Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0046—Ear
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/1605—Excipients; Inactive ingredients
  • A61K9/1629—Organic macromolecular compounds
  • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/16—Otologicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• Stem cells exhibit an extraordinary ability' to generate multiple cell types in the body . Besides embryonic stem ceils, tissue specific stem cells serve a critical role during development as well as in homeostasis and injury repair in the adult. Stem cells renew themselves through proliferation as well as generate tissue specific cell types through differentiation. The characteristics of different stem cells vary from tissue to tissue, and are determined by their intrinsic genetic and epigenetic status. However, the balance between self-renewal and differentiation of different stem cells are all stringently controlled. Uncontrolled self-renewal may lead to overgrowth of stem cells and possibly tumor formation, while uncontrolled differentiation may exhaust the stem cell pool, leading to an impaired ability to sustain tissue homeostasis.
• stem cells continuously sense thdr environment and appropriately respond with proliferation, differentiation or apoptosis. It would be desirable to drive regeneration by controlling the timing and extent of stem cell proliferation and differentiation. Controlling the proliferation with small molecules that are cleared over time would allow for control of the timing and extent of stem cell proliferation and differentiation.
• tissue stem ceils from different tissues share a limited number of signaling pathways for the regulation of thdr self-renewal and differentiation, albdt in a very context dependent maimer. Some of these pathways are the Wnt and GSKS-b pathways.
• LgrS is expressed across a diverse range of ti ssues and has been identified as a biomarker of adult stem cells in a variety of tissues such as the gut epithelia (Barker et al. 2007), kidney, hair follicle, and stomach (Barker et al, 2010; Haegebarth & Clevers, 2009). For example, it was first published in 2011, that mammalian inner ear hair cells are derived from LGR5 + cells (Chai et al, 2011, Shi et al. 2012). LgrS is a known component of the Wnt/ -catenin pathway, which has been shown to play major roles in differentiation, proliferation, and inducing stem ceil characteristics (Barker et al. 2007).
• Hair cells are the receptor cells that transduce the acoustic stimulus. Regeneration of damaged hair ceils would provide an avenue for the treatment of a condition that currently has no therapies other than prosthetic devices. Although hair cells do not regenerate in the mammalian cochlea, new hair cells in lower vertebrates are generated from epithelial cells, called supporting cells, that surround hair cells.
• the present disclosure provides a lyophiiized pharmaceutical composition comprising a gelling agent
• the present disclosure provides a gel pharmaceutical composition, fix example a thermoreversible gel, comprising one or more otic therapeutic agents.
• the lyophiiized pharmaceutical composite ons di sclosed herein are reconstituted to form the gel pharmaceutical composition, for example a thermoreversible gel, disclosed herein.
• the present disci osure provi des, inter alia, a lyophiiized pharmaceutical composition comprising one or more oti c therapeutic agents and a gelling agent
• the present disclosure provides a lyophiiized pharmaceutical composition comprising about 50 to about 500 mg of poloxamer and about 50 to about 500 mg of a compound of formula (I), for example valproic acid or a pharmaceutically acceptable salt thereof.
• a pharmaceutical composition comprising one or more otic therapeutic agents and a gelling agent.
• a pharmaceutical composition may comprise purified poloxamer and an increased concentration of valproic add or a pharmaceutically acceptable salt thereof while maintaining suitable gelling characteristics.
• a pharmaceutical composition may comprise an increased concentration of valproic add or a pharmaceutically acceptable salt thereof and CHIR99021 or a. pharm aceutically acceptable salt thereof, wherein tire increased concentration of valproi c add or a
• pharmaceutically acceptable salt thereof increases the level of CH1R99021 or a pharmaceutically acceptable salt thereof in the inner ear.
• the present disclosure provides comprising a gelling agent and a compound of formula (1):
• the present disclosure provides a pharmaceutical composition
• a pharmaceutical composition comprising a gelling agent, valproic add or a pharmaceutically acceptable salt thereof at a concentration of greater than about 70 mg'ml, and one or more otic therapeutic agents.
• the present disclosure provides a composition that is suitable for intratympanic injection.
• the present disclosure provides a pharmaceutical composition
• a pharmaceutical composition comprising a poioxamer, wherein at least 85% by wt% of the poloxamer has an average molecular weight of greater than about 7250 Da, and valproic acid or a pharmaceutically acceptable salt thereof is present at a concentiation of greater than 70 mg/ml.
• the present disclosure provides a pharmaceutical composition
• a pharmaceutical composition comprising a poloxamer, wherein less than 20% by wt.% of the poloxamer has an average molecular weight less about 7250 Da, and valproic add or a pharmaceutically acceptable salt thereof at a concentration of greater than 70 mg/ml.
• the present disclosure provides a method for preparing a pharmaceutical composition comprising the steps of: (a) having an aqueous sol ution comprising a gelling agent; and (b) adding a solution of one or more otic therapeutic agents or a pharmaceutically acceptable salt thereof
• the present disclosure provides a lyophilized pharmaceutical compositi on comprising a gelling agent and one or more otic therapeutic agents, wherein the composition does not contain an additional bulking agent.
• the present disclosure provides a lyophilized pharmaceutical composition comprising a poloxamer and one or more otic agents, wherein the composition does not contain an antioxidant.
• the present disclosure provides a method of lyophilizing a pharmaceutical composition.
• the present disclosure provides a method of reconstituting a lyophilized pharmaceutical composition.
• the present disclosure provides a reconstituted pharmaceutical composition.
• the one or more otic therapeutic agents are one or more hearing loss treatment agents.
• the one or more otic therapeutic agents are modulators of one or more biological pathways and biological targets associated with hearing loss.
• the one or more otic therapeutic agents are hair cell regeneration agents and/or otoprotective agents
• the one or more otic therapeutic agents are selected from the group consisting of the agents described in Tables 1-13, and pharmaceutical salts thereof.
• the one or more otic therapeutic agents are CHIR99021 or a pharmaceutical acceptable salt thereof, and valproic acid or a pharmaceutical acceptable salt thereof [028]
• the composition comprises CHIR99021 or a pharmaceutically acceptable salt thereof, valproic add or a pharmaceutically acceptable salt thereof, and a gelling agent.
• the pharmaceutically acceptable salt of valproic acid is a sodium salt (e.g., sodium valproate).
• the gelling agent is a themioreversibie gdling agent (e.g., a poloxamer).
• the poloxamer i s Poloxamer 407
• the poloxamer is a purified poloxamer (e.g., purified Poloxamer 407).
• the present disclosure provides a method of treating hearing loss, comprising administering to a subject in need thereof a pharmaceutically acceptable amount of a reconstituted solution, wherein the reconstituted solution is prepared by a reconstitution process using the lyophilized pharmaceutical composition of any one of the preceding claims.
• the present disclosure provides a pharmaceutical composition, compri sing:
• CHIR99021 or a pharmaceutically acceptable salt thereof being present at a concentration ranging from 0.025 mgml to about 25 mg/ml;
• valproic acid or a pharmaceutically acceptable salt thereof being present at a concentration ranging from 0.5 mg/ml to about 500 mg/ml
• poloxamer 407 being present at a concentration ranging from 1 vvl% to about 25 wt%; and iv) dimethyl sulfoxide (DMSO) being present at a concentration below 7.5 wt%.
• DMSO dimethyl sulfoxide
• the present disclosure provides a pharmaceutical composition, comprising:
• CHIR99021 or a pharmaceutically acceptable salt thereof being present at a concentration ranging from 0.025 mg/ml to about 25 mg'ml;
• valproic acid or a pharmaceutically acceptable salt thereof being present at a concentration ranging from 1 mg'ml to about 500 mg’ml;
• poloxamer 407 being present at a concentration ranging from 1 wt% to about 25 wt%; and iv) dimethyl sulfoxide (DMSO) being present at a concentration below 7.5 wt%.
• DMSO dimethyl sulfoxide
• the present disclosure provides a pharmaceutical composition, amprising:
• CHIR99021 or a pharmaceutically acceptable salt thereof being present at a concentration ranging from 0.025 mg'ml to about 25 mg/ml;
• valproic acid or a pharmaceutically acceptable salt thereof being present at a concentration ranging from 0.5 mg'ml to about 500 mg'ml;
• poloxamer 407 being present at a concentration ranging from 1 wt% to about 25 wt%; and iv) dimethyl sulfoxide (DMSO) being present at a concentration below' 7.5 wt%.
• DMSO dimethyl sulfoxide
• the present disclosure provides a method of processing the pharmaceutical composition of the present disclosure to form a lyophilized phannaceutical composition.
• the present disclosure provides a lyophilized pharmaceutical composition being prepared by iyophi!izing the pharmaceutical omposition of the present disclosure.
• the present disclosure provides a lyophilized phannaceutical composition being prepared by the method of the present disclosure.
• the present disclosure provides a reconstituted solution being prepared by adding a diluent to the lyophilized phannaceutical composition of the present disclosure.
• the present di sclosure provides a reconsti luted solution being prepared by adding a diluent to a lyophilized pharmaeeuti cal composition which i s prepared by lycphilizing the phannaceutical composition of the present disclosure.
• the present disclosure provides a reconstituted solution being prepared by adding a diluent to a lyophilized pharmaceutical composition which is prepared by the method of the present disclosure.
• the present disclosure provides a reconstituted solution being prepared by adding a diluent to a lyophilized pharmaceutical composition, comprising one or more otic therapeutic agents and a gelling agent.
• the present disclosure provides a method of facilitating the generation of a tissue and/or a cell, compri sing delivering a pharmaceutically effective amount of the lyophilized phannaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure to the tissue and/or the cell.
• the present disclosure provides a method of treating a subject who has, or is at risk of developing a disease associated with absence or a lack of a tissue and/or a cell, comprising administering to the subject a pharmaceutically effective amount of the lyophilized phannaceutical composition, the pharmaceutical
• the present disclosure provides a method of increasing a population of vestibular ceils in a vestibular tissue, comprising delivering a pharmaceutically effective amount of the lyophilized
• the presort disclosure provides a method of treating a subject who has, or is at risk of developing a vestibular condition, comprising administering to the subject a pharmaceutically effective amount
• the present disclosure provides a method of increasing a population of cochlear cells in a cochlear tissue, comprising delivering a pharmaceutically effective amount of the lyophilized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure.
• the present disclosure provides a method of treating a subject who has, or i s at risk of developing a cochlear condition, comprising administering to the subject a pharmaceutically effective amount of the lyophilized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure.
• the present disclosure provides a method of increasing a population of cells found in the Organ of Cord, comprising delivering a pharmaceutically effective amount of the lyophiiized pharmaceutical composition, the pharmaceutical composition, or the recoastituted solution of the present disclosure to the population.
• tihe present disclosure provides a method of increasing a population of hair cells found in the Organ of Cor i, comprising delivering a pharmaceutically effective amount of the lyophiiized
• the present disclosure provides a method of increasing a population of inner hair cells found in the Organ of Corti, comprising delivering a pharmaceutically effective amount of the lyophiiized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure to the population.
• the present disclosure provides a method of increasing a population of outer hair cells found in the Organ of Corti, compri sing delivering a pharmaceutically effective amount of the lyophiiized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure to the population.
• the present disclosure provides a method of increasing a population of neuronal cells found in the Organ of Corti, comprising delivering a pharmaceutically effective amount of the lyophiiized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure to the population.
