Method for preparing stable peptide nanoparticle formulations

Abstract

The present disclosure is directed to methods of preparing peptide nanoparticle formulations, and in particular, peptide nanosuspensions, using low shear milling. More specifically, the disclosure is directed to methods of preparing peptide nanosuspensions by applying low frequency acoustic energy to an admixture comprising a peptide, an aqueous dispersion medium comprising a surface-active polymer and optionally a surfactant, and milling media, until the peptide has been milled to nanoparticle size. Also described are stable peptide nanosuspensions prepared by the methods.

Description

METHOD FOR PREPARING STABLE PEPTIDE NANOPARTICLE FORMULATIONSCROSS-REFERENCE TO RELATED APPLCIATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 594,576 filed October 31, 2023, the entire contents of which are incorporated herein by reference.BACKGROUND OF THE DISCLOSURE

[0002] The present disclosure is directed to methods of preparing peptide nanoparticle formulations, and in particular, peptide nanosuspensions, using low shear milling. More specifically, the disclosure is directed to methods of preparing peptide nanosuspensions by applying low frequency acoustic energy to an admixture comprising a peptide, an aqueous dispersion medium comprising a surface-active polymer and optionally a surfactant, and milling media, until the peptide has been milled to nanoparticle size. Also described are stable peptide nanosuspensions prepared by the methods.

[0003] Peptides have emerged as a distinct therapeutic modality compared to typical small molecule drugs and large molecule proteins (Henninot, et al., “The Current State of Peptide Drug Discovery: Back to the Future?”, J. Med. Chem. 2018, 61, 1382-1414; Wang, et al., “Therapeutic Peptides: Current Applications and Future Directions,” Signal Transduct. Target. Ther. 2022, 7, 48; Lau, et al., “Therapeutic Peptides: Historical Perspectives, Current Development Trends, and Future Directions,” Bioorg. Med. Chem. 2018, 26, 2700-2707). However, peptide therapeutics exhibit unique challenges that make their successful development difficult. These can consist of pharmacokinetic liabilities such as short half-life and limited absorption across physiological barriers as well as poor chemical and physical stability (Di, L., “Strategic Approaches to Optimizing Peptide ADME Properties,” AAPS J. 2015, 17, 134-143). In some cases, structural modification can address these risks to some extent, while formulation development can enable improved delivery.

[0004] In general, protein and peptide drug products are formulated at relatively low concentration for intravenous (IV) administration (Garidel, et al., “High-Concentration Protein Formulations: How High Is High?,” Eur. J. Pharm. Biopharm. 2017, 119, 353-360).On the other hand, subcutaneous (SC) administration would provide significant benefits to patients including the ability to self-administer the medication at home, reducing costs and increasing compliance. However, SC injections are limited to 1-1.5 mL dosing volume, potentially resulting in high peptide dose concentrations of >100 mg / mL (Garidel, et al., Eur. J. Pharm. Biopharm. 2017, 119, 353-360). This can result in significant challenges including solubility limitations, high viscosity, and physical instability leading to aggregation (Bak, et al., “Physicochemical and Formulation Developability Assessment for Therapeutic Peptide Delivery — A Primer,” Aaps J 2015, 17, 144-155; Evers, et al., “Peptide Optimization at the Drug Discovery-Development Interface: Tailoring of Physicochemical Properties Toward Specific Formulation Requirements,” J Pharm Sci 2019, 108, 1404-1414). Higher concentrations often exacerbate these effects and lead to increased risk of peptide-peptide interactions potentially leading to conformational changes, aggregation, precipitation, or gelation (Zapadka, et al., “Factors Affecting the Physical Stability (Aggregation) of Peptide Therapeutics,” Interface Focus 2017, 7, 20170030). This can cause loss of activity as well as toxicity and immunogenicity risks gelation (Zapadka, et al., “Factors Affecting the Physical Stability (Aggregation) of Peptide Therapeutics,” Interface Focus 2017, 7, 20170030).

[0005] Additionally, peptides are generally preferred to be formulated in aqueous solution (Nugrahadi, et al., “Designing Formulation Strategies for Enhanced Stability of Therapeutic Peptides in Aqueous Solutions: A Review,” Pharmaceutics 2023, 15, 935). Unlike larger proteins, peptides are smaller and often lack a strong secondary structure. As a result, hydrophobic residues can have a disproportionately large effect on solubility since the lack of a strong secondary structure results in their surface exposure. Additionally, peptides often exhibit sharp pH-dependent solubilities that are difficult to control effectively with buffers. In some cases, non-aqueous solvents have been explored, such as the use of ethanol and Cremophor EL in formulating cyclosporine A, although their use remains relatively limited due to their potential for unfolding and denaturation as well as having limited pharmaceutical acceptability (Stevenson, C., “Characterization of Protein and Peptide Stability and Solubility in Non-Aqueous Solvents,” Curr. Pharm. Biotechnol. 2000, 1, 165-182).

[0006] In contrast, stable suspension formulations of peptides in water may provide an attractive alternative (DeFelippis, et al., “Peptides and Proteins as Parenteral Suspensions:An Overview of Design, Development, and Manufacturing Considerations,” In Pharmaceutical Formulation Development of Peptides and Proteins, Hovgaard, et al., Eds.; CRC Press: Boca Raton, FL, USA, 2013; p. 193). There have been some recent advances in the use of suspensions for long-acting injectable depot formulations, although these have been largely limited to lipid-based approaches or aqueous suspensions of small molecules (Sharma, et al., “Recent Advances in Lipid-Based Long-Acting Injectable Depot Formulations,” Adv. Drug Deliv. Rev. 2023, 199, 114901; Johnson, et al., “Retrospective Analysis of Preclinical and Clinical Pharmacokinetics from Administration of Long- Acting Aqueous Suspensions,” Pharmaceut Res 2023, 1-16). Suspension formulation approaches may be able to overcome challenges with solubility limitations when formulating at higher concentrations. In addition, the chemical stability of the peptide may be improved in the solid state. However, due to their relatively large molecular weight and conformational flexibility, peptides often exist in disordered amorphous forms in the solid state and control over physical stability and particle size can be difficult. Thus, a general approach for formulating stable peptide suspensions has been lacking.

[0007] Nanosuspensions have been demonstrated as an effective enabled formulation strategy for small molecules (Merisko-Liversidge, et al., “Drug Nanoparticles: Formulating Poorly Water-Soluble Compounds,” Toxicol Pathol 2008, 36, 43-48; Merisko- Liversidge, et al., “Nanosizing for Oral and Parenteral Drug Delivery: A Perspective on Formulating Poorly-Water Soluble Compounds Using Wet Media Milling Technology,” Adv Drug Deliver Rev 2011, 63, 427-440; Kesisoglou, et al., “Nanosizing — Oral Formulation Development and Biopharmaceutical Evaluation,” Adv Drug Deliver Rev 2007, 59, 631-644; Rabinow, B.E., “Nanosuspensions in Drug Delivery,” Nat Rev Drug Discov 2004, 3, 785-796; Muller, et al., “State of the Art of Nanocrystals - Special Features, Production, Nanotoxicology Aspects and Intracellular Delivery,” Eur J Pharm Biopharm 2011, 78, 1-9; Yadollahi, et al., “Nanosuspension Technologies for Delivery of Poorly Soluble Drugs,” J Nanomater 2015, 2015, 1-13; Gao, et al., “Drug Nanocrystals for the Formulation of Poorly Soluble Drugs and Its Application as a Potential Drug Delivery System,” J Nanopart Res 2008, 10, 845-862; Liu, etal., “A Mini Review of Nanosuspensions Development,” J Drug Target 2011, 20, 209-223; Chandra, et al., “Nanosuspension: An Overview,” J Drug Deliv Ther 2013, 3, 162-167). These formulations generally consist of a suspension of drug nanocrystals and thus exhibit particularly high drug loading incomparison to other engineered nanoparticle delivery systems such as liposomes and lipid or polymer nanoparticles. Nanosuspensions often consist of nanoparticles of >75% drug load and can often be prepared at high overall drug concentration in aqueous suspension (i.e., >100 mg / mL). Due to their small particle size and large surface area, nanosuspension formulations exhibit dramatically increased dissolution rate and potentially saturation solubility (Patel, et al., “Nanosuspension: An Approach to Enhance Solubility of Drugs,” J Adv Pharm Technology Res 2011, 2, 81-87), enabling improved absorption in vivo when administered orally. In addition, nanosuspensions can also be administered via parenteral delivery routes as well (Ma, et al., “Nanosuspensions Technology as a Master Key for Nature Products Drug Delivery and In Vivo Fate,” Eur J Pharm Sci 2023, 185, 106425; Marques, et al., “Factors Affecting the Preparation of Nanocrystals: Characterization, Surface Modifications and Toxicity Aspects,” Expert Opin Drug Del 2023, ahead-of-print, 1-24; Pinar, et al., “Formulation Strategies of Nanosuspensions for Various Administration Routes,” Pharm 2023, 15, 1520), including intravenous (IV) delivery (Chiang, et al., “Nanosuspension Delivery of Paclitaxel to Xenograft Mice Can Alter Drug Disposition and Anti-Tumor Activity,” Nanoscale Res Lett 2014, 9, 156; Gao, et al., “Preparation, Characterization, Pharmacokinetics, and Tissue Distribution of Curcumin Nanosuspension with TPGS as Stabilizer,” Drug Dev Ind Pharm 2010, 36, 1225-1234; Gao, et al., “Studies on Pharmacokinetics and Tissue Distribution of Oridonin Nanosuspensions,” Int J Pharmaceut 2008, 355, 321-327).

