure and the nanofiber structure of peptide, which could then result in the undesirable change of its rheological properties. Thus, there exists a need for new sterilization methods that work advantageously with self-assembling peptides. For the reasons cited above, sterilization by irradiation has been avoided so far.
• Mass spectra of PuraStat ® (RADA162.5%) (A) after e-beam irradiation at 25 kGy and (B) after e-beam irradiation at 40 kGy.
• Figure 5. Mass spectra of IEIK131.3% (A) before irradiation; (B) after gamma irradiation at 40 kGy; (C) after X-ray irradiation at 25 kGy; (D) after X-ray irradiation at 40 kGy; (E) after e-beam irradiation at 25 kGy; and (E) after e-beam irradiation at 40 kGy.
• the method of sterilizing a self-assembling peptide solution comprises: a) placing one or more containers with a solution of self-assembling peptide into an irradiation machine, said self-assembling peptide capable of forming a hydrogel when applied to a biological tissue at about neutral pH; and b) exposing the container to gamma ray, X-ray and/or e-beam irradiation at a predetermined dose so that the peptide solution is sterilized without substantial degradation of the peptide while its desired biological and/or rheological property(ies) is/are maintained at the same level or improved.
• the peptides are selected from the group consisting of RADA16, KLD12, and IEIK13.
• such peptides are exposed to the dose is 15 – 50 kGy, preferably 15 – 40 kGy, more preferably, to a minimum dose resulting in a desired sterility assurance level (SAL), without substantial degradation and/or substantial negative change in biological properties of these peptides.
• the peptide solution is irradiated by gamma-rays, X-rays, or e-beam.
• the over-all degradation of the total peptides in solution after irradiation does not exceed 20%, more preferably, 10%, most preferably 5%, of the amount of peptides prior to irradiation.
• the desired biological or physical property(ies) is/are selected from the group consisting of: hemostatic, anti- adhesion, prevention of re-bleeding, anti-stenosis, tissue occlusion, storage modulus (e.g., in some embodiments, the storage modulus of the gelled solution is increased at least by 10%, at least by 15% or at least by 20% post-irradiation, and viscosity, and tissue void filling property are maintained within acceptable or improved parameters after irradiation.
• irradiation dose achieves sterility assurance level (SAL) of at least 10 -5 , preferably 10 -6 , or less.
• SAL sterility assurance level
• the acceptable level of contamination of the peptide solution pre-irradiation is 1000, 500,100, 15, 10, 9, 5, 2, 1.5, 1 CFU, or less.
• the concentration of the degradation products of the intact (“major” or “full-length”) peptide in the solution post-irradiation ranges from 0.1% to 5%.
• the pH of the peptide solution post-irradiation ranges from about 1.8 to 3.5.
• the solution container is a plastic syringe, with or without an adapter nozzle.
• care is taken to ensure that the plastic and rubber parts of the packaging also maintain their desired physical properties. While some yellowing of the plastic syringes may be expected and is normal, any rubberized material must preserve its e plastic properties at an acceptable level.
• the gelled solution is further subjected to sheering to reduce or restore its storage modulus.
• the invention provides a sterilization method for self-assembling peptides.
• the solution is further applied the solution to a biological tissue, for example, during surgery, or after trauma involving bleeding.
• the method of sterilizing a self-assembling peptide solution comprises: a) placing one or more containers with a solution of self-assembling peptide into an irradiation machine, said self-assembling peptide capable of forming a hydrogel when applied to a biological tissue (e.g., in situ) at about neutral pH; and b) exposing the container to gamma ray, X-ray and/or e-beam irradiation at a predetermined dose so that the peptide solution is sterilized to a pre- determined Sterility Assurance Level (SAL) without substantial degradation of the peptide while its desired biological and/or physical property(ies) is/are maintained substantially at the same level or improved.
• SAL Sterility Assurance Level
• the composition and methods of the invention maintain or improve the desired biological property(ies) such as hemostatic, anti-adhesion, prevention of re-bleeding, anti-stenosis, tissue occlusion, storage modulus, viscosity, and tissue void filling property, etc.