• the present disclosure provides a method of treating a subject w o has, or is at risk of developing a hearing condition, comprising administering to the subject a pharmaceutically effective amount of the lyophiiized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure.
• the present disclosure provides the lyophiiized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, for use in facilitating the generation of a tissue and/or a cell.
• the present disclosure provides the lyophiiized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, for use in in treating a subject who has, or is at risk of developing a disease associated with absence or a lack of a tissue and/or a cell.
• the present disclosure provides the lyophiiized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, for use in increasing a population of vestibular cells in a vestibular tissue
• the present disclosure provides the lyophiiized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, for use in treating a subject who has, or is at risk of developing a vestibular condition.
• the present disclosure provides the lyophilized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, for use in increasing a population of cochlear cells in a cochlear tissue.
• the present disclosure provides the lyophilized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, for use in treating a subject who has, or i s at risk of developing a cochlear condition.
• the present disclosure provides the lyophilized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, for use in increasing a population of cells found in the Organ of Corti.
• the present disclosure provides the lyophilized pharmaceutical composition, the pharmaceutical composition, or the reconstitute! solution of the present disclosure, for use in increasing a population of Mr cells found in the Organ of Corti .
• the present disclosure provides the lyophilized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, for use in increasing a population of inner hair cell s found in the Organ of Corti .
• the present disclosure provides the lyophilized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, for use in increasing a population of outer hair cells found in the Organ of Corti.
• the present disclosure provides the lyophilized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, for use in increasing a population of neuronal cells found in the Organ of Corti .
• the present disclosure provides the lyophilized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, for use in treating a subject who has, or i s at risk of developing a hearing condition.
• the present disclosure provides for the use of the lyophilized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of tire present disclosure, in the manufacture of a medicament for facilitating the generation of a tissue and/or a cell.
• the present disclosure provides for the use of the lyophilized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, in the manufacture of a medicament for in treating a subj ect who has, or i s at risk of developing, a disease associate! with absence or a lack of a tissue and/or a cell.
• the present disclosure provi des for the use of the lyophilized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, in the manufacture of a medicament for increasing a population of vestibular cells in a vestibular tissue.
• the present disclosure provides for the use of the lyophilized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, in the manufacture of a medicament for treating a subject who has, or is at risk of developing a vestibular condition.
• the present disclosure provides for the use of the lyophilized pharmaceutical composition, the phannaceutical composition, or the reconstituted solution of the present disclosure, in the manufacture of a medicament for increasing a population of cochlear cells in a cochlear tissue.
• the presort disclosure provides for the use of the lyophilized phannaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, in the manufacture of a medicament for treating a subj ect who has, or is at risk of developing a cochlear condition.
• the present disclosure provides for the use of the lyophilized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, in the manufacture of a medicament for increasing a population of ceils found in the Organ of Corti.
• the present disclosure provi des for the use of the lyophilized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, in the manufacture of a medicament for increasing a population of hair cells found in the Organ of Corti.
• the present disclosure provides for the use of the lyophilized pharmaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, in the manufacture of a medicament for increasing a population of inner hair cells found in the Organ of Corti.
• the present disclosure provides for the use of the lyophilized pharmaceutical
• the present disclosure provides for the use of the lyophilized pharmaceutical composition, the phannaceutical composition, or the reconstituted solution of the present disclosure, in the manufacture of a medicament for increasing a population of neuronal cells found in the Organ of Corti.
• the present disclosure provides for the use of the lyophilized phannaceutical composition, the pharmaceutical composition, or the reconstituted solution of the present disclosure, in the manufacture of a medicament fix treating a subj ect who has, or is at risk of developing a hearing condition.
• Figure 1 Shows an analysis of auditory brainstem responses (ABR) for the treatment in a noise- damage model for induced hearing loss. Treatment with CH1R99021 + VPA leads to hearing improvement in an in vivo noise damage model.
• ABR auditory brainstem responses
• ABR auditory brainstem responses
• C At 5wks after inj ectteai treated animals ha significantly lower hearing thresholds relative to control animals for 4 of the 5 frequencies tested.
• D The distribution of individual healing recoveries was analyzed.
• Values represent the change in dB needed to elicit an ABR response, with positive values representing further threshold increases (further hearing less) and negative values representing threshold decreases (improved hearing).
• Figure 2 shows an analysis of hair cell count for treatment in a noise-damage model for indeed hearing loss.
• A Low magnification view of a healthy isolated cochlear section showing complete rows of inner hair cells (IHCs) and outer hair cells (OHCs).
• B High magnification view of the region highlighted in a) showing intact IHCs and OHCs in mid frequency' regions.
• C Cochleae of vehicle injected animals show' widespread hair cell loss throughout the cochlea (apex and mid region shown).
• D High magnification view' of the region highlighted in (C) showing substantial absence of hair ceils in mid frequency' regions, where a single IHC can be seen in the field of view (solid arrow).
• C V treated cochlea show significantly higher percentage of total hair cells, IHCs, and OHCs relative to vehicle treated cochleae (grey).
• Figure 7 CHIR99021 logarithmic mean concentrations.
• Figure 8 Lyophilized test composition without use of an appropriate lyophilization cycle.
• Figure 9 Lyophilized test composition manufactured using the developed lyophilization cycle.
• Figure 10 Test composition time course stability' .
• Figure 13 The chromatogram P407 Lot GNAC 17521 C before (red trace) and after purification (blue trace).
• HWM High molecular ⁇ weight
• Figure 15 A zoomed in portion of Figure 12,
• Figure 16 Molecular ⁇ weight calibration curve for PEG standards analyzed by SEC.
• Figure 17 Cumulative molecular weight distribution.
• Figure 18 A typi cal CAD chromatogram for a blank EbO inj ection compared to a 1 % P407 sample.
• Figure 19 RPLC-CAD chromatogram of P407 with impurities are divided into“zones” in the chromatogram.
• Figure 20 lyophilized test composition A (entry' 2, Table 35).
• Figure 21 lyophilized test composition B (entry 3, Table 35).
• Figure 24 lyophrtized test composition E (entr 6, Table 35).
• Figure 25 reconstituted compositions A (At ), B (B-l), C (C-l), D (F-l), and E (G-l) from Table 35.
• Figure 26 Aldehyde content in liquid placebo before and after lyophilization.
• the present di sclosure provides, inter alia , a lyophilized pharmaceutical compositi on comprising one or more otic therapeutic agents (e.g., CH1R99021 and sodium valproate) and a gelling agent (e.g., Pdoxamer 407).
• otic therapeutic agents e.g., CH1R99021 and sodium valproate
• a gelling agent e.g., Pdoxamer 407
• the present disclosure provides a lyophilized pharmaceutical composition
• a lyophilized pharmaceutical composition comprising one or more otic therapeutic agents (e.g., CHIR99021 or a pharmaceutically acceptable salt thereof and sodium valproate or a pharmaceutically acceptable salt thereof) and a gelling agent (e.g., a pdoxamer).
• one or more otic therapeutic agents e.g., CHIR99021 or a pharmaceutically acceptable salt thereof and sodium valproate or a pharmaceutically acceptable salt thereof
• a gelling agent e.g., a pdoxamer
• the present disclosure provides a lyophilized pharmaceutical composition comprising one or more otic therapeutic agents (e.g., LY2090314 or a pharmaceutically acceptable salt thereof and sodium valproate or a pharmaceutically acceptable salt thereof) and a gelling agent (e.g , a pdoxamer).
• a gelling agent e.g., a poloxamer
• a compound of formula (I) e.g., an HD AC inhibitor, such as valproic add or a pharmaceutically acceptable salt thereoi).
• the present disclosure provides a pharmaceutical composition comprising one or more otic therapeutic agents (e.g., CHIR99021 or a pharmaceutically acceptable salt thereof, and valproic add or a pharmaceutically acceptable salt thereof), wherein the increased concentration of one of the one or more otic therapeutic agents (e.g., valproic add or a pharmaceutically acceptable salt thereof), increases the level of the other one or more otic therapeutic agents (e.g., CHIR9902I or a pharmaceutically acceptable salt thereof) in the inner ear.
• one or more otic therapeutic agents e.g., CHIR99021 or a pharmaceutically acceptable salt thereof, and valproic add or a pharmaceutically acceptable salt thereof
• the increased concentration of one of the one or more otic therapeutic agents e.g., valproic add or a pharmaceutically acceptable salt thereof
• increases the level of the other one or more otic therapeutic agents e.g., CHIR9902I or a pharmaceutically acceptable salt thereof
• the present disclosure provides a pharmaceutical composition
• a pharmaceutical composition comprising a gelling agent (e.g., a poloxamer) at a certain purity and one or more otic therapeutic agents (e.g., valproic acid or a pharmaceutically acceptable salt thereof) at a certain concentration.
• a gelling agent e.g., a poloxamer
• one or more otic therapeutic agents e.g., valproic acid or a pharmaceutically acceptable salt thereof
• the present disclosure provides a lyophilized pharmaceutical composition
• a lyophilized pharmaceutical composition comprising one or more otic therapeutic agents (e.g. , CHIR99021 or a pharmaceutically acceptable sal t thereof and val proic add or a pharmaceutically acceptable salt thereof) and a gelling agent (e.g., poloxamer), where the composition does not comprise an additional bulking agent
• the present disclosure provides a lyophilized pharmaceutical composition
• a lyophilized pharmaceutical composition comprising one or more otic therapeutic agents (e.g., CHIR99Q21 or a pharmaceutically acceptable salt thereof and valproic add or a pharmaceutically acceptable salt thereof) and a gelling agent (e.g. poloxamer), where the composition does not comprise an antioxidant.
• one or more otic therapeutic agents e.g., CHIR99Q21 or a pharmaceutically acceptable salt thereof and valproic add or a pharmaceutically acceptable salt thereof
• a gelling agent e.g. poloxamer
• the present disclosure provides a method of preparing the pharmaceutical composition of the present disclosure.
• the present disclosure provides a method for preparing a pharmaceutical composition
• a method for preparing a pharmaceutical composition comprising the steps of; (a) having a solution comprising a gelling agent (e.g. a poloxamer) and one or more otic therapeutic agents (e.g. valproic acid or a pharmaceutically acceptable salt thereof); an (b) adding a solution of one or more otic therapeutic agents (e.g. CHIR99021 or a pharmaceutically acceptable salt thereof).
• a gelling agent e.g. a poloxamer
• one or more otic therapeutic agents e.g. valproic acid or a pharmaceutically acceptable salt thereof
• adding a solution of one or more otic therapeutic agents e.g. CHIR99021 or a pharmaceutically acceptable salt thereof.
• the present disclosure provides a method for lyophilizing a pharmaceutical composition.
• the present disclosure provides a pharmaceutical composition (e.g, a pre-lyophilized pharmaceutical composition) comprising one or more otic therapeutic agents (e.g., CHIR99G21 and sodium valproate) and a gelling (e.g., Poloxamer 407 and other polyethylene oxide-polypropylene oxide block copolymers, including triblock polymers) or other tiiermoreversible (also called“thermosetting” gelling agents) such as polylactic add (PLA) --- polyethylene oxide block copolymers (including PEO-PLA-PEO triblock copolymers).