[0008] Small molecule nanosuspensions are often manufactured using top-down wet milling, high pressure homogenization, or ultrasonication (Eerdenbrugh, et al., “Top- down Production of Drug Nanocrystals: Nanosuspension Stabilization, Miniaturization and Transformation into Solid Products,” Int J Pharmaceut 2008, 364, 64-75). Bottom-up precipitation methods have been investigated, although these require initial solubility in organic solvents, presenting challenges with purification and isolation as well as stability (Ma, et al., “Nanosuspensions Technology as a Master Key for Nature Products Drug Delivery and In Vivo Fate,” Eur J Pharm Sci 2023, 185, 106425). Once prepared, the drug nanoparticles themselves are inherently high energy and unstable, presenting a risk of aggregation. In order to mitigate this risk, small amounts of polymer and / or surfactant excipients are added to stabilize the nanoparticles and prevent aggregation from occurring. This requires the selection of the optimal stabilizer combination, which can be drug specificand must be identified through empirical screening (Ma, et al., “Nanosuspensions Technology as a Master Key for Nature Products Drug Delivery and In Vivo Fate,” Eur J Pharm Sci 2023, 185, 106425; Marques, et al., “Factors Affecting the Preparation of Nanocrystals: Characterization, Surface Modifications and Toxicity Aspects,” Expert Opin Drug Del 2023, ahead-of-print, 1-24; Pinar, et al., “Formulation Strategies of Nanosuspensions for Various Administration Routes,” Pharm 2023, 15, 1520), although recent computational work has started to elucidate an understanding of the drug stabilizer interactions involved (Ferrar, et al., “Towards an Improved Understanding of Drug Excipient Interactions to Enable Rapid Optimization of Nanosuspension Formulations,” Int J Pharmaceut 2020, 578, 119094).

[0009] Nevertheless, these types of manufacturing processes involve high shear forces used to generate the small particle size, which has made these approaches prohibitive for sensitive large molecules such as peptides and proteins.

[0010] Thus, there still remains a need for a general approach to formulate peptides at high concentrations.BRIEF DESCRIPTION OF THE DISCLOSURE

[0011] In one aspect, the present disclosure is directed to a method of preparing a peptide nanosuspension, the method comprising applying low frequency acoustic energy at a frequency of from about 10 to about 20,000 Hertz to an admixture comprising (i) a peptide; (ii) an aqueous dispersion medium comprising a surface-active polymer; and (iii) milling media.

[0012] In another aspect, the disclosure is directed to a method of screening aqueous dispersion media for use in a peptide nanosuspension, the method comprising: (a) admixing a peptide, milling media, and a plurality of aqueous dispersion media in one or more slurry containers to form a plurality of admixtures; and (b) applying low frequency acoustic energy at a frequency of from about 10 to about 20,000 Hertz to the admixtures.

[0013] In another aspect, the disclosure is directed to a kit for preparing a peptide nanosuspension comprising a slurry container, milling media, a peptide, and an aqueous dispersion media comprising a surface-active polymer.

[0014] Other aspects of the disclosure are set forth hereinafter.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGs. 1 A-1C depict the structures of insulin (a large peptide hormone) (1A), GNE-A (a cystine-knot peptide) (IB), and cyclosporine A (CsA, a macrocyclic peptide) (1C).

[0016] FIG. 2 is a chart depicting the average particle size as measured by dynamic light scattering of insulin nanosuspension formulations prepared from 1) 25% PVP K29-32 and 1% SDS, or 2) 25% HPC-SL and 1% SDS (wt% to insulin). The formulations were prepared using resonant acoustic milling.

[0017] FIGs. 3 A and 3B are charts depicting the physical stability as measured by size exclusion chromatography of insulin nanosuspension formulations prepared from 1) 25% PVP K29-32 and 1% SDS (3 A), or 2) 25% HPC-SL and 1% SDS (3B) (wt% to insulin). The formulations were prepared using resonant acoustic milling.

[0018] FIGs. 4A and 4B are charts depicting the chemical stability as measured by reverse phase chromatography of insulin nanosuspension formulations prepared from 1) 25% PVP K29-32 and 1% SDS (4A), or 2) 25% HPC-SL and 1% SDS (4B) (wt% to insulin). The formulations were prepared using resonant acoustic milling.

[0019] FIG. 5 is a chart depicting the average particle size as measured by dynamic light scattering of GNE-A nanosuspension formulations prepared from 1) 25% Tween80, or 2) 25% Pluronic F127 (wt% to GNE-A). The formulations were prepared using resonant acoustic milling.

[0020] FIGs. 6A and 6B are charts depicting the physical stability as measured by size exclusion chromatography of GNE-A nanosuspension formulations prepared from 1) 25% Tween80 (6 A), or 2) 25% Pluronic Fl 27 (6B) (wt% to GNE-A). The formulations were prepared using resonant acoustic milling.

[0021] FIGs. 7A and 7B are charts depicting the chemical stability as measured by reverse phase chromatography of GNE-A nanosuspension formulations prepared from 1)25% Tween80 (7 A), or 2) 25% Pluronic Fl 27 (7B) (wt% to GNE-A). The formulations were prepared using resonant acoustic milling.

[0022] FIG. 8 is a graph comparing the 4-week stability as determined using size exclusion chromatography of a GNE-A nanosuspension formulation prepared with Pluronic Fl 27 using resonant acoustic milling and a GNE-A solution formulation at 100 mg / mL concentration at 37°C.

[0023] FIGs. 9A and 9B depict transmission electron microscopy (TEM) images of a cyclosporine A (CsA) nanosuspension formulation prepared with 25% HPC-SL and 1% SDS (wt% to CsA) using resonant acoustic milling.

[0024] FIG. 10 is a chart depicting the average particle size as measured by dynamic light scattering of cyclosporine A (CsA) nanosuspension formulations prepared from 1) 25% HPC-SL and 1% SDS, or 2) 25% Tween80 (wt% to CsA). The formulations were prepared using resonant acoustic milling.

[0025] FIG. 11 is a chart depicting the average particle size as measured by dynamic light scattering of cyclosporine A (CsA) nanosuspension formulations prepared with increasing ratios of SDS at a 10 mg / mL concentration. The formulations were prepared using resonant acoustic milling.

[0026] FIG. 12 is a chart depicting the average particle size as measured by dynamic light scattering of cyclosporine A (CsA) nanosuspension formulations prepared with 25% SDS (wt% to CsA) over 28 days at a 10 mg / mL concentration. The formulations were prepared using resonant acoustic milling.

[0027] FIG. 13 is a graph depicting the physical stability as measured by size exclusion chromatography of cyclosporine A (CsA) nanosuspension formulations with 25% SDS (wt% to CsA). The formulations were prepared using resonant acoustic milling.

[0028] FIG. 14 is a graph depicting the chemical stability as measured by reverse phase chromatography of cyclosporine A (CsA) nanosuspension formulations with 25% SDS (wt% to CsA). The formulations were prepared using resonant acoustic milling.

[0029] FIG. 15 is a graph depicting the injection forces of the Sandimmune® lipid- based formulation as compared to an aqueous nanosuspension of cyclosporine A (CsA) prepared using resonant acoustic milling by using a BD 1-mL syringe attached with 25G needle. The maximum injection forces of the Sandimmune® formulation and the CsA nanosuspension were 26.97 N and 4.33 N, respectively.

[0030] FIG. 16 is a graph depicting the PK profile of the commercial Sandimmune® formulation as compared to aqueous nanosuspension formulations prepared by resonant acoustic milling of cyclosporine A (CsA).

[0031] FIG. 17 is a graph depicting the average particle sizes of various peptide formulations prepared using ultrasonication.