• the storage modulus in increased by at least 5%, 10%, 15%, 20%, or more. If such increase is undesirable for certain applications, the gel can be thinned further by dilution or sheering by methods known in the art, turning it into the solution or otherwise reducing its storage modulus.
• the irradiated solution of SAPs remains clear and viscous.
• the SAPs comprise a sequence of amino acid residues conforming to one or more of Formulas I-IV: ((Xaa neu -Xaa + ) x (Xaa neu -Xaa ⁇ ) y ) n (I) ((Xaa neu -Xaa ⁇ ) x (Xaa neu -Xaa + ) y ) n (II) ((Xaa + -Xaa neu ) x (Xaa ⁇ -Xaa neu ) y ) n (III) ((Xaa ⁇ -Xaa neu ) x (Xaa + -Xaa neu ) y ) n (IV) Xaa neu represents an amino acid residue having a neutral charge; Xaa + represents an amino acid residue having a positive charge; Xaa ⁇ represents an amino acid residue having a negative charge; x and y are integers having a value of 1, 2, 3, or 4, independently
• the SAPs further comprise an amino acid sequence that interacts with the extracellular matrix, wherein the amino acid sequence anchors the SAPs to the extracellular matrix.
• the amino acid residues in the SAPs can be naturally occurring or non-naturally occurring amino acid residues.
• Naturally occurring amino acids can include amino acid residues encoded by the standard genetic code as well as non-standard amino acids (e.g., amino acids having the D-configuration instead of the L-configuration), as well as those amino acids that can be formed by modifications of standard amino acids (e.g., pyrolysine or selenocysteine).
• Suitable non-naturally occurring amino acids include, but are not limited to, D-alloisoleucine(2R,3S)-2-amino- 3-methylpentanoic acid, L-cyclopentyl glycine (S)-2-amino-2-cyclopentyl acetic acid.
• another class of materials that can self-assemble are peptidomimetics.
• Peptidomimetics refers to molecules which mimic peptide structure. Peptidomimetics have general features analogous to their parent structures, polypeptides, such as amphiphilicity. Examples of such peptidomimetic materials are described in Moore et al., Chem. Rev.101(12), 3893-4012 (2001).
• the peptidomimetic materials can be classified into four categories: ⁇ -peptides, ⁇ -peptides, ⁇ -peptides, and ⁇ -peptides. Copolymers of these peptides can also be used.
• ⁇ -peptide peptidomimetics include, but are not limited to, N,N′-linked oligoureas, oligopyrrolinones, oxazolidin-2-ones, azatides and azapeptides.
• Examples of ⁇ -peptides include, but are not limited to, ⁇ -peptide foldamers, ⁇ -aminoxy acids, sulfur-containing ⁇ -peptide analogues, and hydrazino peptides.
• ⁇ -peptides include, but are not limited to, ⁇ -peptide foldamers, oligoureas, oligocarbamates, and phosphodiesters.
• ⁇ -peptides include, but are not limited to, alkene-based ⁇ -amino acids and carbopeptoids, such as pyranose-based carbopeptoids and furanose-based carbopeptoids.
• the SAP is AC5®, AC5-V®, AC5-GTM or TK45, also known as AC1, made by Arch Therapeutics, Inc. (see www.archtherapeutics.com).
• the SAP solution is contained in "storage and/or drug delivery system", such as, for example, storage and/or delivery systems suitable for peptide compositions described herein, for example, vials, bottles, beakers, bags, syringes, ampules, cartridges, reservoirs, or LYO-JECTS ® .
• Storage and/or delivery systems need not be one in the same and can be separate.
• SAPs is provided in plastic syringe, containing, about 10 ml, about 7.5 ml, about 5, about 2.5., about 1, or about 0.5 ml of a SAP solution.
• the plastics may acquire a yellowish tint after irradiation, which is normal, and does not affect biomedical characteristics of the therein contained SAP solution.
• such storage and delivery system may further contain a "nozzle” which refers to a generally thin, cylindrical object, often with a narrow end and a wide end, which is adapted for fixing onto a delivery device described herein.
• nozzle refers to a generally thin, cylindrical object, often with a narrow end and a wide end, which is adapted for fixing onto a delivery device described herein.
• the terms "nozzle” and "cannula” are used interchangeably.
• Nozzles are composed of two connection points or ends, a first connection point or end to connect to a delivery system (e.g., a syringe) and a second connection point which may serve as the point where delivery of pharmaceutical composition is administered or as a point to connect to a secondary device (e.g., a catheter).