• a pharmaceutical composition e.g, a pre-lyophilized pharmaceutical composition
• a gelling e.g., Poloxamer 407 and other polyethylene oxide-polypropylene oxide block copolymers, including triblock polymers
• other tiiermoreversible also called“thermosetting” gelling agents
• the present disclosure provid es a method of processing the pharmaceutical composition of the present disclosure to form
• the present disclosure provides a reconstituted solution comprising one or more otic therapeutic agents (e.g., CHIR99021 and sodium valproate) and a gelling (e.g , Poloxamer 407)
• one or more otic therapeutic agents e.g., CHIR99021 and sodium valproate
• a gelling e.g , Poloxamer 407
• the present disclosure provides a Iyophilized pharmaceutical composition
• a Iyophilized pharmaceutical composition comprising Poloxamer 407, CH1R99021 or a pharmaceutically acceptable salt thereof and valproic acid or a
• the present disclosure provides a iyophilized pharmaceutical composition
• a iyophilized pharmaceutical composition comprising Poloxamer 407, CHR99021 or a pharmaceutically acceptable salt thereof and 24iexyl-5-pentynoic add or a pharmaceutically acceptable salt thereof (e.g. sodium 2-hexyl-5-pentynoic add).
• the present disclosure provides a Iyophilized pharmaceutical composition
• a Iyophilized pharmaceutical composition comprising Poloxamer 407, CH0199021 or a pharmaceutically acceptable salt thereof and linol c add or a
• pharmaceutically acceptable salt thereof e.g. sodium lineofate.
• the present disclosure provides a Iyophilized pharmaceutical composition
• a Iyophilized pharmaceutical composition comprising Poloxamer 407, LY2090314 or a pharmaceutically acceptable salt thereof and valproic a d or a
• pharmaceuti cally acceptable salt thereof e.g. sodium valproate
• the present disclosure provides a iyophilized pharmaceutical composition
• a iyophilized pharmaceutical composition comprising Poloxamer 407, AZD 1080 or a pharmaceutically acceptable salt thereof and valproic acid or a pharmaceutically acceptable salt thereof (e.g. sodium valproate).
• the present disclosure provides a iyophilized pharmaceutical composition
• a iyophilized pharmaceutical composition comprising Poloxamer 407, GSK3 XXI f or a phairnaceutically acceptable salt thereof and valproic add or a
• the present disclosure provides a Iyophilized pharmaceutical composition
• a Iyophilized pharmaceutical composition comprising Poloxamer 407, Compound 1-7 or a pharmaceutically acceptable sal t thereof and valproic acid or a phairnaceutically acceptable salt thereof (e.g. sodium valproate).
• the present disclosure provides a iyophilized pharmaceutical composition
• a iyophilized pharmaceutical composition comprising Poloxamer 407, Compound I- 1 or a pharmaceutically acceptable salt thereof and valproic add or a pharmaceutically acceptable salt thereof (e.g. sodium valproate).
• the present disclosure provides a iyophilized pharmaceutical composition
• a iyophilized pharmaceutical composition comprising Poloxamer 407, Compound 1-3 or a phairnaceutically acceptable salt thereof and valproic add or a pharmaceutically acceptable salt thereof (e.g. sodium valproate).
• the present disclosure provides a Iyophilized pharmaceutical composition
• a Iyophilized pharmaceutical composition comprising Poloxamer 407 and valproic add or a pharmaceutically acceptable salt thereof (e.g sodium valproate).
• the present disclosure provides a pharmaceutical composition suitable for intratympanic injection comprising Poloxamer 407, valproic acid or a pharmaceutically acceptable salt thereof (e.g. sodium valproate) at a concentration of at least about 120 mg/ml, and CHIR99Q21 or a pharmaceutically acceptable salt thereof
• the present disclosure provides a pharmaceutical composition
• a pharmaceutical composition comprising at least 85 wt% .% Poioxamer 407 having an average molecular weight greater than about 7250, and valproic acid or a pharmaceutically acceptable salt thereof (e.g. sodium valproate) at a concentration of greater than 120 mg/ml, and CHIR99021 or a pharmaceutically acceptable salt thereof
• the present disclosure provides a lyophilized pharmaceutical composition
• a lyophilized pharmaceutical composition comprising Poioxamer 407, valproic add or a pharmaceutically acceptable salt thereof (e.g sodium val proate), and CHIR99021 or a pharmaceutically acceptable salt thereof wherein in the composition does not comprise an additional bulking agent
• the present disclosure provides a lyophilized pharmaceutical composition
• a lyophilized pharmaceutical composition comprising Poioxamer 407, valproic acid or a pharmaceutically acceptable salt thereof (e.g. sodium valproate), and CHER99Q21 or a pharmaceutically acceptable sal t thereof, wherein in the composition does not. comprise an antioxidant
• the present disclosure provides a method for preparing a pharmaceutical composition comprising the steps of: (a) having an aqueous solution comprising Poioxamer 407 and valproic add or a pharmaceutically acceptable salt thereof (e.g. sodium valproate), and (b) adding a solution compri sing DMSO and CHIR99021 or a pharmaceutically acceptable salt thereof
• a pharmaceutically acceptable salt thereof e.g. sodium valproate
• the present disclosure provides a method for lyophilizing a pharmaceutical composition
• a pharmaceutical composition comprising Poioxamer 407, valproic add or a pharmaceutically acceptable salt thereof (e.g. sodium valproate), and CHIR99021 or a pharmaceutically acceptable salt thereof, wherein the method comprises:
• the present disclosure provides a method for lyophilizing a pharmaceutical composition
• a pharmaceutical composition comprising Poioxamer 407, valproic add or a pharmaceutically acceptable salt thereof (e.g. sodium valproate), and CH1R99021 or a pharmaceutically acceptable salt thereof wherein the method comprises:
• a way to provide a pharmaceutical composition is in a dry or non-hydrated form, e.g. as a tablet, since this typically renders the pharmaceutically active ingredients) in the composition stable for a useful time period that may elapse between the composition being manufactured and to when composition is administered.
• the pharmaceutically active ingredients) is usually stable in the dry composition at varying conditions (temperature, humidity etc.) over the time period that it may be subjected to.
• the degradation problem can be further exacerbated when the components of the composition are slow to dissolve into the solution (i.e. have poor solubility). For example, with the extended time period time taken to dissolve the components in the solution, degradation can occur. In addition, components can precipitate out of the solution over periods of time. Lyophilizaiion of the composition does not necessarily solve the degradation problem in this scenario where the components) also has poor solubility because the composition has two instances, one when the composition is being manufactured and another when the composition is being reconstituted, where the composition is in the form of a solution for an extended period of time, which can lead to degradation of the components.
• the present disclosure offers a solution to the problem described above.
• a lyophilized composition comprising a gelling agent and a salt of an organic acid reconstitutes (i.e. dissolves into solution) more quickly than the time taken to dissolve its constituent parts prior to lyophilizaiion.
• the composition can be manufactured, lyophilized to produce a stable composition, stored, and then reconstituted quickly prior to administration.
• the components of the lyophilized composition are stable for extended periods of time, unlike the composition in solution form.
• the present di sclosure provides compositions with improved reconstitution time, for example relative to its constituent parts prior to lyophilizaiion.
• the present disclosure provides compositions with improved reconstitution time relative to its constituent parts without lyophilization (for example as non-iyophilized powders, crystals or other forms).
• a lyophilized composition comprising a poloxamer and valproic acid or a pharmaceutically acceptable salt thereof can be reconstituted about three times faster than a lyophilized poloxamer alone or powdered poloxamer (i e. non- lyophilized poloxamer).
• Ibis result is unexpected and enables the fast reconstitution of pharmaceutical compositions.
• the fast reconstitution time is especially useful where it is not practical to either freshly prepare the composition, or to wait for long periods time for the composition to reconstitute e.g. because this would lead to the degradation of components of the composition.
• the present invention offers a solution to the problem described above.
• a pharmaceutical composition comprising high concentrations of an organic acid as defined herein by Formula (I), for example valproic acid, or a pharmaceutically acceptable salt thereof increases the levels of otic therapeutic agent(s) in the cochlea.
• composition comprising CH99Q21 or a pharmaceutically acceptable salt thereof and an increased amount of valproic add or a pharmaceutically acceptable saltthereof, e.g. greater than 100 mg/mL, leads to a non-linear increase in the levels of CH99Q21 found in the cochlea after administration.
• a -50% increase in the amount of valproic acid or a pharmaceutically acceptable salt thereof in the composition car result in far more than a 50% increase of CHR99Q21 in the cochlea.
• the increase of CFDR99021 in the cochlea can be in region of 4- 14 fold.
• the increased concentration of valproic add or a pharmaceutically acceptable salt thereof in the composition can increase the concentration of valproic add or a pharmaceutically acceptable salt thereof in cochlea by at least an order of magnitude.
• the present invention describes a pharmaceutical composition in the form of a solution, which comprises a poloxamer.
• Tire poloxamer when dissolved in the composition at a certain concentration, may impart various properties to the composition, such as a certain viscosity and/or a certain
• the present invention requires a pharmaceutical composition with a viscosity to fonn an immobile gel when heated to about body temperature.
• compositions may perturb the composition’ s viscosity and/or gelation in a manner such that the ability to form an immobile gel when hsited to about body temperature is diminished (for example where the gel is a thermoreversible gel). Therefore, there may be an upper limit of the concentrations) of the further components), e.g. therapeutic components), that can be tolerated by the composition while retaining physical properties that are suitable for use. Accordingly, there is a need to provide a pharmaceutical composition with an increased amount of a further components), e.g. therapeutic components), while maintaining gelling characteristics in order to manufacture pharmaceutical compositions.
• the present invention offers a solution to the problem described above. Surprisingly, it has been discovered that purifying a poloxamer prior to manufacture of a pharmaceutical composition enables an increased concentration of the other component® to be tolerated while maintaining the composition’ s gelling characteristics. For example, the composition comprising purified poloxamer can tolerate increased
• the purified poloxamer can be prepared or characterized by any of the methods anchor measures set out herein, in any combination, including those disclosed in the numbered embodiments and examples.
• compositions comprising Poloxamer 407 will have a certain gelation temperature.
• the composition desirably forms a gel at about body temperature.
• oilier components in the composition can perturb the temperature that the composition forms a gel.
• a concentration of about 80 mg/mL of sodium valproate can be achieved.
• the gelation temperature may be perturbed and the composition’s desirable characteristics, such as gelation temperature, diminish.
• a concentration of greater than about 80 mg'rnL of sodium valproate can be achieved, while the desirable gelation temperature is maintained.
• the gel compositions may be lyophilized as set out herein.
• Those lyophilized compositions will therefore have hi gher concentrations of further components), such as therapeutic components, than would otherwi se be possible (e.g. with unpurified poloxamer) while retaining favorable gel properties when reconstituted.
• the lyophilized composition made from that gel provides a number of benefits.
• such a lyophilized composition can be reconsti tuted, for example with the same or similar gi ven amount of water, to provide the compositions disclosed herein that retain their gel properties despite the increased levels of further components).
• me aspect of the present invention is a composition comprising a poloxamer having an increased amount of VP A, or pharmaceutically acceptable salt thereof ’ as disclosed herein.
• one approach to achieve the increased level of VPA, or pharmaceutically acceptable salts thereof is to purify the poloxamer as disclosed herein.
• the composition may, for example, be lyophiiized or reconstituted with water.