[0032] FIGs. 18A and 18B depict peptide formulations of insulin, cyclosporine A, and GNE-A milled using ultrasonication at formation (18A) and at day 3 (18B). For each peptide formulation, the formulation in the left vial was subjected to ultrasonicated for 5 minutes, and the formulation in the right vial was subjected to ultrasonicated for 10 minutes. The formulations exhibited settling behavior from the initial timepoint.DETAILED DESCRIPTION OF THE DISCLOSURE

[0033] The present disclosure is directed to methods of preparing peptide nanoparticle formulations, and in particular, peptide nanosuspensions, using low shear milling. In particular, the disclosure is directed to methods of preparing peptide nanosuspensions by applying low frequency acoustic energy to an admixture comprising a peptide, an aqueous dispersion medium comprising a surface-active polymer and optionally a surfactant, and milling media, until the peptide has been milled to nanoparticle size. The nanosuspension formulations of the present disclosure are thus aqueous colloidal dispersions of peptide nanoparticles.

[0034] Because many peptides are sensitive to instability, and susceptible to aggregation, it was previously believed that nanomilling processes, including acoustic milling, were unsuitable for use with sensitive proteins and peptides. It has now surprisingly been discovered that acoustic milling, such as resonant acoustic milling, can be used to formulate stable peptide nanosuspensions, and as demonstrated in the examples, is generally applicable to a wide variety of peptide types. Advantageously, the methods of the presentdisclosure allow for formation of stable nanosuspensions, even at high concentrations (e.g., including 50-300 mg / mL) above the solubility limit, and without the need for harsh excipients. Because the peptides in the nanosuspensions remain in a solid state, the formulations have improved stability.

[0035] More specifically, the method of the disclosure comprises admixing in a slurry-container an aqueous dispersion medium, a peptide, and milling media, and subjecting the admixture to acoustic energy of sufficient frequency and amplitude, and for a sufficiently sustained period, to provide a nanoparticle suspension (nanosuspension) of the peptide dispersed in the aqueous dispersion medium. In certain aspects, after the nanosuspension has been milled to the desired particle size the nano-suspension is separated from the milling media.

[0036] In the methods of the present disclosure, the low frequency acoustic energy is applied to the admixture until the peptide has been milled to nanoparticle size. In one aspect, the admixture is subjected to acoustic energy for from about 0.25 to about 24 hours, including from about 0.5 to about 10 hours, or from about 1 to about 5 hours, or from about 1 to about 2 hours. In one aspect, the admixture is subjected to acoustic energy for about 2 hours.

[0037] As will be appreciated, recovery of the nanosuspension (separation from the milling media) can be accomplished by any known physical separation method, for example, where the media permits, separation can be accomplished by decantation, for example, as in the case of the Examples presented herein, a nanosuspension prepared using YTZ milling media can be recovered using a 18 gauge needle and syringe since the milling media is of sufficient density and size to permit such separation. Where decantation is not possible, for example where the milling media is polyester prill, size exclusion separation can be employed, for example, by centrifuging through a properly sized sieve.

[0038] Milling media suitable for use in the method of the present disclosure include any particulate material compatible with acoustic mixing processes. Examples of suitable milling media include glass beads, polyester prill; polystyrene milling beads, and yttria-stabilized zirconia milling beads (e g., YTZ grinding media from Tosoh).

[0039] As the term is used herein, acoustic energy is linear or spherical energy propagation through a tangible medium which is within the frequency range of 10 hertz to 20,000 hertz. In some embodiments of the process of the present disclosure, it is preferred to employ linear acoustic energy at a frequency of from about 10 hertz up to about 100 hertz, more preferably the acoustic energy is supplied at a frequency of about 60 hertz. It will be appreciated that in certain aspects, in accordance with known principles, the exact frequency will be selected to provide a standing wave in the slurry from which a nanosuspension is being provided. The frequency required to achieve a standing wave will vary according to known principles depending upon the nature of the slurry and the dimensions of the slurry to which acoustic energy is applied. The acoustic energy is advantageously propagated uniformly throughout the slurry container.

[0040] In one embodiment, the methods of the present disclosure utilize resonant acoustic mixing. Unlike techniques such as ultrasound, resonant acoustic mixing delivers energy to a sample at its resonant frequency, typically only between 58-62 Hz. This is an extremely efficient mechanism for mixing while, at the same time, the low frequencies used result in less stressful shear forces to the materials being mixed. As a result, resonant acoustic mixing results in more stable materials compared to earlier high shear techniques as well as an approach to mill sensitive compounds.

[0041] Acoustic energy can be supplied to an admixture using any known source, however, in general it is preferred to supply the energy by cyclic linear displacement of a container filled with the admixture. In processes of the invention, preferably the acoustic energy supplied by linear displacement exerts between about 10 times G-force (where “G” is the force of gravity) and about 100 times G-force. In one aspect, the acoustic energy supplied by linear displacement exerts about 50 times G-force. Although it will be appreciated that numerous mechanical or electronic transducer arrangements can be utilized to supply the cyclic linear displacement required to generate the desired G-force within the desired frequency range, one example of commercially available equipment suitable for supplying the necessary acoustic energy is the Resodyn™ acoustic mixer (Resodyn Acoustic Mixers, Inc ), which makes equipment available in a range of capacities from bench-scale to multi-kilogram capacity.

[0042] It was previously known that an acoustic mixer such as a Resodyn™ acoustic mixer could be used to efficiently provide suspensions by mixing pre-formed nanoparticulate materials and an aqueous dispersion medium, however, acoustic mixing has not been previously employed to prepare peptide nanoparticles from bulk powdered solid materials (i.e., macro-particulate materials defined herein as having a D5O>1 micron). Surprisingly, it has been discovered that the use of acoustic energy to prepare a nanosuspension from macro-particulate solid peptide materials provides a nanosuspension product which has stability not obtainable in a nanosuspension prepared using high-energy mixing, such as ultrasonication. Additionally, the methods of the present disclosure advantageously have been found to be highly general and have been demonstrated to be effective across a wide range of different peptide structures and molecular weights. Peptide nanosuspensions produced by the methods of the present disclosure additionally exhibit improved chemical and physical stability compared to corresponding solution formulations, particularly at high concentrations. In addition, certain nanosuspensions produced by the methods of the disclosure have been shown to exhibit similar exposures compared to current commercial formulations when dosed in vivo, with the additional benefit of not requiring high concentrations of lipids and surfactants.

[0043] As the term is used herein, a nanosuspension comprises sub-micron particles (i.e., particles yielding a D50 measurement of <1 micron) of a peptide present as a colloidal dispersion in an aqueous dispersion medium. In one embodiment, the methods of the present disclosure produce nanosuspensions comprising peptide particles having a D50 measurement of less than 500 nm. The term “macro-particulate” material is used herein to distinguish nano-particulated materials provided by the method of the present disclosure from powdered material provided by ordinary milling or precipitation techniques, for example, those which in general have a D50 value well in excess of 1 micron.

[0044] As discussed herein and as demonstrated in the Examples, the methods of the present disclosure are generally applicable to structurally diverse peptides. As used herein, the term “peptide” refers to molecules comprising between 2 and 50 amino acids and having a molecular weight ranging from about 0.5 kDa to about 100 kDa, including from about 1 kDa to about 50 kDa, or from about 1.2 kDa to about 7.0 kDa. The peptide present in a nanosuspension of the present disclosure may be any peptide including, but not limited to, macrocyclic peptides, cystine-knot peptides, and large peptide hormones. In oneparticular aspect, the peptide is selected from the group consisting of insulin, cyclosporine A, and cystine-knot peptides.

[0045] In general, the aqueous dispersion medium employed in nanosuspensions of the disclosure comprises primarily water (based on wt. %) and one or more water-miscible surface-active constituents. In nanosuspensions of the present disclosure, preferably the surface-active constituents are water-miscible polymers (also referred to herein as surfaceactive polymers), described in detail herein, and optionally, comprises also a surfactant. When a surfactant is employed in aqueous dispersions medium used to prepare a nanosuspension of the disclosure, preferably the surfactant is an ionic surfactant, described in detail herein.

[0046] It will be appreciated that it is advantageous for peptide nanosuspensions to: (i) demonstrate physical and chemical stability; (ii) avoid the use of harsh excipients and organic solvents that may disrupt the peptide structure; (iii) demonstrate good absorption in vivo after subcutaneous injection; and (iv) retain high drug loading and good stability at high concentrations.