• a delivery system e.g., a syringe
• a second connection point which may serve as the point where delivery of pharmaceutical composition is administered or as a point to connect to a secondary device (e.g., a catheter).
• a delivery system e.g., a syringe
• a second connection point which may serve as the point where delivery of pharmaceutical composition is administered or as a point to connect to a secondary device (e.g., a catheter).
• the invention provides methods of making sterilized solutions of the self- assembling peptides.
• the invention also provide use of such sterilized solutions can be applied to a biological tissue, e.g.,
• the invention provides the use of a sterilized solution of the self- assembling peptide for treating or preventing aforementioned disease or condition, wherein the sterilized solution is obtained by the methods of the invention.
• the self-assembling peptide solution exhibits a post-irradiation mass spectrometric (MS) profile substantially as shown in corresponding post-irradiation profiles of FIGS.2-6 and/or as described in the Examples.
• MS mass spectrometric
• the additional major Mz peaks at are observed at 836/1670, 1100, and 1513 m/z.
• An average bioburden ⁇ 1,000 CFU is the typical sterility of PuraStat ® before sterilization.
• SAL of 10 -6
• the range of irradiation dose should be between 25 kGy and 40 kGy.
• the acceptable level of contamination of the peptide solution pre-irradiation may be ⁇ 1000, ⁇ 500, ⁇ 100, ⁇ 15, ⁇ 10, ⁇ 9, ⁇ 5, ⁇ 2, ⁇ 1.5, ⁇ 1 CFU, or less per product unit.
• the preferred bioburden pre-sterilization is ⁇ 9 CFU.
• the range of irradiation dose can be selected between 15 kGy and 24 kGy.
• the low end of the irradiation dose range may be determined by the bioburden of the pre-irradiated product
• the high end of the range is chosen based on the configuration and choice of the irradiator machine and set at a minimum required to achieve desirable sterility assurance level (SAL).
• SAL sterility assurance level
• gamma and X-ray methods up to 10% of the peptides may degrade during sterilization process.
• e-beam method the peptides may not significantly degrade during sterilization process.
• PuraStat ® rheology increases after gamma-ray and X-ray sterilization, which may somewhat change its hemostatic efficacy positively or negatively.
• PuraStat ® rheology does not change after e-beam sterilization and thus may be preferred, if no or minimal change in rheological properties and its hemostatic properties is desirable. Therefore, e-beam irradiation may be particularly preferred for PuraStat ® .
• the concentration of degraded full-length peptide (“major peptide”) in the solution post-irradiation ranges from 0.1% to 5%, 0.1% to 4%, 0.1% to 3%, 0.1% to 2.5%, 0.1% to 2%, or 0.1% or 1.5% or less.
• a total dose which may depend on its pre-irradiation bioburden, including, for example, 15-50 kGy, 25 kGy +/- 15 kGy, 40 kGy - 10 kGy, 35 kGy -10 kGy, 30 kGy -10 kGy, 15 kGy – 24 KGy, 25 kGy -10 kGy, 20 kGy -10 kGy, 10 kGy -15 kGy, and 12 kGy -14 kGy.
• the "storage and/or drug delivery system" for example, a plastic syringe contained in a blister pack, is irradiated in batches of at least: 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000 units or more at a time.
• the total exposure of any sample in an irradiated batch does not exceed 100 kGy, more preferably 60 kGy, most preferably, 50 kGy, or less as described herein.
• the major peptide’s degradation also referred to as “full- length peptide’s degradation”
• the total dose is achieved over a period of time sufficient to achieve necessary sterility of the solution.
• the dose of 40 kGy can be delivered by radiation intensity 6.3 kGy/hr for about 6 hrs and 21 min. Other combination can be found in Example 1.
• a constant dose was delivered over a number of hours, e.g., about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 hours.
• samples are irradiated with gamma ray doses, for example, of about 23, about 25, about 28 or about 40 kGy, or other doses indicated above with an instrument, for example, such as Gammacell 220 ® High Dose Rate Co-60 Irradiator in VPTrad (www.vptrad.com; MDS Nordion, Ottawa, Canada).
• samples are irradiated with X-ray with doses of about 25 kGy to about 40 kGy, or the doses as indicated above, with a Mevex accelerator (with the following settings: 10 MeV, 20 kW).