• An additional bulking agent such as a polysaccharide
• a pharmaceutical compositi on prior to !yophilization in order to help control the maphology of the lyophiiized composition.
• the additional bulking agent such as a polysaccharide
• the characteristics may be the improved morphology of the lyophiiized product, in the form of a cake. It is also advantageous if the lyophiiized cake is porous, has a large volume, and/or is a fluffy cake.
• the present invention offers a solution to the problem described above. Surprisingly, it has been discovered that a lyophiiized composition of the present invention can be successfully lyophiiized even when the composition does not comprise an additional bulking agent.
• compositions comprise an antioxidant to increase the stability' of the composition over an extended period of time.
• an antioxidant is required where the composition contains, or degrades over time to produce, a reactive species that may react further with other components, thereby affecting the stability' the composition.
• a species in a composition that contains an aldehyde functional group can be a reactive species, for example reacting through undesired redox pathways, which may cause degradation of the other components.
• the inclusion of an antioxidant may increase stability of the composition by inhibiting the redox pathways. Balanced with the need to provide a stable pharmaceutical composition, there is a need to provide a pharmaceutical composition with minimal components since the composi tions are administered to subjects in need thereof
• the present invention offers a solution to the problem described above. Surprisingly, it has been discovered that a lyophiiized composition of the present disclosure, that comprises a poloxamer, is stable when the composition dees not comprise an antioxidant even though the poloxamer component can degrade to produce aldehydes.
• compositions of the present disclosure comprise a poloxamer, which may degrade to produce aldehydes.
• lyophilization removed substantially all of the aldehydes from the composition anchor resulted in a composition that does not produce further aldehydes once lyophiiized. This result means that an antioxidant not required in the composition.
• a pharmaceutical composition that is suitable for administration as a solution or a gel typically comprises an aqueous component, such as water.
• an aqueous component such as water.
• the actives can take extended periods of time to dissolve, precipitate out of solution and/or be unstable in solution. Accordingly, there remains a need to provide further methods of making a pharmaceutical composition as an aqueous solution in less time while maintaining the integrity of the components.
• the present disclosure offers a solution to the problem described above.
• a pharmaceutically acceptable active(s) in the form of a concentrated solution of a polar aprotic solvent results in a pharmaceutical composition where the pharmaceutically acceptable agent(s) has been solubilized in the aqueous solution.
• the time taken to form the composition is reduced in comparison to alternative orders of addition, and the time that any potentially unstable components are in solution is minimized.
• CHIR99021 may exhibit low solubility in aqueous solutions and manufacturing is especially problematic where large quantities of an aqueous solution and long durations of time are required to dissolve CHIR99021 or its salts.
• pre-dissolving CHIR99021 in a polar aptotic solvent and adding that solution to the aqueous component of tire composition successfully solvates CHIR99021 in an aqueous system. This result is unexpected since it occurs on a relatively short timescale, dees not lead to precipitation of CHIR99021, is amenable to scale up, and is reproducible. This result is useful since it allows the formation of previously inaccessible compositions.
• Lyophiiizing a pharmaceutical composition to produce an acceptable fomi of the !yophilized product, such as a porous cake may be challenging. Many factors affect the outcome of the method, and the factors are amenable to a wide range of variation. For example, temperature, rate of temperature change, pressure, and duration at various temperatures and/or pressures all require careful consideration. Thus, obtaining a suitable lyophilized product from a method is no small endeavor and there remains a need to provide more lyophilization methods.
• the present disclosure offers a solution to the problem described above. Surprisingly, it has been discovered that a particular method gives a suitable lyophilized composition in the form of a lyophilized cake.
• the lyophilization method of the present disclosure is particularly advantageous because it is requires mild auditions, achievable on ommercial iyophilizers, which results in a lyophilized product with good characteristics, e.g. the product cake is porous.
• otic therapeutic agenf refers to an agent capable of treating or preventing a disease associated with the ear (e.g., Meniere's disease, hearing loss, a disease of the yesitubular system, vertigo, ear inflammation, or ear infection) or a condition associated with (e.g., resulting into or resulting from) the disease.
• a disease associated with the ear e.g., Meniere's disease, hearing loss, a disease of the yesitubular system, vertigo, ear inflammation, or ear infection
• a condition associated with e.g., resulting into or resulting from
• the otic therapeutic agent i s a hearing loss treatment agent
• hearing loss treatment agent refers to an agent capable for treating or preventing hearing loss or a condition associated with (e.g, causing or developing into or resulting from) hearing loss.
• the one or more otic therapeutic agents are one or more hearing loss treatment agents.
• the one or more otic therapeutic agents are modulators of one or more biological pathways and/or biological taigets associated with hearing loss.
• Each of the modulators may independently be an agonist (e ., activator) or antagonist (e.g, inhibitor) of one or more biological pathways and/or biological taigets.
• one or more of the modulators are agents that increase or activate the activity of one or more biological pathways and/or biological targets.
• one or more of the modulates are agents that decrease or eliminate the activity of one or more biological pathways and/or biological targets.
• the one or more otic therapeutic agents are selected from the group consisting of Wnt pathway agonists, histone deacetylase (HD AC) inhibitors, Dkkl inhibitors, Axin inhibitors, SFRP1 inhibitors, bone morphogenetic protein (BMP) inhibitors, beta-catenin agonists, CyciinDl activates, REST corepressor 1 (CoKEST) inhibitors, NOTCH agonists, TGF-beta inhibitors, cAMP response element binding protein (CREB) activators, cyclin-dependent kinase (CDK) activators, CDK inhibitors, PBK-AKT activators, PI3K-AKT inhibitors, PTEN inhibitors, ATOH1 agonists, ATOH1 antagonists, POU4F3 agonists, POU4F3 antagonists, GFIl agonists, GFl !
• Wnt pathway agonists Wnt pathway agonists
• HD AC histone deacetylase
• Dkkl inhibitors A
• ERK/MAPK agonists ERK/MAPK antagonists
• FGF agonists FGF antagonists
• FGF antagonists y-aminobutyric adds (GAB As)
• voltage- gated Na+ channel antagonists inositol, PKC agonists, PKC antagonists, FOXO inhibitors, FOXO agonists, Kv3 channel antagonists, p27Kipl inhibitors, IL-Ib, N-Methyl-D-aspartate (NMD A) receptor antagonists, NADPH quinone oxidoreductase 1, gamma secretase inhibitors, gamma seeretase activators, MCI receptor antagonist, NK 1 receptor agonist, AMPA receptor agonist, AMPA receptor antagonist, Toll-Like Receptor (TLR) agonist, Toll-Like Receptor (TLR) antagonist, histamine H4 receptor agonist, H4 receptor antagonist, 5- HT3 receptor agonist, 5-HT3 receptor
• the one or more otic therapeutic agents are hair cell regeneration agents and/or otoprotective agents.
• the one or more otic therapeutic agents are selected from the group consisting of the agents described in Tables 1-13, and pharmaceutical salts thereof
• the one or more otic therapeutic agents in any embodiment disclosed could be one or more of the following hair cell regeneration agents
• a hair cell regeneration agent 1 s an agent that promotes regeneration of hair cells.
• a single agent may be used as a hair cell regeneration agent or a combination of agents may provide the hair cell regenerative function.
• the hair cell regeneration agent is a single agent !n other embodiments the hair cell regeneration agent is a combination of agen ts.
• the combination of agents may be compounded together in a single composition. In other embodiments, the combination of agents may be provided to a patient separately.
• a hair cell regeneration agent may promote regeneration of hair cells by stimulating
• a hair cell regeneration agent may activate a proliferative response in the sensory epithelium of the cochlea, thereby providing a new population of cells that can subsequently differentiate into supporting cells.
• the hair cell regeneration agent stimulates proliferation of cochlear supporting cells in which proliferation is stimulated expresses Lgr5 (Leucine-rich repeat-containing G-protein coupled receptor 5). However the hair cell regeneration agent may also stimulate proliferation of supporting cells with little or no Lgr5 expression. In some embodiments, the hair cell regeneration agent produces an expanded population of cochlea cells. In some embodiments, the expanded cells are enriched for Lgr5 expression (i .e. a greater percentage of the expanded cell population express Lgr5 compared to the starting cell population).
• Lgr5+ stem cells are the precursors for sensory hair cells that are present in the cochlea. Increasing the population of Lgr5+ cochlear cells is therefore beneficial because it increases the population of precursor cells which may differentiate into sensory hair cells.
• the hair cell regeneration agent is a Wnt agonist and an epigenetic
• Any Wnt agonist and epigenetic modulator described herein may be used.
• the hair cell regeneration agent is a Wnt agonist and two or more epigenetic modulator. Any Wnt agonist and epigenetic modulator described herein may be used
• the hair cell regeneration agent is a Wnt agonist alone.
• a Wnt agonist may be used alone in line with any of the treatments di sclosed herein that relate to Wnt agonists and/or epigenetic modulators in which both the Wnt agonist and epigenetic modulator are administered to the patient.
• the epigeneti c modulator is not included. Any Wnt agonist described herein may be used.
• the hair cell regeneration agents is a GSK3 inhibitor. Any GSK3 inhibitor described herein may be used.
• the hair cell regeneration agent is gamma secretase inhibitor.
• gamma secretase inhibitors are described in WO 2018007331 A! ; WO 2018111926 A2; WO 2018065340
• the hair cell regeneration agent is an Atohl acti vator.
• Atohl acti vator Sui table Atohl activators are described in US 20160030445 Al; WO 2018172997 Al; WO 2016022776 A2;
• the hair cell regeneration agent is a Notch inhibitor.
• Notch inhibitors ai-e described in W02017007702-A1; WO2016056999-A1 ; WO2014039781A1;
• WO2014047369A1 WQ2014047372A1; W02014047390A1; WO2014047391A1; WO2014G47397A1; WQ2014047392A1; W02014047370A1; WQ2014047374A1 ; W02013093885A1; W02013178821 Al and W 02013016081 Al , each of which is incorporated by reference.
• the hair cell regeneration agent is a Wnt agonist and a Notch inhibitor. Any Wnt agonist and Notch inhibitor may be used as described herein. In certain such embodiments the Wnt agonist is a GSK3 inhibitor. Any GSK3 inhibitor described herein may be used. [0193] !n some embodiments, the hair cell regeneration agent is a Wnt agonist and a gamma secretase inhibitor. Any Wnt agonist and gamma secretase inhibitor may be used as described herein. In certain such embodiments, the Wnt agonist is a GSK inhibitor. Any GSK3 inhibitor described herein may be used.
• At least one hearing loss treatment agent is CHIR99Q21 :
• Pharmaceutically acceptable salts include, for example salts formed by reacting any of the weakly basic active agents described herein, such as CHER99021, with a pharmaceutically acceptable acid known in the art.
• a non-limiting list of suitable acid salts include hydrochloride, hydrobromide, citrate, malate, mesylate, phosphate, tartrate, hydrochloride, tosylate, glucuronate, ethanesulfonate, fumarate, sulfate, napthalene-2- sdfonate, ascorbate, oxalate, napthalene-1, 5-disulfonate, malonate, aminosalicylate, benzenesulfonate, isethionate, genistate, l-hydroxy-2-na.pthoate, dichloroacetate, cyclamate, and ethane-1, 2-disulfonate.