[0047] Two important metrics of a nanosuspension are: (i) the ratio of peptide to aqueous dispersion medium (i.e., “drug loading”) which can be employed in a slurry from which the nanosuspension is prepared; and (ii) the ratio of peptide to the surface-active constituents (water-miscible polymer and optional surfactant) employed in the slurry from which the nanosuspension is prepared to stabilize the nanoparticles formed. As will be understood from the Examples which follow, selection of suitable ratios of peptide to surface-active constituents allow for formation of peptide nanosuspensions having good chemical and physical stability, even at high drug loading. Thus, the method of the present disclosure advantageously permits preparation of peptide nanosuspensions in which the slurry can employ a high weight percentage (wt. %) of the peptide relative to the aqueous dispersion medium (water and surface-active constituents described herein), while maintaining stability.

[0048] As mentioned above, an aqueous dispersion medium utilized in a peptide nanosuspension of the present disclosure comprises water and one or more surface-active constituents which stabilize the particles present in a nanosuspension. Although the ratio of aqueous dispersion medium and peptide will vary depending upon the nature of the peptide,in general the slurry from which a nanosuspension is prepared will comprise from about 0.1 wt% to about 50 wt% peptide relative to the weight of the aqueous dispersion medium employed (i.e., a peptide concentration of from about 1 mg / mL to about 500 mg / mL). In another aspect, the slurry from which a nanosuspension is prepared will comprise from about 1 wt. % to about 40 wt. % peptide relative to the weight of aqueous dispersion medium employed (i.e., a peptide concentration of from about 10 mg / mL to about 400 mg / mL). In certain other aspects, the slurry from which a nanosuspension is prepared will comprise from about 1.3 wt% to about 10 wt% peptide relative to the weight of the aqueous dispersion medium employed (i.e., a peptide concentration of from about 13 mg / mL to about 100 mg / mL). In another aspect, the slurry from which a nanosuspension is prepared will comprise from about 0.5 wt% to about 10 wt% peptide relative to the weight of the aqueous dispersion medium employed (i.e., a peptide concentration of from about 5 mg / mL to about 100 mg / mL).

[0049] With regard to the wt. % ratio of peptide to surface-active constituents present in the aqueous dispersion medium (and therefore associated with the nanoparticles present in a nano-suspension of the invention at the conclusion of the milling process), the ratio will vary depending upon the nature of the peptide employed. In general, it is preferred to prepare a nanosuspension with the highest weight ratio of peptide relative to the weight of surface-active constituent which will provide a stable nanosuspension. In some embodiments, the nanosuspensions produced by the process of the disclosure comprise from about 25 wt. % to about 99 wt. % peptide relative to the weight of surface-active constituents present in the nanosuspension. In some embodiments, the nanosuspensions have in excess of at least about 40 wt. % peptide relative to the weight of surface-active constituents present in the nano-suspension. In some embodiments, the nanosuspensions have more than 90 wt. % peptide relative to the weight of surface-active constituents employed and in some embodiments more than 95 wt. % peptide relative to the amount of surface-active constituents employed. In some embodiments, as much as 98 wt. % peptide is present relative to the amount of surface-active constituents employed. In other embodiments, the methods of the disclosure produce nanosuspensions that comprise from about 1 wt% to about 500 wt%, including from about 1 wt% to about 200 wt% surface-active constituents relative to the weight of peptide present in the nanosuspension.

[0050] In general, surface-active polymers suitable for use in the present disclosure are water miscible polymers, and in particular, include amphiphilic polymers. In the nanosuspensions of the present disclosure, the hydrophobic portion of the polymer adsorbs to the nanoparticle surface, while the hydrophilic portion of the polymer is exposed to water, thus stabilizing the nanoparticles. Examples of suitable polymers include, but are not limited to: polymers based on hydroxypropylcellulose (HPC), for example, HPC-SL (Ashland Chemical); alkoxide block copolymers, for example an ethylene oxide / propylene oxide copolymer, for example, Plurionic F127 from BASF; polyethylenesorbitol polymers, for example Tween 80 (Sigma, article of commerce); polyvinylpyrrolidone (PVP) polymers, for example, Plasdone® K29-32 from Ashland Chemical; methyl Cellulose (article of commerce, for example, available from Sigma) and cellulose derivatives, for example, carboxycellulose derivatives, for example, hydroxypropylmethyl cellulose derivatives, for example, Methocel polymers from Dow Chemical, and polypropylene- and polyethylene glycol polymers and derivatives, for example, Carbowax® available from Dow Chemical. It will be appreciated that other surface-active polymers may be employed, dictated by the nature of the peptide being dispersed.

[0051] In some embodiments, the surface-active polymer is combined with a surfactant to help maintain dispersion. As is known, the nature and amount of surfactant employed will depend upon the polymer employed and the nature of the surface of the particles of peptide dispersed. Examples of surfactants suitable for use in nanosuspensions of the present disclosure include, but are not limited to, dioctyl sulfosuccinates (docusate sodium, DOSS, article of commerce), and sodium dodecyl sulfate (SDS), an ionic surfactant prepared from laurel alcohol, an article of commerce, both available, for example, from Fluka.

[0052] Advantageously, the method of the present disclosure can be applied to any volume of admixture, and thus is scalable. In some aspects, the volume of admixture in the slurry container is at least 1 uL, including at least 10 uL, at least 100 uL, at least 1 mL, at least 10 mL, at least 100 mL, at least 1 L, at least 5 L, at least 10 L, at least 50 L, at least 100 L, or at least 200 L, or from about 1 uL to about 200 L, or from about 10 uL to about 100 L, or from about 100 mL to about 1 L, or from about 100 uL to about 100 mL.

[0053] As mentioned above, a nanosuspension of the disclosure is prepared by applying acoustic energy to a slurry contained in a suitable slurry container. For carrying out the process of the disclosure, any convenient, sealable container may be employed which can be fixed to the carriage of the acoustic mixing equipment utilized to prepare the slurry. Examples of suitable containers are, but not limited to, sealable bottles (of any material, for example, glass, plastic or metal, preferably glass or plastic), a sealable plastic bag, and a sealable micro-titer well plate.

[0054] It will be appreciated from the foregoing that the flexibility of choice in suitable slurry container means that the process of the present disclosure readily affords the ability to provide peptide nanosuspensions under aseptic manufacturing conditions as well as provides “line-of-sight” scalability of a particular set of parameters from “bench-scale” batch size to volumes suitable for commercial preparation of nanosuspension in the provision of a peptide on a commercial scale.

[0055] In one aspect the present disclosure is a peptide nanosuspension prepared by the process of the disclosure. As mentioned above, and exemplified below, peptide nanosuspensions prepared by the method of the disclosure exhibit good physical and chemical stability, even at higher concentrations. Moreover, it will be appreciated that the process of the present disclosure is readily adaptable to either a “batch” or “continuous process” mode for large-scale preparation of nanosuspensions for use in the preparation of medicaments.

[0056] As will be apparent, the selection of surface-active polymer and optional surfactant may vary depending on the peptide included in the nanosuspension. It will be appreciated that the method of the present disclosure is amenable to the provision of multiple simultaneously prepared samples of a peptide nanosuspension using the same mixing conditions. This latter feature permits variations of a formulation to be prepared simultaneously, under the same conditions, for the purposes of comparing or optimizing formulations. It will be appreciated that the methods of the present disclosure provide for the screening and optimization of formulations while consuming only small amounts of peptide when a small sample size is prepared, for example, by the use of a sealable micro-titer well plate as a slurry container.

[0057] Thus, in another aspect, the disclosure is directed to a method of screening aqueous dispersion media for use in a peptide nanosuspension. The method comprises (a) admixing a peptide, milling media, and a plurality of aqueous dispersion media in one or more slurry containers to form a plurality of admixtures; and (b) applying low frequency acoustic energy at a frequency of from about 10 to about 20,000 Hertz to the admixtures. In one embodiment, the slurry container is a multi-well plate, for example, a sealable microtiter well plate. It will be appreciated that each of the plurality of admixtures is in a separate slurry container, or in a separate well of a single slurry container, such as a multi-well plate. The low frequency acoustic energy may be applied to the admixtures simultaneously and / or sequentially.

[0058] In one aspect, following application of the low frequency acoustic energy to the admixtures, the resulting nanosuspensions are analyzed for stability. As used herein, “stability” of the nanosuspensions may refer to physical and / or chemical stability. In some embodiments, physical stability of the nanosuspensions is evaluated using one or more of the following measures: 1) size of the peptide nanoparticles, 2) aggregation of the peptide nanoparticles, and / or peptide monomer content present in the nanosuspension over time. In some embodiments, chemical stability of the nanosuspensions is evaluated by determining the presence of peptide degradation products in the nanosuspension. Stability may be analyzed using any known technique, such as those set forth in the examples. In one embodiment, stability is analyzed after storage of the admixtures for at least 14 days, or at least 28 days at room temperature, 4°C, and / or 37°C, as described in the Examples. It will be appreciated that following stability measurements of the nanosuspensions, the aqueous dispersion media producing a nanosuspension with the best stability may be selected for further development of peptide nanosuspension formulations. In one embodiment, a stable nanosuspension refers to a nanosuspension wherein the smallest particle size of the nanoparticles in the nanosuspension remain unchanged over the measured time period.