• X-ray frequency range is 3x10 16 -3x10 19 Hz, while the frequency range of gamma-rays frequency is 3x10 19 or higher.
• samples are irradiated with e-beam at about 25 to about 40 kGy with a Mevex accelerator (with the following settings: 10 MeV, 20 kW) (see mevex.com/linacs/10mev-system-e-beam-sterilization-for-medical-devices/).
• the desired biological and physical property(ies) is/are selected from the group consisting of: hemostatic, anti-adhesion, prevention of re- bleeding, anti-stenosis, tissue occlusion, storage modulus, viscosity, tissue void filling property, mucosa elevation, and wound healing,
• the irradiation dose achieves sterility assurance level (SAL) of at least 10 -5 , 10 -6 , or less.
• SAL sterility assurance level
• the invention also provide the sterilized solution of self-assembling peptide made by the methods described herein. Such comprising applying the solution to a biological tissue, for example, during surgery or after trauma involving bleeding, to a site with substantially neutral pH, resulting in gelling.
• such site is internal, while in other embodiment the site can be external such surface sutures, cuts, or scrapes.
• Other aspects of the invention would be apparent to those of skill in the art based on the present description, including the Examples and the appended claims.
• Example 1 Irradiation conditions Samples were irradiated with gamma rays at 23, 25, 28 and 40 kGy with VPTrad Gammacell 220 ® High Dose Rate Co-60 Irradiator. In some embodiments, the run dose rate and duration time for 40 kGy irradiation were 6.30 kGy/hr and 6 hours 20 minutes 58 seconds, respectively.
• the run dose rate and duration time for 28 kGy irradiation were 4.40 kGy/hr and 6 hours 22 minutes 31 seconds, respectively.
• the run dose rate and duration time for 25 kGy irradiation were 6.58 kGy/hr and 3 hours 47 minutes 42 seconds, respectively.
• the run dose rate and duration time for 23 kGy irradiation were 6.58 kGy/hr and 3 hours 29 minutes 29 seconds, respectively.
• samples were irradiated with X-ray at 25 kGy and 40 kGy with a Mevex accelerator (10 MeV, 20 kW).
• samples were irradiated with e-beam at 25 and 40 kGy with a Mevex accelerator (10 MeV, 20 kW).
• Example 2 HPLC conditions HPLC tests were performed to evaluate the major peptide content after irradiation tests.
• An Agilent HPLC 1100 (Agilent Technologies) was used for this study. The column temperature was kept at 25 °C.
• solvent A was water with 0.1% TFA and solvent B was 80% acetonitrile with 0.1% TFA. Gradient of solvent B was controlled from 10% to 40% in 20 min and 40% for another 5 min at 25 °C.
• Agilent Zorbax 300SB-C18 column (4.6 mm X 250 mm, 5 ⁇ m, 300 ⁇ ) was used for this test.
• PuraStat ® (RADA162.5%) (40 mg) was mixed with 10 ⁇ L of DH 2 O and the mixture were vortexed. The mixture was further mixed with 500 ⁇ L of formic acid and vortexed. This mixture was then mixed with DH 2 O (4,450 ⁇ L) and vortexed.20 ⁇ L samples were injected using an Agilent autosampler.
• solvent A was water with 0.1% TFA and solvent B was 90% Acetonitrile with 0.1% TFA.
• Example 4 Rheological properties of irradiated self-assembling peptides
• the rheological properties of samples were evaluated using a rheometer (DHR1, TA Instruments) with 40 mm cone and plate. Peptide solution (700 ⁇ L) was placed on the rheometer plate and excess solution was gently removed by a metal spatula. Measurements were performed after 2 minutes of relaxation time at 37 °C. Frequency tests were performed at 0.1% of strain from 0.1 Hz to 10 Hz. Frequency tests after gelation were performed under the same conditions for 20 minutes after 3 mL of DMEM was gently added around the cone and the plate. Thixotropic tests were carried out with the following method.
• the appearance and pH of KLD121.3% after X-ray or e-beam irradiation were not changed.
• the pH of KLD121.3% control was 2.2.
• the pH of KLD121.3% after X- ray or e-beam irradiation at 25 kGy and 40 kGy were 2.2.
• the appearance of QLEL12 after gamma, X-ray or e-beam irradiation was significantly altered.
• the pH of the QLEL12 control was 7.0.
• the pH’s of QLEL12 after gamma irradiation at 25 and 40 kGy were 6.5 and 5.9, respectively, those after X-ray irradiation at 25 and 40 kGy were 6.3 and 6.5, respectively, and those after e-beam irradiation at 25 and 40 kGy were 6.6 and 6.5, respectively.