• the composition of the present disclosure may comprise a compound of formula (I) or a pharmaceutically acceptable salt thereof
• the compound of formula (! may also be an otic therapeutic agent
• the compound of formula (G) is an otic therapeutic agent
• it may be included in compositions of the present disclosure that comprise one or more otic therapeutic agents.
• the compound of formula (I) may also be a hearing loss treatment agent.
• the compound of formula (I) may be an HD AC inhibitor.
• the compound of formula (I) or a pharmaceutically acceptable salt thereof i s included in lyophilized pharmaceutical compositions of the present disclosure
• the compound of formula 0) or a pharmaceutically acceptable salt thereof is included in reconstituted pharmaceutical compositions of the present disclosure.
• R 1 is selected from H, alkyl, alkoxy, halo, eycloalkyl, alkenyl, alkynyl, carbocyclyl, and aryl;
• R 23 is independently selected from H, alkyl, alkoxy', halo, cydoalkyl, alkenyl, alkynyl, carbocyclyl, and aryl;
• R 2b is independently selected fromH, alkyl, alkoxy; halo, cydoalkyl, alkenyl, alkynyl, carbocyclyl, and aryl;
• X is selected from oris not present
• R 3 ⁇ 4 is independently selected from H, alkyl, alkoxy, halo, cydoalkyl, alkenyl, alkynyl, carbocyclyl, and aryl;
• R 3 ⁇ 4 is independently selected fromH, alkyl, alkoxy; halo, cydoalkyl, alkenyl, alkynyl, carbocyclyl, and aryl;
• R 4 is selected from H, alkyl, alkoxy; halo, cydoalkyl, alkenyl, alkynyl, carbocyclyl, and aryl;
• R 3 ⁇ 4 is independently selected from H, alkyl, alkoxy; halo, cydoalkyl, alkenyl, alkynyl, carboeydyl, and aryl;
• R 3 ⁇ 4 is independently selected from H, alkyl, alkoxy, halo, cydoalkyl, alkenyl, alkynyl, carbocyclyl, and aryl;
• Y is selected from s not present
• R M is selected from H, alkyl, alkoxy, halo, cydoalkyl, alkenyl, alkynyl, carbocyclyl, and aryl;
• R ® is selected from H, alkyl, alkoxy; halo, cydoalkyl, alkenyl, alkynyl, carboeydyl, and aryl;
• each R 7 is independently selected from H, alkyl, alkoxy; halo, cydoalkyl, alkenyl, alkynyl, carboeydyl, and aiyl;
• R 3 ⁇ 4 is independently selected from H, alkyl, alkoxy, halo, cydoalkyl, alkenyl, alkynyl, carbocyclyl, and
• R & is independently selected fromH, alkyl, alkoxy, halo, cydoalkyl, alkenyl, alkynyl, carbocyclyl, and aryl;
• Z is selected from not present
• R 1,Ja is independently selected from H alkyl, alkoxy, halo, cydoalkyl, alkenyl, alkynyl, carbocyclyl, and aryl;
• R l0b is independently selected from H, alkyl, aikoxy, halo, cycioalkyl, alkenyl, alkynyl, carbocydyl, and aryl;
• R ila is selected from H, alkyl, alkoxy, halo, cycioalkyl, alkenyl, alkynyl, carbocydyl, and aryl;
• R lib is selected from H, alkyl, alkoxy, halo, cycioalkyl, alkenyl, alkynyl, carbocydyl, and aiyl;
• n ® is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8,
• n b is selected from 0, 1, 2, 3, and 4;
• n c is selected from 0, 1, and 2;
• n d is selected from 0, 1 , and 2;
• n e is selected from 0, 1, 2, 3, 4, 5, and 6.
• R l is H. In some embodiments, R l is alkyl hi some embodiments, R ! is alkoxy. In some embodiments, R 1 is halo. In some embodiments, R 1 is cycioalkyl. In some embodiments, R 1 is alkenyl. In some embodiments, R 1 is alkynyl. In some embodiments, R 1 is carbocydyl. In some embodiments, R 1 is aryl.
• R 2® is H. In some embodiments, R 2a is alkyl. In some embodiments, R 23 is alkoxy. In some embodiments, R 28 is halo. In some embodiments, R 2® is cycioalkyl. In some embodiments, R 2® is alkenyl. In some embodiments, R 2® is alkynyl. In some embodiments, R 2® is carbocydyl. In some embodiments, R 2® is aryl. In some embodiments, * is H In some embodiments, R 2b is alkyl. In some embodiments, R 2b is aikoxy. In some embodiments, R 2b is halo.
• R 2b is cycioalkyl .
• R 3 ⁇ 4 is alkenyl.
• R 3 ⁇ 4 is alkynyl
• R 2b is carbocydyl.
• R 2b is aryl.
• R 3a is H. In some embodiments, R 3a is alkyl In some embodiments, R 3® is alkoxy. In some embodiments, R 3a is halo. In some embodiments, R 3a is cycioalkyl. In some embodiments, R 3a is alkenyl. In some embodiments, R 3a is alkynyl. In some embodiments, R 3a is carbocydyl. In some embodiments, R 3a is aryl. In some embodiments, R 3b is H. In some embodiments, R 3b is alkyl. In some embodiments, R 3b is alkoxy'. In some embodiments, R 3b is halo.
• R 3b is cycioalkyl. In some embodiments, R 3b is alkenyl. In some embodiments, R 3b is alkynyl. In some embodiments, R 3b is carbocydyl. In some embodiments, R 3b is aryl.
• R 4 is H. In some embodiments, R 4 is alkyl. In some embodiments, R 4 is alkoxy. In some embodiments, R 4 is halo. In some embodiments, R 4 is cycioalkyl hi some embodiments, is alkenyl in some embodiments, R 4 is alkynyl. In some embodiments, R 4 is carbocydyl.
• R 4 is aryl
• R 3a is H.
• R 5® is alkyl.
• 3 ⁇ 4 is alkoxy.
• R 3 ⁇ 4 is halo.
• R 5® is cycioalkyl.
• R 5® is alkenyl.
• R 5® is alkynyl.
• R 5a is carbocydyl.
• R 3 ⁇ 4 is aryl.
• R 3 ⁇ 4 is H. In some embodiments, R 5 ’* is alkyl. I some embodiments, R 3 ⁇ 4 is alkoxyr In some embodiments, R 51 ’ is halo. In some embodiments, R 5b is cycioalkyl. In some embodiments, R 51 ’ is alkenyl. In some embodiments, R 3 ⁇ 4 is alkynyl. In some embodiments, R 55 is carbocydyl. In some embodiments, R 3 ⁇ 4 is aryl. [0205] In some embodiments, R 6® is H. In some embodiments, R 6® is alk l. In some embodiments, R 6® is alkoxy'.
• R 6® is halo. In some embodiments, R 6® is cycloalkyl. In some embodiments, R 6® is alkenyl. In some embodiments, R 6® is alkynyi. In some embodiments, R 6® is carboeyclyl. In some embodiments, R 6® is arvi
• R 6 is H. In some embodiments, R 6b is alkyl. In some embodiments, R 65 is alkoxy. In some embodiments, Ii 6b is halo. In some embodiments, R 6b is cycloalkyi. In some embodiments, R 613 is alkenyl. In some embodiments, R* is alkynyi. Ei some embodiments, R* is carbocydyl. Ei some embodiments, R 6 * 5 is aryl.
• R 7 is H. In some embodiments, R 7 is alkyl. In some embodiments, R 7 is alkoxy. In some embodiments, R ; is halo. In some embodiments, R ; is cycloalkyi In some embodiments, R 7 is alkenyl. In some embodiments, R 7 is alkynyi. In sane embodiments, R 7 is carbocydyl. In some embodiments, R 7 is aryl.
• X is * * . In some embodiments, X is not present
• Y is In some embodiments, Y is not present
• R 3 ⁇ 4 is H.
• R 83 is alkyl.
• R 8® is alkoxy.
• R 83 is halo.
• R 8® is cycloalkyl.
• R 8® is alkenyl.
• R 8® is alkynyi.
• R 8® is carboeyclyl.
• R 8® is aryl.
• R 3 ⁇ 4 is H.
• R sb is alkyl.
• R a is alkoxy.
• Ii 8b is halo.
• R sb is cycloalkyi.
• R 8b is alkenyl.
• R sb is alkynyi.
• Il sb is carboeyclyl.
• R a is aiyl.
• R 9a is H.
• R ya is alkyl.
• R 3 ⁇ 4 is alkoxy 7 .
• R 3 ⁇ 4 is halo. In some embodiments, R 3 ⁇ 4 is cydoalkyl. In some embodiments* R 3 ⁇ 4 is alkenyl. In some embodiments, R 9a is alkynyl In some embodiments, R 9a is caibocydyl. ln some embodiments, R 3 ⁇ 4 is arvi
• R* is H.
• R 9b is alkyl.
• R 3 ⁇ 4 is alkoxy.
• R 9b is halo.
• R 9b is cydoalkyl.
• R 9b is alkenyl.
• R 95 is alkynyl.
• R 9b is caibocydyl.
• R 3 ⁇ 4 is aryl.
• Z is . Ei some embodiments, Z is not present
• R 10a is H. In some embodiments, R 10a is alkyl. In some embodiments, R i0a is alkoxy. In some embodiments, R i0a is halo. In some embodiments, R IOa is cycloalkyl. In some embodiments, R 1,Ja is alkenyl. Ei some embodiments, R l,Ja is alkynyl. Ei some embodiments, R 10a is caibocydyl. Ei some embodiments, R 10a is aryl .
• R i0b is H.
• R 1 * is alkyl.
• R !0b is alkoxy.
• R l0b is halo.
• R l!3 ⁇ 4 is cycloalkyl.
• R 10b is alkenyl.
• R 10b is alkynyl.
• R 10b is caibocydyl.
• R i0b is aryl.
• R lia is H.
• R nb is alkyl.
• R lla is alkoxy.
• R lla is halo.
• R lla is cydoalkyl.
• R Iia is alkenyl.
• R lia is alkynyl.
• R Ua is carbocydyi.
• R lla is aiyl.
• R ll is H. In some embodiments, R llb is alkyl. In sane embodiments, R ub is alkoxy . Ei some embodiments, R ilb is halo. Ei some embodiments, R lib is cydoalkyl. In some embodiments, R llb is alkenyl. In some embodiments, R llb is alkynyl. Ei some embodiments, R i lb is caibocydyl. Ei some embodiments, R llb is aryl
• n a is 0. Ei some embodiments, n a is 1. In some embodiments, n a is 2. Ei some embodiments, n a is 3. Ei sane embodiments, n a is 4. Ei some embodiments, n a is 5. Ei some embodiments, n a is 6. In some embodiments, n a is 7. In some embodiments, n a is 8.
• n b is 0. Ei some embodiments, n b is 1. In some embodiments, n b is 2. Ei some embodiments, n b is 3. E some embodiments, n b is 4.
• n c is 0. In some embodiments, n c is 1. In some embodiments, n c is 2. [0222] !n some embodiments, n d is 0. In some embodiments, n d is 1. Ei some embodiments, n d is 2.
• n 8 is 0. In some embodiments, n e is 1. In some anbodirnenis, n e is 2 In some embodiments, n e is 3. In some embodiments, if is 4. In some embodiments, if is 5. In some embodiments, n e is 6.