[0059] In another aspect the present disclosure is directed to kits for preparing a peptide nanosuspension comprising a slurry container, milling media, a peptide, and an aqueous dispersion media comprising a surface-active polymer and optionally a surfactant. The kit may comprise peptide, surface-active polymer, and / or surfactant in amounts suitable to prepare the nanosuspensions described herein.EXAMPLESMaterials

[0060] Compounds, reagents, and solvents were obtained from commercial sources and used as received unless otherwise noted.

[0061] Insulin (recombinant human) was obtained from Millipore Sigma Cat# 91077C-1G. GNE-A is a disulfide constrained peptide and was obtained from Genentech Research Laboratories, South San Francisco, USA. Cyclosporine A was obtained from Toronto Research Chemicals Cat# C988900 Lot# 15-XJZ-47-1. Sandimmune® Injection (cyclosporine, USP) 50 mg / mL was obtained from McKesson Medical NDC #00078010901. Formic acid was obtained from Alfa Aesar CAS #64-18-6. Acetonitrile was obtained from VWR Cat# 099891. Trifluoroacetic acid was obtained from J.T. Baker CAS #76-05-1. Ammonium formate was obtained from Sigma-Aldrich CAS #540-69-2. Sodium Dodecyl sulfate (SDS) was obtained from Spectrum Chemical CAS #151-21-3. Plasdone (PVP) K29- 32 was obtained from Acros Organics CAS #2687-91-4. Pluronic F127 was obtained from Sigma CAS #9003-11-6. Tween 80 was obtained from Sigma CAS #9005-65-6. Hydroxypropyl Cellulose (HPC)-SL was obtained from Alfa Aesar CAS #9004-64-2. Loadings and concentrations are reported as weight percent (wt%) unless otherwise noted.Analytical Characterization Methods for Nanosuspension SamplesParticle Size Analysis Using Dynamic Light Scattering:

[0062] After milling, a 5 pL aliquot of the nanosuspension sample was taken and diluted in 995 pL of D.I. water for analysis using a Wyatt DynaProTM Plate Reader II (Waters Corporation, Santa Barbara, CA, USA) dynamic light scattering instrument. A 30 pL aliquot of the diluted suspension was dispensed into a Coming® low volume black polystyrene 384-well plate for analysis. The particle size of each sample was reported as an average of 10 acquisitions with an acquisition time of 5 sec at 25 °C. Autocorrelation curves were fitted using either the cumulants or regularization method, and the average particle radius and diameters D50 and D90 were obtained and reported. The normalized polydispersity (%Pd) was calculated as the polydispersity divided by the estimated hydrodynamic radius from the cumulants fit of the autocorrelation function multiplied by 100.Stability Analysis Using HPLC:

[0063] Aliquots of the nanosuspension formulation samples were added to Eppendorf tubes and stored at room temperature, 4 °C, and 37 °C. Samples were taken at time points corresponding to day 0, 14, and 28. At each timepoint, the physical and chemical stability of the formulations were investigated by HPLC. The injected sample concentration was 0.5 mg / mL with 200-fold dilution in 30 / 70 acetonitrile / water for GNE-A and cyclosporin A or in 0.0 IN HC1 for insulin.

[0064] An Agilent 1290 Infinity II (Agilent Technologies, Santa Clara, CA, USA) was used for chromatographic analysis. Data were acquired and processed using Empower 3 software (Waters, Milford, MA, USA). Separations were performed in an Xbidge BEH125 SEC column (3.5mm, 7.8 x 150 mm) for physical stability analysis. The injection volume was 8 pL and the column temperature was 30 °C. The detection wavelength was 214 nm and the flow rate was 0.5 mL per minute. 80% of the 5mM Phosphate buffer at pH 7 in acetonitrile was used as the mobile phase.

[0065] For chemical stability, Cyclosporin A was analyzed by using a BEH Cl 8 column (1.7mm, 2.1 x 150 mm). The injection volume was 5 pL and the column temperature was 45 °C. The detection wavelength was 214 nm and the flow rate was 0.5 mL per minute. 0.1% trifluoroacetic acid in water was used as mobile phase A and 0.1% trifluoroacetic acid in acetonitrile was used as mobile phase B.

[0066] GNE-A and insulin were analyzed by using a Halo Peptide ES-CN column (2.7 mm, 3.0 x 150 mm). The injection volume was 5 pL and the column temperature was 35 °C. The detection wavelength was 214 nm and the flow rate was 0.3 mL per minute. 0.1% formic acid in 10 mM ammonium formate (pH 3.2) was used as mobile phase A and 0.1% formic acid in 80 / 20 acetonitrile / lOmM ammonium formate was used as mobile phase B.Dry-State Transmission Electron Microscopy:

[0067] Prior to usage, all buffer and stain aliquots underwent filtration using 0.22 pM spin filters. FCF300-CU grids were glow discharged for 7 seconds at 15 mA using the CEMRC GlowQube. Subsequently, a 3 pL sample was applied to the grid and left for 1 minute. The grids were then washed with 2 x 20 pL drops of dilution buffer, followed by 1x 20 pL drop of a 1% (w / v) uranyl acetate solution as a stain. The grids were allowed to float on the stain for 1 minute before being left to dry. All TEM images were acquired using the Talos L120C microscope (Thermo Fisher Scientific Inc., Waltham, MA, USA), operating at 120 kV with a spot size of 3. The micrographs were recorded on a 4K x 4K Thermo Fisher Scientific Ceta CMOS Camera.Viscosity Measurements:

[0068] The viscosity was measured using a TA instruments HR-30 Discovery Hybrid Rheometer (Waters, New Castle, Delaware), equipped with a 20 mm stainless steel 1° angle cone. All samples were allowed to equilibrate at 25 °C prior to testing and a solvent trap was used to prevent solvent evaporation. The sample volume for each sample was 40 pL. The sample viscosity was measured every 15 s for 2.5 min at a constant shear rate of 1000 / s. The viscosity (mPas or cP) was calculated by shear stress (Pa) divided by shear rate (1 / s).Injection Force Measurements:

[0069] The injection force was measured using an Instron Materials Testing System (Model 5542; Norwood, MA) with an 100N load cell, a syringe holder fixture, a syringe plunger compression plate, and a glass vial to collect the expelled solution.

[0070] The samples (25 mg / mL) were prepared by attaching a 25G BD PrecisionGlide Needle (P / N 305122) to a BD 1 mL Luer-Lok syringe (P / N 309628) and extracting approximately 0.5 mL of nanosuspension or solution into the syringe. The syringe and needle were primed to 0.3 mL, removing any air bubbles in the syringe. The syringe was placed into the syringe holder and the Instron crosshead was lowered to contact the syringe plunger rod. The program was initiated, displacing the instron crosshead 17.299 mm at 192 mm / min speed, while recording the associated injection force.Example 1: Nanosuspension Screening Using Resonant Acoustic Milling

[0071] Resonant acoustic milling was evaluated as a technique for preparing stable nanoparticle suspensions using three structurally diverse peptides: cyclosporine A (CsA, a macrocyclic peptide), GNE-A (a cystine-knot peptide), and insulin (a large peptide hormone) (see FIGs. 1A-1C). In order to determine the appropriate formulation composition for thenanoparticles, a nanomilling screen was conducted for each peptide, using the process described below. Select formulations were subjected to a scale-up process for further evaluation.

[0072] Nanosuspension Screening Using Resonant Acoustic Milling: A UV-Star clear, flat-bottom 96-well plate was used as a high throughput mixing container. Each well was charged with 500 pm YTZ grinding media from Tosoh (800 mg, 175 L by volume) (Tosoh USA, Inc., Grove City, OH, USA), 2 mg of peptide powder (1.3% drug loading), and 148 pL of an aqueous excipient solution. The concentrations of the polymer and / or surfactant excipients varied between 0.006% and 1.95% within each well. The plate was sealed with a Thermo Fisher Scientific ALPS 50 V Manual Heat Sealer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The sealed plate was then placed on a Resodyn LabRAM II Resonant Acoustic mixer (Resodyn Acoustic Mixers, Butte, MT, USA) and milled at 50 G acceleration for 2 hours.