• QLEL12 solutions became watery after gamma, X-ray or e-beam irradiation.
• RADA16, IEIK13 and KLD12 remained unchanged after gamma, X- ray and e-beam irradiation at around 25 ⁇ 40 kGy, but QLEL12 was not. Therefore, unlike QLEL12, RADA16, IEIK13, and KLD12 can be sterilized using a gamma, X-ray and e- beam irradiation techniques.
• Example 7 Characterization by HPLC and Mass Spectrometry The HPLC tests for RADA16, IEIK13, and QLEL12 were performed before and after gamma, X-ray and e-beam irradiation, and the results for their major peptide content were listed in Tables 7-10. Table 7.
• the major peptide contents of RADA16 after X-ray irradiation at 25 and 40 kGy were 73.8 and 71.9%, respectively.
• the major peptide contents of RADA16 after e-beam irradiation at 25 and 40 kGy were 76.0 and 70.0%, respectively.
• the major peptide content of IEIK13 control was 99.6%.
• the major peptide content of IEIK13 after gamma irradiation at 40 kGy was 99.8%.
• the major peptide contents of IEIK13 after X-ray irradiation at 25 and 40 kGy were 99.8 and 99.9%, respectively.
• the major peptide contents of IEIK13 after e-beam irradiation at 25 and 40 kGy were 99.6 and 99.8%, respectively.
• the major peptide content of QLEL12 control was 90.5%.
• QLEL12 showed significant decrease in its major peptide content after irradiation sterilization.
• the major peptide content of QLEL12 after gamma irradiation at 40 kGy was 40.7%.
• the major peptide contents of QLEL12 after X-ray irradiation at 25 and 40 kGy were 57.0 and 47.3%, respectively.
• Control PuraStat® also exhibited other M z peaks at 502, 665/1329, 916, 1143, and 1229, which are estimated as ARADA-NH 2 (SEQ ID NO:5), ARADARADARADA-NH 2 (SEQ ID NO:6), ARADARADA-NH 2 (SEQ ID NO:8), ARADARADARA (SEQ ID NO:10), Ac- RADARADARAD (SEQ ID NO:11), respectively.
• Table 12 shows the pattern of degradation of Ac-(RADA) 4 -NH 2 (SEQ ID NO:1) when PuraStat® is stored at 2-8 °C for about 4 years. From the pattern, we found that degradation mainly occurs at the points between ⁇ RAD and A ⁇ .
• the irradiated PuraStat® showed additional Mz peaks at 836/1670, 1100, and 1513, which are estimated as(RADA) 4 -NH 2 (SEQ ID NO:7), ADARADARADA-NH 2 (SEQ ID NO:9), and ADARADARADARADA-NH 2 (SEQ ID NO:12), respectively.
• Table 13 shows the additional pattern of degradation of Ac-(RADA) 4 -NH 2 (SEQ ID NO:1) when PuraStat® is sterilized with irradiation.
• the peaks at 836 and 1670 are ones of major additional peaks, which represent RADARADARADARADA-NH 2 (SEQ ID NO:7).
• Example 7 Rheological properties Based on ISO 11137 (Sterilization of health care products - Radiation), radiation sterilization methods can be used with 25 kGy or 15 kGy irradiation as the sterilization dose to achieve a sterility assurance level, SAL, of 10 -6 .
• SAL sterility assurance level
• the rheology results are shown in Figures 7 and 8 for PuraStat® with gamma irradiation sterilization before and after gelation, respectively.
• the determined rheological results are listed in Tables 14-15. Table 14.
• PuraStat® gamma- irradiated at 40 kGy showed significantly higher storage modulus than PuraStat® gamma-irradiated at 23 and 25 kGy. Also, PuraStat® gamma-irradiated at 25 kGy showed slightly higher storage modulus than PuraStat® gamma-irradiated at 23 kGy. This indicates that gamma-irradiation positively affected the rheological properties of PuraStat®.
• PuraStat ® X-ray-irradiated at 40 kGy showed significantly higher storage modulus that PuraStat ® X-ray-irradiated at 25 kGy. This indicates X-ray irradiation positively affected the rheological properties of PuraStat ® .
• PuraStat ® irradiated at 25 kGy (449.8 ⁇ 21.2) and 40 kGy (607.2 ⁇ 27.0 Pa) exhibited 39% and 88% increases in their storage moduli, respectively, compared to PuraStat ® control (322.9 ⁇ 21.0 Pa) (Table 17). Table 17.
• PuraStat ® e-beam-irradiated at 25 kGy and 40 kGy did not show significant difference (i.e., the p values were higher than 0.05) in their storage moduli compared to PuraStat ® control (Table 19).
• PuraStat ® demonstrated shear thinning property at high shear rate and thixotropic behavior suggesting slow rheological property recovery when high shearing stopped ( Figure 9). From these properties of PuraStat® , the stiffness of PuraStat ® can be lowered for easier handling during application to patients and stiffness can then slowly recover to initial values after application.