• R 1 is Me. In some anbodiments, R h is Me. In seme embodiments, R a is Me. In some embodiments, R 3 ⁇ 4 is Me. In some embodiments, R* is Me. In some anbodiments, R 4 is Me. In some embodiments, R 3 ⁇ 4 is Me. In some embodiments, R* is Me. In some embodiments, R 63 is Me. In some embodiments, some embodiments, R 7 is Me. In some anbodiments, R & is Me. In some embodiments, R 3 ⁇ 4 is Me. In some embodiments, R 3 ⁇ 4 is Me. In some embodiments, R 3 ⁇ 4 is Me. In some embodiments, some embodiments, R u3 ⁇ 4 is Me. In some embodiments, R i !a is Me. Ei sane embodimen ts,
• R 3 is F. In some embodiments, R 3 ⁇ 4 is F. in some embodiments, R 2b is F. In some embodiments, R 1 ⁇ 2 is F. In s ne embodiments, R 3 ⁇ 4 is F. Ei some embodiments, R 4 is F. In some embodiments, R 33 is F. In some anbodiments, R 51 ’ is F. In some embodiments, R 68 is F. In sane anbodiments, R 60 is F. In some embodiments, R-' is F. In some embodiments, R 8® is F. in some embodiments, R sb is F. In some embodiments, R 3 ⁇ 4 is F. In some embodiments, R 3 ⁇ 4 is F. In some embodiments, R 3 ⁇ 4 is F. In some embodiments, R l0a is F. E some embodiments, R l0b is F. In some embodiments, R lla is F. In some anbodiments, R llb is F.
• R 1 is alkyl.
• R 2a is alkyl.
• R 2b is alkyl
• Il 3a is alkyl.
• R 3 ⁇ 4 is alkyl.
• R 4 is alkyl.
• R 3a is alkyl.
• R 3 ⁇ 4 is alkyl
• R 63 is alkyl.
• R 6 is alkyl.
• R 7 is alkyl.
• R 83 is alkyl.
• R sb is alkyl.
• Il 9a is alkyl.
• Il 9b is alkyl.
• R 10a is alkyl.
• R l0b is alkyl.
• R 31a is alkyl.
• R llb is alkyl.
• alkyl is methyl. In some embodiments, alkyl is ethyl. In some embodiments, alkyl is n-propyl. Ei some embodiments, alkyl is iso-propyl. Ei some embodiments, alkyl is n- biityl. In some anbodiments, alkyl is sec-butyl. In some embodiments, alkyl is iso-butyl. In some embodiments, alkyl is tert-butyl.
• alkoxy is methoxy. In some embodiments, aikoxy is ethoxy. In some embodiments, alkoxy is n-propoxy. In some embodiments, alkoxy is iso-propoxy. In some embodiments, alkoxy- is n-butoxy. In some embodiments, alkoxy is seo-butoxy. In some embodiments, alkoxy is iso-butoxy. In some embodiments, alkoxy is tert-butoxy.
• halo is F. In some embodiments, halo is Cl. In some anbodiments, halo is Br. In some embodiments, halo is I.
• cydoalkyl is cydopropyl. In some embodiments, cycioalkyl is cyclobutyl. In some embodiments, cydoalkyl is eydopentyl. In some embaliments, cycioalkyl is cyclohexy!.
• aryl is phenyl. In some embodiments, aryl is tolyl. In some embodiments, aryl is xylyl. [0232] Jn some embodiments, one of R 1 , R 23 , R 2b , R 3 ⁇ 4 , R ® , R 4 , R 53 , R ® , R 63 , R ® , R 7 , R 83 , R ® , R 3 ⁇ 4 , R ® , R l0a , R i ° b , R lla , and R llb , is further substi tuted with methyl.
• one of R 1 , R 23 , Il 2b , R 3a , R ® , R 4 , R ® , R ® , R 63 , R ® , R 7 , R 83 , R ® , R 3 ⁇ 4 , R ® , R f0a , g lib [ s further substituted with sec-butyl.
• R ® , R ® , R ® , R ® , R ® , R 7 , R ® , R ® , R 3 ⁇ 4 , R ® , R 10a , R 10b , R lla , and R llb is further substitute.! with iso-butyl.
• one of R 1 , R 23 , R 2b , R 38 , R ® , R 4 , R ® , R ® , R 63 , R ® , R 7 R 83 , R ® , R 93 , R ® , R 10a , R 1® , R lfa and R nb is further substituted with tert-butyl .
• one of R 1 , R 23 , R 2b , R ® , R 3b , R 4 , R 38 , R ® , R 63 , R ® , R 7 , R 83 , R sb , R 93 , R ® , R i0a , R 1® , R lja , and R ub , is further substituted with ethoxy.
• R 63 , R ® , R 7 , R 83 , R ® , R 9a , R 9b , R 10a , R 10b , R lla , and R nb is further substituted with n-propoxy.
• one of R 1 , R 23 , R 2b , R 3 ⁇ 4 , R ® , R 4 R 58 , R ® , R ® , R 6b , R 7 , R 8a , R sb , R ® , R 9b , R l0a , R !0b , R Ua , and R ub is further substituted with iso-propoxy.
• one of R 1 , R 23 , R 2b , R 3 ⁇ 4 , R ® , R 4 , R 53 , R ® , R 63 , R ® , R 7 , R ® , R ® , R ® , R ® , R 10a , R 1® , R lia , and R ub is further substituted with n-butoxy.
• one of R 3 , R 23 , R ® , R 3a , R ® , R 4 , R ® , R ® , R 63 , R ® , R 7 , R 83 , R ® , R ® , R 9b , R l a , R !0b , R lia , and Il !!b is further substituted with sec-butoxy.
• one of R 1 , R 23 , R ® , R 33 , R 3b , R 4 , R 53 , R ® , R 63 , R ® , R 7 , R 83 , R ® , R 3 ⁇ 4 , R 9b , R lfts , R 10b , R ila and R iib is further substituted with iso- butoxy.
• one of R 1 , R 2a , R 2b , R 38 , R ® , R 4 , R ® , R ® , R 63 , R ® , R 7 , R 83 , R ® , R 93 R ® , R 103 , R i0b , R 1 !a and R 3 !b is further substituted with tert-butnxy.
• one of R 1 , R 23 , R 2b , R 3a , R 3b , R R ® , R ® , R ® , R 7 , R 83 , R a , R 93 , R ® , R 10a , R 1® , R 113 , and R llb is further substituted with F.
• one of R 1 , R 23 , R ® , R ® , R ® , R 4 , R ® , R ® , R 03 , R ® , R 7 , R ® , R ® , R ® , R ® , R i0a , R 3® , R lia , and R llb is further substituted with Cl.
• one of R 3 , R 23 , R 2b , R 3a , R 3b , R 4 , R 53 , R ® , R 63 , R ® , R 7 , R 83 , R sb , R 93 , R 9 * 5 , R 10a , R 1® , R lla , and R ilb is further substituted with Br.
• one of R 1 , R 2a , R ® , R ® , R ® , R 4 , R ® , R ® , R 63 , R ® , R 7 R 83 , R ® , R ® , R ® , R 10a , R i0b , R 1 !a and R 3 !b is further substituted with I.
• one of R 1 , R ® , R 2b , R 3a , R 3b , R R ® , R ft , R ® , R ® , R 7 , R 83 , R a , R 93 , R ® , R 10a , R l0b , R lia , and R ub is further substituted with cycloalkyl.
• one of R 3 , R 3 ⁇ 4 , R 3 ⁇ 4 , R 3a , R 3b , R 4 , R ® , R ® , R 63 , R ® , R ; , R 8a , R ® , R 9a , R ® , R 1 *, R 30b , R ila , and R l3b is further substituted with alkenyl.
• one of R 3 , R 23 , R ® , R 3a , R ® , R 4 , R ® , R ® , R ® , R 7 , R 83 , R sb , R ® , R ® , R i0a , R i0b , R Ua , and R ub is fuifher substituted with alkynyf.
• one ofR 1 , R 28 , R ® , R ® , R ® , R 4 R ® , R ® , R 68 , R ® , R 7 , R 83 , R ® , R ® , R ® , R 10a , R 1® , R lla and R llb is further substituted with carbocyclyl.
• one of R 1 , R 23 , R 2b , R 3 ⁇ 4 , R 3b , R 4 , R 3 ⁇ 4 , R 3 ⁇ 4 , R & , R 65 , R 7 , R 83 , R a , R 3 ⁇ 4 , R 3 ⁇ 4 , R lfti , R 10b , R i la , and R ub is further substituted with aryl.
• the compound of formula (I) is valproic acid or a pharmaceutically
• the compound of formula (1) is 2-(prop-2-yn-l-yl)-octanoic add or a pharmaceutically acceptable salt thereof.
• the compound of formula (1) is linoleic add or a pharmaceutically acceptable salt thereof
• the compound of formula (1) is phenylbutyric acid or a pharmaceutically acceptable salt thereof
• At least one hearing loss treatment agent is valproic add:
• a pharmaceutical acceptable salt thereof e.g., sodium valproate
• a non-limiting list of other suitable valproate salts includes potassium valproate, lithium valproate, etc.
• a further non-limiting list of other suitable of valproate salts includes sodium valproate, valproate semisodium, magnesium divalproate (magnesium valproate), calcium divalproate (calcium valproate).
• Valproic add is also referred to as VP A.
• Sodium valproate is al so referred to as NaVPA.
• At least one healing loss treatment agent is CHER99Q21 or a pharmaceutical acceptable salt thereof ’ and at least one hearing loss treatment agent is valproic add or a pharmaceutical acceptable salt thereof (e.g., sodium valproate).
• the one or more otic therapeutic agents are CH1R99021 or a pharmaceutical acceptable sal t thereof, and valproic acid or a pharmaceutical acceptable salt thereof (e.g., sodium valproate).
• the pharmaceutically acceptable salt of valproic add is a sodium valproate.
• the one or more otic therapeutic agents are CHIR99021 and sodium valproate.
• the at least one otic therapeutic agent is LY2090314 or a pharmaceutically acceptable salt thereof
• At least one hearing loss treatment agent is LY2090314 or a pharmaceutical acceptable salt thereof
• at least one hearing loss treatment agent is LY2090314 or a pharmaceutical acceptable salt thereof
• at least one healing loss treatment agent is valproic acid or a pharmaceutical acceptable salt thereof (e.g., sodium valproate).
• the one or more otic therapeutic agents are e.g., hearing loss treatment agents.
• the tenn“gelling agent” refers to an agent capable of imparling a gel-like or thickening quality to the pharmaceutical composition or reconstituted solution of the present disclosure upon being subjected to a gelling condition (e.g., a particular temperature or temperature range, the presence of an ion, a pH value or range, or a concentration of gelling agent that causes the gelling agent to undergoing a change or transition from low viscosity to high viscosity, or the reverse).
• a gelling condition e.g., a particular temperature or temperature range, the presence of an ion, a pH value or range, or a concentration of gelling agent that causes the gelling agent to undergoing a change or transition from low viscosity to high viscosity, or the reverse).
• the gelling condition is a particular temperature (e.g., about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, or about 40 °C).
• a particular temperature e.g., about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, or about 40 °C.