[0073] Nanosuspension Scale-Up: A 4-mL clear glass vial was used as a scale-up container. The vial was charged with 9.12 grams of 500 pm YTZ grinding media from Tosoh, 175 mg of peptide powder (10% drug loading), and 1.575 mL of an aqueous excipient solution. The vial was then placed on a Resodyn LabRAM II Resonant Acoustic mixer and milled at 50 G acceleration for 2 hours. The resulting nanosuspension was recovered by using a syringe equipped with an 18G needle.Results and Discussion:

[0074] Insulin Nanosuspensions

[0075] Insulin is a large peptide hormone that regulates glucose metabolism in vivo. It consists of 51 amino acid residues in two linear peptide chains (21 peptide and 30 peptide chains) with an overall molecular weight of 5.8 kDa. The insulin monomer is the physiologically active agent but is sensitive to instability (Brange, et al., “Stability and Characterization of Protein and Peptide Drugs, Case Histories,” Pharm. Biotechnol. 1993, 5, 315-350). Insulin is particularly susceptible to aggregation, forming oligomers such as dimers, tetramers, and hexamers, as well as uncontrolled amyloid fibrils (Das, et al., “Molecular Aspects of Insulin Aggregation and Various Therapeutic Interventions,” ACS Bio Med Chem Au 2022, 2, 205-221). As a result, historically, insulin has been prepared asa zinc complex, which exists in a more stable hexamer form (Souto, et al., “Nanoparticle Delivery Systems in the Treatment of Diabetes Complications,” Molecules 2019, 24, 4209; Dunn, et al., “Zinc-Ligand Interactions Modulate Assembly and Stability of the Insulin Hexamer - A Review,” Biometals 2005, 18, 295-303). Thus, most formulation work on insulin has been focused on mitigating aggregation in solution, particularly of the active monomeric form.

[0076] There have been a number of reports around the use of nanoparticle approaches for the delivery of insulin, although these are often complex nanocarrier systems such as polymeric and lipid nanoparticles (Merisko-Liversidge, et al., “Insulin Nanoparticles: A Novel Formulation Approach for Poorly Water Soluble Zn-Insulin,” Pharm. Res. 2004, 21, 1545-1553). An initial report from Merisko-Liversidge and coworkers demonstrated nanomilling on an insulin-zinc complex, although this was limited to the more stable and inactive insulin hexamer species rather than the active and more sensitive insulin monomer, which is more desirable for its rapid time of action (Wang, et al., “Enhancement of Oral Bioavailability of Cyclosporine A: Comparison of Various Nanoscale Drug-Delivery Systems,” Int J Nanomed 2014, 9, 4991-4999). Thus, we evaluated whether the softer resonant acoustic mixing approach could be used to prepare stable nanoparticles of the insulin monomer itself.

[0077] In order to determine the appropriate formulation composition for the insulin nanoparticles, a nanomilling screen was conducted. As seen in Table 1, insulin monomer solid was suspended in water and was milled under resonant acoustic mixing conditions in the presence of different commonly used polymer and surfactant stabilizers. The formulations prepared using HPC-SL / SDS and PVP K29-32 / SDS resulted in small nanoparticles and were thus selected for scale up for further study. The average radius of the nanoparticles in both formulations were translatable in the scale-up batch and remained consistent at -110-130 nm (Table 2).Table 1: Nanomilling screen conducted on insulin monomer milled at 100 mg / mL concentrationTable 2: Particle size of the insulin nanosuspensions prepared at larger scale at 100 mg / mL concentration

[0078] The chemical and physical stability of the two nanosuspensions with HPC- SL / SDS and PVP K29-32 / SDS were evaluated. The insulin nanosuspensions were stored at room temperature (RT) as well as at 4 °C and 37 °C for up to 1 month. As can be seen in FIG. 2, the particle size of the insulin nanoparticles with PVP K29-32 / SDS remained in the accepted nano-scale range for up to a month even when stored at 37 °C. Impressively, no significant particle size growth was observed after being stored for 28 days at 4 °C or even room temperature. In contrast, the insulin nanosuspensions with HPC-SL / SDS appeared to thicken over time and experienced some particle growth at 4 °C and room temperature as well as significant aggregation at 37 °C.

[0079] In order to more closely quantitate the aggregation state and monomer content of the insulin nanosuspensions, the samples were also analyzed by size exclusion chromatography (SEC). As can be seen in FIGs. 3A and 3B, the insulin monomer concentration remains quantitative for both formulations even after 28 days for all of the samples with no presence of higher order oligomers such as dimers, trimers, or hexamers. The chemical stability of the insulin nanosuspension was also investigated using reverse-phase (RP) chromatography (FIGs. 4A and 4B). Both of the formulations remained relatively chemically stable with only a small amount of degradation observed for the samples stored under the accelerated condition of 37 °C for 28 days.

[0080] These results establish that resonant acoustic milling could be used to identify and prepare discrete nanoparticles of insulin monomer that can remain stable even under accelerated conditions

[0081] GNE-A Nanosuspensions

[0082] GNE-A is a disulfide constrained peptide developed at Genentech. It is composed of a 30-residue linear peptide having three internal disulfide bonds with an overall molecular weight of 3.4 kDa. This peptide is highly prone to aggregation via multiple pathways, forming both amorphous non-covalent aggregates as well as oligomers formed from covalent disulfide scrambling (Chen, et al., “Discovery of a Dual Pathway Aggregation Mechanism for a Therapeutic Constrained Peptide,” J Pharm Sci 2021, 110, 2362-2371). As a result, the ability to overcome these risks and develop a stable high concentration formulation of GNE-A would be highly valuable.

[0083] Similar to the insulin results, an initial nanomilling screen of GNE-A was conducted in order to identify the optimal formulation composition. GNE-A solid was suspended in water and was milled under resonant acoustic mixing conditions in the presence of commonly used polymer and surfactant stabilizers. In this case, stable nanoparticles of GNE-A were observed for a wide range of formulation compositions (Table 3). Two formulations, GNE-A with 25% Pluronic F127 and in 25% Tween 80, were selected due to their compatibility for parenteral administration for preparation at a larger scale to conduct more rigorous stability studies. As with the insulin samples, the selected nanosuspension formulations of GNE-A could be successfully prepared at larger scale (Table 4).Table 3: Nanomilling screen conducted on disulfide constrained peptide GNE-A milled at 100 mg / mL concentrationTable 4: Particle size of the scale-up nanomilling on cystine-knot peptide GNE-A at 100 mg / mL concentration

[0084] As can be seen in FIG. 5, nanosuspensions prepared with Tween80 and Pluronic Fl 27 retained their nanoparticle size after 28 days at room temperature and 4 °C conditions. However, a significant increase in the average particle size was observed for the Tween80 samples at the 37 °C conditions after 6 days. In contrast, the Pluronic F127 formulations remained relatively stable even after an extended period of time at 37 °C, with only a slight increase in average particle size with no excessive aggregation observed. In order to more closely quantitate the aggregation state and monomer content of GNE-A, the samples were also analyzed by SEC. As can be seen in FIGs. 6A and 6B, the GNE-A monomer concentration remains relatively stable under these conditions, although a reduction is seen for the samples stored at 37 °C.

[0085] GNE-A is also sensitive to chemical stability liabilities, particularly oxidation. The nanosuspension samples were also analyzed by RP chromatography using a method to quantify the presence of the oxidative degradation product (FIGs. 7A and 7B). For these samples, the Tween80 formulations exhibited increasing amounts of oxidation degradation. This is likely due to the presence of small amounts of residual peroxide products in the Tween80 material (Ha, et al., Peroxide Formation in Polysorbate 80 and Protein Stability,” J. Pharm. Sci. 2002, 91, 2252-2264). In contrast, the Pluronic F127formulation samples remained highly chemically stable with no significant oxidation degradation observed even after 28 days at 37 °C. Thus, the nanosuspension formulations appeared to be highly chemically stable as well.

[0086] The stability benefits of the nanosuspension formulations of GNE-A are particularly striking when compared to a corresponding solution formulation prepared at the same concentration. GNE-A can form a high concentration aqueous solution at pH levels >6. A solution of GNE-A in 60 mM phosphate buffer at pH 7 was prepared at 100 mg / mL. However, in solution, GNE-A rapidly begins to undergo aggregation and loss of monomer as determined by SEC (see FIG. 8). In contrast, the nanosuspension formulation also at 100 mg / mL concentration remains stable with 100% monomer over 28 days. Thus, a nanoparticle suspension of GNE-A remains physically stable while reducing the tendency of the peptide to directly self-associate and undergo aggregation. The active monomer form remains intact under these conditions.