• samples were collected as follows: 10 samples for bioburden tests inside the packages (collecting 4 at the beginning, each 3 at the middle and the end of the packaging operation); 10 samples for bioburden tests of the filling liquid in the syringe (collecting 4 at the beginning, each 3 at the middle and the end of the packaging operation); spare samples, e.g.30 samples, may be collected in case extra testing may be required.
• a syringe was used to inject 50 mL of rinsing fluid for the sterilization test (USP Fluid D) into the inside of the package. A sample were shaken thoroughly and allowed to stand to reduce bubbles. Then, the outside opening of the package was sterilized by lightly passing the opening through a flame without heating the contents. Then, a syringe or another suitable method was used to extract all the injected solution from the package, which was then collected in a heat-resistant bottle.
• USP Fluid D rinsing fluid for the sterilization test
• a recovery rate and a correction coefficient based on the recovery rate were calculated in advance from the viable bacteria count and the added bacteria count from a sample which was prepared in the same manner as above.
• a correction coefficient was calculated as 1/recovery rate (%) x 100. This correction coefficient was recalculated when the sample preparation procedure was changed.
• For testing content fluid 1 mL of the content solution was mixed with 9 mL of Soybean-Casein Digest Medium agar medium (SCD agar medium), and the gel finely dispersed to make 10 mL of sample solution.
• Example 9 Experimental design of PuraStat® sterilization by Gamma irradiation Irradiation conditions--PuraStat ® samples (Lot# 17C09A30) were irradiated with gamma rays at 25, 28 and 40 kGy with Gammacell 220 ® High Dose Rate Co-60 Irradiator (MDS Nordion, Ottawa, Canada).
• the run dose rate and duration time for 40 kGy irradiation were 6.30 kGy/hr and 6 hours 20 minutes 58 seconds, respectively.
• the run dose rate and duration time for 28 kGy irradiation were 4.40 kGy/hr and 6 hours 22 minutes 31 seconds, respectively.
• the run dose rate and duration time for 25 kGy irradiation were 6.58 kGy/hr and 3 hours 47 minutes 42 seconds, respectively.
• Methods and test results The appearance of PuraStat ® was observed after each test.
• the pH of PuraStat ® was tested using an Accumet AB15 pH meter (Fisher Scientific). HPLC tests were performed to evaluate the major peptide content after irradiation tests.
• Agilent HPLC 1100 Agilent HPLC 1100 (Agilent Technologies) was used for this study. Column temperature was kept at 25 °C. Solvent A was water with 0.1% TFA and solvent B was 80% Acetonitrile with 0.1% TFA.
• the biological reactivity of a mammalian cell monolayer, L929 mouse fibroblast, in response to the test article extract was determined.
• the test article extract was obtained with serum-supplemented Minimum Essential Medium (MEM) at the ratio 0.2 g of article per mL. Extraction was done for 24 ⁇ 2 hours at 37 ⁇ 1 °C. Positive control (Natural Rubber) and negative control (Negative Control Plastic) articles and an untreated control (blank) were prepared to verify the proper functioning of the test system.
• the test article and control article extracts were used to replace the maintenance medium of the cell culture.
• the test article extract was tested at the 100% (neat) concentration. All cultures were incubated in at least 6 replicates for 24 to 26 hours.
• PuraStat® (40 mg) were mixed with 10 ⁇ L of DH2O and 500 ⁇ L of formic acid and vortexed. And the mixture was mixed with DH 2 O (4,450 ⁇ L) and vortexed.20 mL of samples were injected using an Agilent autosampler. Results are shown in following Table 23. Table 23. Findings--Overall, the major peptide content of PuraStat ® decreased with gamma irradiation methods and the extent of decrease was more significant with higher dose.
• PuraStat ® ’s degradation did not exceed 15% of the Overall, the major peptide content of PuraStat® decreased with gamma irradiation methods and the extent of decrease was more significant with higher dose.
• PuraStat®’s degradation did not exceed 15% of the amount of peptide prior to the irradiation.
• PuraStat®’s degradation did not exceed 5% of the amount of peptide prior to the irradiation. From the pattern of degradation of control PuraStat®, we found that degradation mainly occurs at the points between ⁇ RAD and A ⁇ .