• the gelling condition is a particular temperature range (e.g, about 26 °C or higher, about 27 °C or higher, about 28 °C or higher, about 29 °C or higher, about 30 °C or higher, about 31 °C or higher, about 32 °C or higher, about 33 °C or higher, about 34 °C or higher, about 35 °C or higher, about 36 °C or higher, about 37 °C or higher, about 38 °C or higher, about 39 °C or higher, or about 40 °C or higher).
• the gelling agent provides a viscosity of between about 1,000 and 10,000,000 eentipoise, between about 5,000 and 5,000,000 eentipoise, or between about 100,000 and 4,000,000 eentipoise, to the
• the gelling agent provides a viscosity of between about 50,000 and 2,000,000 eentipoise to tine pharmaceutical composition or reconstituted solution of the present disclosure.
• the gelling agent prior to gelling (e.g, at ambient temperature (e.g., between about 20 °C and about 26 °C)), provides a viscosity of less than about 100,000 eentipoise, less than about 50,0( * eentipoise, 20,000 eentipoise, less than about 10,000 eentipoise less than about 8,000 eentipoise, less than abaft 7,000 eentipoise, less than about 6,000 eentipoise, less than about 5,000 eentipoise, less than about 4,000 eentipoise, less than about 3,000 eentipoise, less than about 2,000 eentipoise, or less than about 1,000 eentipoise to the pharmaceutical composition or reconstituted solution of the present disclosure.
• the gelling agent upon gelling (e.g., at the temperature of a human body (e.g., between about 35 °C to about 39 °C, between abort 36 °C to about 38 °C, or at about 37 °C)), provides a viscosity of greater than about 1 ,000 eentipoise, greater than about 5,000 centi poi se, greater than about 10,000 centi poi se, greater than about 20,000 eentipoise, greater than about 50,000 eentipoise, greater than about 60, (XX) eentipoise, greater than about 70,000 eentipoise, greater than about 80,000 eentipoise, greater than about 90,000 eentipoise, or greater than about 100,000 eentipoise.
• the viscosity 7 of the pharmaceutical composition or reconstituted solution of the present disclosure upon gell ing (e.g , at the temperature of a human body (e.g., between about 36 °C to about 39 °C, or at about 37 °C)), the viscosity 7 of the pharmaceutical composition or reconstituted solution of the present disclosure, as measured in units of eentipoise, being about 2 fold or greater, about 5 fold or greater, about 10 fold or greater, about 20 fold or greater, about 50 fold or greater, about 60 fold or greater, about 7 fold or greater, about 80 fold or greater, about 90 fold or greater, about 100 fold or greater as compared to the viscosity of the pharmaceutical composition or reconstituted solution prior to gelling (e.g., at ambient temperature (e.g., at about 25 °C)).
• the gelling condition e.g , gelling temperature
• the gelling temperature is determined using a commercially available rheomoeter having a parallel plate geometry (e.g., with plate distance ranging from 0.5 mm to 1.0 mm).
• the analysis is perfonned over a continuous temperature range (e.g., 15 °C to 40 °C) at a constant rate (e.g., 2 to 3 °C/min) and a deformation frequency of 0.74 Hz to 1 Hz.
• the gelation temperature is determined at the temperature whereby the shear storage modulus (G’) and the shear loss modulus (G’’) are equal.
• the gelling agent comprises acacia, alginie add, bentonite, poiy(aaylic add) (Carbomer), carboxymethyl cellulose, ethylcellulose, gelatin, hydroxyethyl cellulose, hydroxypropyl cellulose, magnesium aluminum silicate (Veegum), methyicellulose, poloxamer, hyaluronic add sodium, polylacticglycoiic acid sodium, chitosan, polyvinyl alcohol, sodium alginate, tragacanth, xanthan gam, or any combination thereof.
• the gelling agent comprises poloxamer.
• the gelling agent comprises hyaluronic acid.
• the gelling agent is hyaluronic acid. In some embodiments the hyaluronic has a MW average of between 7.0 x KhS Daltons and 8.5 I CDS Daltons. In some embodiments the hyaluronic has a MW average of 8.23 x I0 A 5 Daltons. In some embodiments, the hyaluronic add is ⁇ A1M provided by Lifecore Bio. In some embodiments the hyaluronic add is a 0.5-5% aq. solution.
• the hyaluronic acid is a 1 -3% aq. solution. In some embodiments, the hyaluronic acid has an average MW of 823 x ICKd Daltons and is prepared as a 1-3% aq. solution.
• the gelling agent comprises aeada. In some embodiments, the gelling agent comprises alginie add. In some embodiments, the gelling agent compri ses bentonite. In some embodiments, the gelling agent comprises poly(acrylic add) (Carbomer). In some embodiments, the gelling agent comprises caiboxymethyi cellulose. In some embodiments, the gelling agent comprises ethylcellulose. In some embodiments, the gelling agent comprises gelatin. In some embodiments, the gelling agent comprises hydroxyethyl cellulose. In some embodiments, the gelling agent comprises hydroxypropyl cellulose. In some embodiments, the gelling agent comprises magnesium aluminum silicate (Veegum).
• the gelling agent comprises methyicellulose. In some embodiments, the gelling agent comprises poloxamer. In some embodiments, the gelling agent comprises hyaluronic add sodium. In some embodiments, the gelling agent comprises hyaluronic add. In some embodiments, the gelling agent comprises polylacticglycoiic acid sodium. In some embodiments, the gelling agent comprises chitosan. In some embodiments, the gelling agent comprises polyvinyl alcohol. In some embodiments, the gelling agent comprises sodium alginate in some embodiments, the gelling agent comprises tragacanth. In some embodiments, the gelling agent comprises xanthan gum.
• the gelling agent comprises a cellulosic derivative (e.g., carboxymethyleellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl meihylcellulose, and/or methylcellulose).
• a cellulosic derivative e.g., carboxymethyleellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl meihylcellulose, and/or methylcellulose.
• the gelling agent is a thermoreversible gelling agent
• thermosible refers to a capability of being reversible by the application of heat
• The“thermoreversible gelling agent” refers to an agent capable of reversibly imparting a gel-like or thickening quality to the pharmaceutical composition or reemstituted solution of the present disclosure upon application of heat
• the thennoreversib!e gelling agent comprises a poloxamer.
• poloxamer forms a thermoreversible gel.
• the viscosity of the solution increases.
• the viscosity of the solution can increase to the extent that the solution forms a gel.
• the solution of poloxamer forms a gel at about body temperature (37 °C).
• the solution of poloxamer forms an immobile gel at about body temperature.
• the solution of poloxamer is a composition comprising further components, such as one or more otic therapeutic agents and/or valproic acid or a pharmaceutically acceptable salt thereof.
• thermoreversible gelling agent disclosed herein it can be useful for a thermoreversible gelling agent disclosed herein to be a gel when at body temperature but a liquid when below body temperature.
• it may be a liquid in order for it to be inj ected into the ear (for example the middle ear).
• Themioreversible gelling agents are known in the art, for example those polymers that reversibly impart a gel-like or thickening quality upon application of heat disclosed in Shalaby et al. W ater-Sduble Polymers, ACS Symposium Series, American Chemical Society, 1991 (Chapter 33). Those include those polymers that have those properties are also disclosed in Molyneaux, P "Water-Soluble Polymers: Properties and Behavior", CRC Press, Vol.
• the gelling agent may also be a bulking agent of the pharmaceutical composition or reconstituted solution of the present disclosure.
• a poloxamer e.g., poloxamer 407
• Poloxomers are a general class of commercially available and pharmaceutically acceptable triblock copolymers of polyethylene oxide- polypropylene oxide-polyethylene oxide which exhibit relatively low 7 viscosity at low temperatures (e.g., room termpature or below) but much high viscosities at elevated temperatures (e.g., body temperatures of approximately 37°C) whereby compositions containing such thermoreversible gelling agents effectively solidify in place.
• Other thermoreversible gelling agents such as polyethylene oxide - polylactic acid- polyethylene oxide polymers are also suitable in various embodiments of the present invention.
• Poloxamers are a general class of commercially available triblock copolymers that in certain embodiments can be used as the gelling agent. More specifically, such poloxamers can comprise a central hydrophobic chain of polyoxypropyiene (polypropylene oxide) or PPO) flanked by two hydrophilic chains of polyoxyethylene (polyethylene oxide) or PEG). This forms an A-B-A structure, shown below:
• a is 10-120. In some embodiments, a is 20-120. In some embodiments, a is 30-120. In some embodiments, a is 40-120. In some embodiments, a is 50-120. In seme embodiments, a is
• a is 60-120. In some embodiments, a is 70-120. In some embodiments, a is 80-120. I some embodiments, a is
• a is 100-120. In some embodiments, a is 110-120. In some embodiments, a is 10—110. In some embodiments, a is 20-110. In some embodiments, a is 30-110. In some embodiments, a is 40-110. In some embodiments, a is 50-110. In some embodiments, a is 60-110. In some embodiments, a is 70-110. In some embodiments, a is 80-110. In some embodiments, a is 90-110. In some embodiments, a is 100-110. In some embodiments, a is 10-100. In some embodiments, a is 20-100. In some embodiments, a is
• a is 40-100. In some embodiments, a is 50-100. In some embodiments, a is
• a is 60-100. In some embodiments, a is 70-100. In some embodiments, a is 80-1 (X). In some embodiments, a is 90-100. In some embodiments, a is 95—105. In some embodiments, a is 95—115. In some embodiments, a is
• a 85-105.
• a 85-115.
• b is 25-70. I some embodiments, b is 35— 70. In some embodiments, b is 45-70. In some embodiments, b is 55-70. In sane embodiments, b is 60-70. In some embodiments, b is 65-70. In some embodiments, b is 56 +/- 10%, and each a is 101 +/- 10%. In some embodiments, b is 61 +/- 15%, and each a is 101 +/- 10%. In some embodiments, b is 70 +/- 20%, and each ais
• b is 56 +/- 10%, and each ais 100 +/- 10%. In some embodiments, b is 61 +/- 15%, and each ais 100 +/- 10%. In some embodiments, b is 70 +/- 20%, and each ais 100 +/- 10%.
• Poloxamers are also known by the tradenames of: Synperonics, Pluronics, and Koliiphor.
• Poloxamer 407 the approximate lengths of the two PEG blocks is about 100 repeat units while the approximate length of the propylene glycol block is about 56-67 repeat units (where about is +i ⁇ 10%).
• P407 is also known by the BASF trade name Pluronic FI 27 or by the Croda trade name Synperonie PEP 127
• Poloxamers can also be composed of a central hydrophilic chain of polyoxyethylene
• Oilier PPO-PEG block copolymers exist, such as those that comprise four PPO-PEO chains, which extend outward from an amine-terminated central chain (e.g. N-C3 ⁇ 4- ( 1 I N) and in certain embodiments the disclosed compositions can comprise one or more of such four block polymers (either in addition to or instead of the poloxamers otherwise disclosed herein).
• the poloxamer (e.g., poloxamer 407) is the gelling agent and the bulking agent of the pharmaceutical composition or reconstituted solution of the present disclosure.
• the presence of the poloxamer (e.g., poloxamer 407) in the phamiaceutieal composition alleviates the need for any other excipient (eg., additional bulking agent). Such alleviation may provide one or more advantages to the pharmaceutical composition (e ., enhanced stability and/or reduced reconstitution time).
• the pharmaceutical composition of the present disclosure does not comprise an additional bulking agent.