[0087] Cyclosporine A Nanosuspension Formulations

[0088] Cyclosporine A (CsA) is a macrocyclic peptide consisting of 11 amino acids and a molecular weight of 1.2 kDa. It has low solubility and low permeability, resulting in significant challenges in absorption (Pinar, et al., “Formulation Strategies of Nanosuspensions for Various Administration Routes,” Pharm 2023, 15, 1520). Due to aqueous solubility limitations, CsA is typically formulated by being dissolved in a mixture of lipids, surfactants, and cosolvents. Often, high concentrations of lipid-based excipients are required (Tatou, et al., “Effects of Cyclosporin and Cremophor on Working Rat Heart and Incidence of Myocardial Lipid Peroxidation,” Pharmacology 1996, 52, 1-7; Chan, et al., “Cholestatic Effects of Cyclosporine in the Rat,” Transplantation 1997, 63, 1574-1578; Jin, et al., “Cremophor EL Releases Cyclosporin A Adsorbed on Blood Cells and Blood Vessels, and Increases Apparent Plasma Concentration of Cyclosporin A,” Int J Pharmaceut 2005, 293, 137-144; Wassef, et al., “Pharmacokinetic Profiles of Cyclosporine in Rats,” Transplantation 1985, 40, 489-493). A current commercial oral formulation of CsA, Sandimmune®, consists of an oral solution or liquid filled capsules with alcohol, com oil, glycerol, and Labrafil (Lemoine, et al., “Dose and Timing of Injections for Effective Cyclosporine A Pretreatment before Renal Ischemia Reperfusion in Mice,” Pios One 2017, 12, e0182358). The corresponding injectable formulation consists of a large amount ofCremophor EL (a polyethoxylated castor oil) and alcohol to achieve the desired solubility. This has resulted in safety issues such as potential risk of intolerability at the injection site as well as anaphylaxis reactions (Yan, et al., “Cyclosporine A Nanosuspensions for Ophthalmic Delivery: A Comparative Study between Cationic Nanoparticles and Drug-Core Mucus Penetrating Nanoparticles,” Mol. Pharm. 2021, 18, 4290-4298; Pinar, et al., “A New Nanosuspension Prepared with Wet Milling Method for Oral Delivery of Highly Variable Drug Cyclosporine A: Development, Optimization and in Vivo Evaluation,” Eur J Pharm Sci 2022, 171, 106123; Pinar, et al., “Optimization and Evaluation of Cyclosporine A Nanosuspension Stabilized by Combination Stabilizers Using High Pressure Homogenization Method,” Journal of Research in Pharmacy 2019, 23, 1009-1021; Pinar, et al., “Development of Cyclosporine A Nanosuspension: Cytotoxicity and Permeability on Caco-2 Cell Lines,” Pharm. Dev. Technol. 2022, 27, 52-62; Gelderblom, et al., “Cremophor EL the Drawbacks and Advantages of Vehicle Selection for Drug Formulation,” Eur. J. Cancer 2001, 37, 1590-1598). Thus, there is a strong desire to develop alternative formulations of CsA without the need for excipients such as Cremophor EL.

[0089] There has been some recent work conducted on aqueous CsA nanosuspensions (Sangalli, et al., “Cyclosporine Pharmacokinetics in Rats and Interspecies Comparison in Dogs, Rabbits, Rats, and Humans,” Dmg Metabolism Dispos Biological Fate Chem 1988, 16, 749-753; “Tissue Distribution, Disposition, and Metabolism of Cyclosporine in Rats” at ASPET Journals on January 31, 2023 Dmd.Aspetjoumals.Org). However, these studies used anti-solvent precipitation, high pressure homogenization, or high shear wet milling processes, making it difficult to translate to other more sensitive peptides. Due to its small size and cyclic structure, CsA acts more like a small molecule and remains stable through these processes. This example evaluates whether resonant acoustic milling could be used as a general process for CsA as well.

[0090] A nanomilling screen was conducted on CsA in order to identify the optimal formulation composition for forming stable nanoparticles. In the initial screen, only the HPC-SL / SDS and Tween80 compositions gave stable nanoparticles (Table 5). Those two formulations were then prepared at a larger scale for further analysis, which exhibited a slight increase in average particle size (Table 6).Table 5: Nanomilling screen conducted on macrocyclic peptide CsA milled at 100 mg / mL concentrationTable 6: Particle size of the scale-up nanomilling on CsA at 100 mg / mL concentration

[0091] Further characterization of the nanoparticles was done by negative-stain EM imaging to review the size and morphology of the nanosuspension. The CsA nanosuspension with HPC-SL / SDS was prepared at a concentration of 100 mg / mL and stored at 4 °C over 1 month before the image was taken. An aliquot of the formulation was taken and diluted to 1 mg / mL with deionized water for imaging. The TEM images (FIGs. 9A and 9B) suggest the particle size of the CsA nanoparticles is <200 nm in diameter.

[0092] The longer term stability of these formulations was then investigated (FIG. 10). With respect to average particle size, both formulations appeared relatively stable for up to 1 month at 4 °C as well as room temperature. However, some larger aggregation was observed over time in the samples stored at 37 °C, potentially leading to settling of the larger particles (explaining the smaller average particle sizes observed in the samples after 1month). While this suggested that cold storage could be used to mitigate physical stability risks, we hoped to identify more stable nanosuspension formulations.

[0093] The challenges in the nanomilling, particularly during scale-up, were likely due in part to the high lipophilicity of CsA, resulting in wettability issues. In order to optimize the process, higher concentrations of surfactant stabilizers were used to aid in wetting and milling performance. As can be seen in FIG. 11, when increasing ratios of SDS surfactant was added to CsA, the milling efficiency increased and the resulting particle sizes continued to reduce. The stability of the CsA nanosuspension with 25% SDS (wt% to CsA) was investigated (FIG 12). Although some variability was observed, no significant increase in aggregation was observed over 28 days. The concentration of the monomer was characterized by SEC, which showed no change over 28 days even at conditions up to 37 °C (FIG. 13). Similarly, no chemical degradation or growth of degradants was observed by RP chromatography (FIG. 14).

[0094] In addition, while the average particle size decreased with increasing SDS concentration, the solubility of the formulation was also measured to see if there was an additional effect with the addition of more surfactant. CsA is practically insoluble in water (0.04 mg / mL) (Berton, et al., “Solubility Studies of Cyclosporine Using Ionic Liquids,” ACS Omega 2019, 4, 7938-7943). However, increasing concentrations of SDS dramatically increase the solubility of CsA in a proportional manner (Table 7).Table 7: Solubility of CsA nanosuspensions with different ratios of SDS (wt% to CsA)

[0095] The viscosities of the CsA nanosuspension formulation with 25% SDS (wt% to CsA) was measured and compared to the commercial Sandimmune® solution to determine the feasibility of processing and injection at high concentrations. The CsA nanosuspension was prepared at 100 mg / mL and diluted to 25 mg / mL and 5 mg / mL with water. The commercial Sandimmune was purchased as a 50 mg / mL lipid-based formulation and was diluted to 25 mg / mL and 5 mg / mL with saline based on the package injection instructions. The two concentrations were chosen as the low and high doses for subcutaneous injection administration. The CsA nanosuspension exhibits a low viscosity of 2.1 Pa*s evenat a high concentration of 100 mg / mL, whereas the commercial Sandimmune® formulation only shows a similar viscosity at a much lower concentration of 5 mg / mL. Moreover, the Sandimmune® formulation shows significantly higher viscosities even at 25 mg / mL (Table 8). Interestingly, the viscosity at 25 mg / mL was observed to be higher than the viscosity at 50 mg / mL in the commercial Sandimmune formulation. The measurement was repeated twice with the same results. This may be due to the interaction of differing ratios of the lipid and aqueous phases at this shear range. Nevertheless, the viscosities of the CsA nanosuspension at all concentrations are dramatically lower and within the acceptable range with no injection concerns.Table 8: Viscosities of CsA nanosuspension (SDS at 25 wt% to CsA) and Sandimmune® solution formulations

[0096] The injection force and syringeability of both the CsA nanosuspension formulation and the commercial Sandimmune® solution formulation was also evaluated at a concentration of 25 mg / mL. The purpose of this study was to mimic the scenario of subcutaneous injection at high dose by using the same equipment. There was a strong back pressure when drawing up the Sandimmune® solution formulation with a 25G needle, but the formulation could be pushed out without any resistance. Therefore, the formulation was initially extracted using a larger 18G needle and then pushed out after switching back to a 25G needle. Consistent with the higher viscosity values, the Sandimmune® lipid-based solution formulation exhibits a significantly higher injection force compared to the aqueous nanosuspension formulation (FIG. 15). Over a 5-second injection, the maximum forces experienced for Sandimmune® solution formulation and the nanosuspension formulation were 26.97 N and 4.33 N, respectively.Example 2; Cyclosporine A In Vivo Pharmacokinetic Studies