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Organic Chemistry (AREA)
• Epidemiology (AREA)
• Engineering & Computer Science (AREA)
• Medicinal Chemistry (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biochemistry (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Zoology (AREA)
• Biomedical Technology (AREA)
• Wood Science & Technology (AREA)
• Biotechnology (AREA)
• Biophysics (AREA)
• Molecular Biology (AREA)
• Pharmacology & Pharmacy (AREA)
• Dermatology (AREA)
• Microbiology (AREA)
• Oral & Maxillofacial Surgery (AREA)
• Cell Biology (AREA)
• General Engineering & Computer Science (AREA)
• Transplantation (AREA)
• Immunology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Peptides Or Proteins (AREA)
• Medicinal Preparation (AREA)

Priority Applications (11)

Applications Claiming Priority (2)

Publications (1)

Family

ID=77855156

Family Applications (1)

Country Status (8)

Families Citing this family (2)

Citations (19)

Family Cites Families (22)

• 2021
  • 2021-03-30 WO PCT/US2021/024954 patent/WO2021202577A1/en not_active Ceased
  • 2021-03-30 CN CN202180026116.0A patent/CN115397454A/zh active Pending
  • 2021-03-30 US US17/217,911 patent/US11534528B2/en active Active
  • 2021-03-30 JP JP2022559333A patent/JP7335025B2/ja active Active
  • 2021-03-30 KR KR1020227037089A patent/KR20220161384A/ko active Pending
  • 2021-03-30 IL IL304283A patent/IL304283B2/en unknown
  • 2021-03-30 EP EP21780526.6A patent/EP4125995A4/en active Pending
  • 2021-03-30 AU AU2021246472A patent/AU2021246472B2/en active Active
  • 2021-03-30 IL IL296772A patent/IL296772B2/en unknown
• 2022
  • 2022-11-06 US US17/981,445 patent/US11738113B2/en active Active
  • 2022-12-02 US US18/074,244 patent/US11738114B2/en active Active
• 2023
  • 2023-07-05 US US18/346,895 patent/US12083243B2/en active Active
  • 2023-07-12 JP JP2023114502A patent/JP7482564B2/ja active Active
  • 2023-12-21 AU AU2023285888A patent/AU2023285888A1/en not_active Abandoned
• 2024
  • 2024-07-14 US US18/772,217 patent/US20240366836A1/en active Pending

Patent Citations (20)

Non-Patent Citations (5)

Also Published As

Similar Documents

Legal Events