[0097] The in vivo pharmacokinetics of cyclosporine A (CsA) prepared as either the lipid-based commercial Sandimmune® solution formulation consisting of (w / w) 32.9Ethanol / 67.1 Cremophor EL or a nanosuspension formulation prepared by resonant acoustic milling were evaluated following a single subcutaneous dose (SC) of 10 mg / kg (5 mg / mL formulation concentration) or 50 mg / kg (25 mg / mL formulation concentration) to male Sprague Dawley rats with a dose volume of 2 mL / kg. The needle switching strategy was implemented on the high dose of Sandimmune® formulation due to its high injection force, whereas there was no injection concern for the aqueous nanosuspension formulations. Male SD rats (6-9 weeks old) ranging from 237 to 251 g obtained from Charles River Laboratories (Hollister, CA) were used in the study with 4 rats per dose group. Animals were not fasted before subcutaneous dose administration. Blood samples (approximately 0.15 mL) were collected from each animal via jugular vein into tubes containing K2EDTA at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after dose administration. Blood was centrifuged at 12,851 x g for 5 minutes to harvest plasma. Compound concentration in each plasma sample was determined by a non-validated LC-MS / MS assay at Genentech, Inc. The lower limit of quantitation (LLOQ) of CsA in plasma was 9.14 ng / mL or 0.00760 pM. Mean CsA concentrations measured plasma were used to construct a semi logarithmic plasma concentration-time curve. PK analysis was performed using nominal time, noncompartmental analysis, linear up log down calculation, and the extravascular input model (Model type: Plasma 200-202), Phoenix™ WinNonlin®, version 8.3 (Certara L.P.).Results and Discussion

[0098] Over a period of 24 hours, all of the formulations exhibited a sustained release with steady plasma concentrations with no sign of dropping (FIG. 16). This suggests that a longer study may be needed to identify the total AUC of the formulations. The nanosuspension at both 10 mg / kg and 50 mg / kg show lower Cmax and absorptions compared to the commercial Sandimmune® formulations (Table 9). The lower steady state plasma concentrations of the nanosuspensions is likely due to solubility-limited absorption of CsA in the formulation. As measured in Table 6, the solubility of CsA in the 25% SDS (wt% to CsA) nanosuspension is 3.848 mg / mL, while the Sandimmune® formulations consisted of fully dissolved peptide at 5 and 25 mg / mL concentrations. Nevertheless, the absorption of the nanosuspensions remains constant over the time period tested and the plasma concentrations achieved provide sufficient coverage over the target concentrations for efficacy for liver transplant patients without the need for harsh excipients (Mourik, et al.,“Comparison of Pharmacokinetics of Neoral and Sandimmune in Stable Pediatric Liver Transplant Recipients,” Liver Transplant. Surg. 1999, 5, 107-111).Table 9: Pharmacokinetic parameters of commercial Sandimmune® and nanosuspension formulations dosed via subcutaneous injection in rat at 10 and 50 mg / kg.Example 3: Comparison of Resonant Acoustic Milling with High Energy Milling Approaches

[0099] As demonstrated in Examples 1 and 2, resonant acoustic milling can be used to prepare stable nanosuspension formulations of a wide variety of peptides with diverse sizes and structures. These peptide nanoparticles exhibit improved physical and chemical stability as well as benefits in formulation development and in vivo performance. In order to demonstrate that this is due to the unique nature of the resonant acoustic mixing effect, the same peptide materials were subjected to more traditional top-down nanomilling approaches as a comparison. In this example, preparation of nanosuspensions of the peptides was attempted using an ultrasonic probe (see Mehmood, et al., “Developing Nanosuspension Loaded with Azelastine for Potential Nasal Drug Delivery: Determination of Proinflammatory Interleukin IL-4 MRNA Expression and Industrial Scale-Up Strategy,” ACS Omega 2023, 8, 23812-23824; El-Badry, et al., “Formulation and Evaluation of Nanosuspension of Albendazole for Dissolution Enhancement,” Nanosci. Nanotechnol. Lett. 2013, 5, 1024-1029; Gulsun, et al., “Preparation and Characterization of Furosemide Nanosuspensions,” J. Drug Deliv. Sci. Technol. 2018, 45, 93-100; lurian, et al., “Optimization of the Sonication Process for Meloxicam Nanocrystals Preparation,” Clujul Med. 2015, 88, 366-372). Unlike resonant acoustic mixing, an ultrasonic probe supplieshigh intensity, high frequency waves (often above 20 kHz) to a sample in order to induce homogenization and reduce particle size (Sandhya, et al., “Ultrasonication an Intensifying Tool for Preparation of Stable Nanofluids and Study the Time Influence on Distinct Properties of Graphene Nanofluids - A Systematic Overview,” Ultrason. Sonochem. 2021, 73, 105479). All three peptides were subjected to ultrasonication at 5 and 10 minutes (Table 10). In these studies, after ultrasonication the resulting suspensions consisted of heterogeneous particle sizes that were larger than those obtained from resonant acoustic mixing (FIG. 17). Furthermore, the suspensions visibly settled overtime, showing that they were not small enough or stable enough to remain in suspension (FIGs. 18A and 18B). When the peptides were sonified beyond 10 minutes in an attempt to further reduce the particle size of the samples, they appeared to noticeably gel due to the high intensity of the ultrasonication.Table 10: Nanosuspension behavior after preparation using ultrasonicationConclusion

[0100] The results of these examples indicate that low shear resonant acoustic mixing can be used as a method for preparing stable peptide nanoparticles. This approach appears to be highly general and has been demonstrated to be effective across a wide range of different peptide structures and molecular weights. These peptide nanosuspensions exhibit improved chemical and physical stability compared to corresponding solution formulations, particularly at high concentrations. In addition, cyclosporine A nanosuspensions were dosed in vivo and exhibit similar exposures compared to the current commercial formulation of Sandimmune, with the benefit of not requiring high concentrations of lipids and surfactants. As a result, this is a general strategy that should enable the formulation of peptides at high concentrations for a number of applications.

[0101] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing a peptide nanosuspension, the method comprising applying low frequency acoustic energy at a frequency of from about 10 to about 20,000 Hertz to an admixture comprising (i) a peptide; (ii) an aqueous dispersion medium comprising a surface-active polymer; and (iii) milling media.

2. The method of claim 1, wherein the acoustic energy is applied as a standing wave.

3. The method of claim 1 or 2, wherein the frequency is from about 10 to about 100 Hertz.

4. The method of claim 3, wherein the frequency is about 60 Hertz.

5. The method of any one of claims 1-4, wherein the acoustic energy imparts a force of from about 10 G to about 100 G.

6. The method of claim 5, wherein the acoustic energy imparts a force of about 50 G.

7. The method of any one of claims 1-6, wherein the surface-active polymer is selected from the group consisting of hydroxypropylcellulose, alkoxide block copolymers, polyethylenesorbitol polymers, polyvinylpyrrolidone polymers, methyl cellulose, hydroxypropylmethyl cellulose derivatives, and polypropylene- and polyethylene glycol polymers and derivatives.

8. The method of any one of claims 1-7, wherein the aqueous dispersion medium further comprises a surfactant.

9. The method of claim 8, wherein the surfactant is selected from the group consisting of dioctyl sulfosuccinates and sodium dodecylsulfate.

10. The method of any one of claims 1-9, wherein the low frequency acoustic energy is applied for from about 0.25 to about 24 hours.

11. The method of any one of claims 1-10, wherein the concentration of the peptide in the nanosuspension is from about 0.1 wt% to about 50 wt%, relative to the weight of the aqueous dispersion medium.

12. The method of claim 11, wherein the concentration of the peptide in the nanosuspension is from about 1 wt% to about 40 wt% relative to the weight of the aqueous dispersion medium.

13. The method of claim 12, wherein the concentration of the peptide in the nanosuspension is about 10 wt% relative to the weight of the aqueous dispersion medium.

14. A method of screening aqueous dispersion media for use in a peptide nanosuspension, the method comprising:(a) admixing a peptide, milling media, and a plurality of aqueous dispersion media in one or more slurry containers to form a plurality of admixtures; and(b) applying low frequency acoustic energy at a frequency of from about 10 to about 20,000 Hertz to the admixtures.

15. The method of claim 14, wherein the slurry container is a multi-well plate.

16. The method of claim 14 or 15, further comprising analyzing the stability of the admixtures.

17. The method of claim 16, wherein the stability of the admixtures is analyzed after storage for at least 14 days.

18. The method of any one of claims 14-17, wherein the acoustic energy imparts a force of from about 10 Gto about 100 G.

19. The method of any one of claims 14-18, wherein the aqueous dispersion media further comprises a surfactant.

20. A kit for preparing a peptide nanosuspension comprising a slurry container, milling media, a peptide, and an aqueous dispersion media comprising a surface-active polymer.

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