• the lyophilized pharmaceutical composition of the present disclosure does not comprise an additional bulking agent.
• the reconstituted lyophilized pharmaceutical composition of the present disclosure does not comprise an additional bulking agent
• the poloxamer is purified. In some embodiments, the poloxamer is not purified. In some embodiments, the poloxamer (eg., Poloxamer 407) has an average molecular weight of about 725 KDa or greater, about 9 kDa or greater, about 92 kDa or greater, about 94 kDa or greater, about 96 kDa or greater, about 9.8 kDa or greater, about 10 kDa or greater, about 10.2 kDa or greater, about 10.4 kDa or greater, about 10.6 kDa or greater, about 10.8 kDa or greater, about 11 kDa or greater, about 11.2 kDa or greater, about 11.4 kDa or greater, about 11.6 kDa or greater, about 11.8 kDa or greater, about 12 kDa or greater, or about 12.1 kDa or greater. In some embodiments, the poloxamer comprises at least 50% polyethylene oxide
• the poloxamer comprises at least 55% polyethylene oxide by molecular mass. In some embodiments, the poloxamer comprises at least 60% polyethylene oxide by molecular mass. In some embodiments, the poloxamer comprises at least 65% polyethylene oxide by molecular mass. In some embodiments, the poloxamer comprises at least 66% polyethylene oxide by molecular mass. In some embodiments, the poloxamer comprises at least 67% polyethylene oxide by molecular mass. In some embodiments, the poloxamer comprises at least 68% polyethylene oxide by molecular mass. In some embodiments, the poloxamer comprises at least 69% polyethylene oxide by molecular mass. In some embodiments, the poloxamer comprises at least 70% polyethylene oxide by molecular mass. In some embodiments, the poloxamer comprises 60-80% polyethylene oxide by molecular mass. In some embodiments, the poloxamer comprises 65-75% polyethylene oxide by molecular mass.
• the poloxamer has an average molecular weight of about 7250 to about 17350 Daltons. In some embodiments, the poloxamer has an average molecular weight of about 8000 to about 17000 Daltons In some embodiments, the poloxamer has an average molecular weight of about 8000 to about 16000 Daltons In some embodiments, the poloxamer has an average molecular weight of about 9000 to about 16000 Daltons In some embodiments, the poloxamer has an average molecular weight of about 9000 to about 15000 Daltons. In some embodiments, the poloxamer has an average molecular weight of about 9800 to about 14600 Daltons.
• the poloxamer has an average molecular weight of about 10000 to abart 14000 Daltons. In some embodiments, the poloxamer has an average molecular weight of about 10500 to about 14000 Daltons. In some embodiments, the poloxamer has an average molecular weight of about 10500 to about 13500 Daltons. In some embodiments, the poloxamer has an average molecular weight of about 11000 to about 14000 Daltons. In some embodiments, the poloxamer has an average molecular weight of abort 11000 to about 13500 Daltons. In some embodiments, the poloxamer has an average molecular weight of about 11500 to about 14000 Daltons.
• the poloxamer has an average molecular weight of about 11500 to about 13000 Daltons. In some embodiments, the poloxamer has an average molecular weight of about 12000 to about 14000 Daltons. In some embodiments, the poloxamer has an average molecular weight of about 12000 to about 13000 Daltons. In some embodiments, the poloxamer has an average molecular weight of about 10500 to about 12500 Daltons. In some embodiments, the poloxamer has an average molecular weight of about 10500 to about 11500 Daltons. In some embodiments, the poloxamer has an average molecular weight of about 11500 to about 12500 Daltons.
• At least 85%s by wt of the poloxamer has an average molecular weight of abart 7250 to about 17350 Da
• at least 86% by weight of the poloxamer has an average molecular weight of about 7250 to about 17350 Da
• at least 87% by weight of the poloxamer has an average molecular weight of about 7250 to about 17350 Da
• at least 88% by weight of the poloxamer has an average molecular weight of about 7250 to about 17350 Da.
• at least 89% by weight of the poloxamer has an average m ol ecular weight of about 7250 to about 17350 Da.
• At least 90% by weight of the poloxamer has an average molecular weight of about 7250 to about 17350 Da. In some embodiments, at least 91% by weight of the poloxamer has an average molecular weight of about 7250 to about 17350 Da. In some embodiments, at least 92% by weight of the poloxamer has an average molecular weight of about 7250 to about 17350 Da. In some embodiments, at least 86% by weight of the poloxamer has an average molecular weight of greater than about 7250 Da. In some embodiments, at least 87% by weight of the poloxamer has an average molecular weight of greater than about 7250 Da.
• the poloxamer may have the following properties. In some embodiments, the poloxamer has a peak molecular weight of about 12,000 to about 12,500 Da.
• the poloxamer has a number average molecular weight of about 11,500 to about 12,000 Da In some embodiments, the poloxamer has a weight average molecular weight of about 11 ,750 to about 12,250 Da In some embodiments, the poloxamer has a poiydispersity index of about 1.02
• less than 19% by weight of the poloxamer has an average molecular weight less about 7250 Da. In some embodiments, less than 18% by weight of the poloxamer has an average molecular weight less about 7250 Da. In some embodiments, less than 17% by weight of the poloxamer has an average molecular weight less about 7250 Da. In some embodiments, less than 16% by weight of the poloxamer has an average molecular wdght less abort 7250 Da. In some embodiments, less than 15% by weight of the poloxamer has an average molecular wdght less about 7250 Da.
• less than 14% by weight of the poloxamer has an average molecular wdght less about 7250 Da. In some embodiments, less than 13% by weight of the poloxamer has an average mdecular weight less about 7250 Da In some embodiments, less than 12% by weight of the poloxamer has an average molecular weight less about 7250 Da In some embodiments, less than J 1 % by weight of the poloxamer has an average molecular weight less about 7250 Da. In some embodiments, less than 10% by wdght of the poloxamer has an average molecular wdght less about 7250 Da.
• the poloxamer may have the following properties.
• the poloxamer has a peak molecular wdght of about 5,000 to about 5,500 Da.
• the poloxamer Iras a number average molecular wdght of about 5,000 to about 5,500 Da.
• the poloxamer has a wdght average molecular wdght of about 5,000 to about 5,500 Da.
• the poloxamer has a poiydispersity index of about 1 02.
• the entire poloxamer distribution has a number average molecular wdght of about 10,800 to about 11,200 Da In some embodiments, the poloxamer distribution has a wdght average molecular wdght of about 11,500 to about 11 ,700 Da. In some embodiments, the poloxamer distribution is from 0 to about 16,600 Da. In some embodiments, the poloxamer has a poiydispersity index of about less than 1.07.
• the poloxamer is selected from the group consisting of Poioxamer 101, Poloxamer 105, Poloxamer 108, Poloxamer 122, Poloxamer 123, Poloxamer 124, Poloxamer 181, Poloxamer 182, Poloxamer 183, Poloxamer 184, Poloxamer 185, Poloxamer 188, Poloxamer 212, Poloxamer 215, Poloxamer 217, Poloxamer 231, Poloxamer 234, Poloxamer 235, Poloxamer 237, Poloxamer 238, Poloxamer 282, Poloxamer 284, Poloxamer 288, Poloxamer 331, Poloxamer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338, Poloxamer 401, Poloxamer 402, Poloxamer 403, and Poloxamer 407.
• the poloxamer is Poloxamer 188 or Poloxamer 407.
• the poloxamer i s Poloxamer 407.
• the poloxamer comprises Poloxamer 407.
• the Poloxamer 407 is at least 10% by weight of the poloxamer. In some embodiments, the Poloxamer 407 is at least 20% by weight of the poloxamer. In some embodiments, the Poloxamer 407 is at least 30% by weight of the poloxamer. In some embodiments, the Poloxamer 407 is at least 40% by weight of the poloxamer. In some embodiments, the Poioxamer 407 is at least 50% by weight of the poloxamer. In some embodiments, the Poloxamer 407 is at least 60% by weight of the poloxamer.
• the Poloxamer 407 is at least 70% by weight of the poloxamer. In some embodiments, the Poloxamer 407 is at least. 75% by weight of the poloxamer. In some embodiments, the Poloxamer 407 is at least 80% by weight of the poloxamer. in some embodiments, the Poloxamer 407 is at least 90% by weight of the poloxamer. In some embodiments, the poloxamer is Poloxamer 407.
• the poloxamer is purified Poioxamer 407.
• the poloxamer is a purified poloxamer (e.g., purified Poloxamer 407).
• the solubility of the otic agent(s) may be usefully increased.
• the purified poloxamer (e.g., purified Poloxamer 407) has an average molecular weight of about 9 kDa or greater, about 92 kDa or greater, about 9.4 kDa or greater, about 9.6 kDa or greater, about 9.8 kDa or greater, about 10 kDa or greater, about 10.2 kDa or greater, about.
• 10.4 kDa or greater about 10.6 kDa or greater, about 10.8 kDa or greater, about 11 kDa or greater, about 11.2 kDa or greater, about 11 A kDa or greater, about 11.6 kDa or greater, about 11.8 kDa or greater, about 12 kDa or greater, or about 12.1 kDa or greater.
• the purified poloxamer e.g., purified Poloxamer 407
• the polymer chains with molecular weight below 7250 Da may be regarded as impurities.
• the purified poloxamer (e.g., purified Poloxamer 407) has about 99% or less, about 98% or less, about 95% or less, about.90% or less, about 80% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, or about 10% or less of polymer chains with molecular weight below 9 kDa as compared to the unpurified poloxamer (e.g., unpurified Poloxamer 407).
• the purified poloxamer contains less than about 15% by weight of polymer having a molecular weight below about 9 kDa (e.g., PEO homopolymer or PEO- PPO copolymer), for example less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11 %, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1 %, less than about 0.9%, less than about 0.8%, less than about 07%, less than about 0.6%, less than about 0.5%, less than about 04%, less than about 0.3%, less than about 0.2%, or less than about 0.1%, (by weight) of polymer with a molecular weight below about 9 kDa, inclusive of all ranges between any of these values.
• polymer having a molecular weight below about 9 kDa e.g., PEO homopolymer or PEO- PPO copolymer
• the purified poloxamer (e.g., purified Poloxamer 407) is prepared by liquid- liquid extraction or size exclusion chromatography.
• the liquid-liquid extraction procedure involves the fractionation of the poloxamer (e.g., Poloxamer 407) between two aqueous phases containing wi th different salt concentration.
• the purified poloxamer e.g., Poloxamer 407
• the size exclusion chromatography provides separation basal on hydrodynamic radius. The fractions containing purified poloxamer (e.g., Poloxamer 407) with the desired molecular weight range are collated.
• about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 98% or more, or about 99% or more of the one or more impurities having molecular weights below 9 kDa are removed from the poloxamer (e.g , Poloxamer 407) during the purification.
• the poloxamer e.g , Poloxamer 407
• about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or- more, about 90% or more, about 95% or more, about 98% or- more, or about 99% or more of the one or more diblock copolymers (e.g., PEQ-PPO), single block polymers (e.g., PEO), and/or aldehydes are removed from the poloxamer (e.g., Poloxamer 407) during the purification.
• the one or more diblock copolymers e.g., PEQ-PPO
• single block polymers e.g., PEO
• aldehydes e.g., Poloxamer 407
• the one or more diblock copolymers e.g., PEO-PPO
• single block polymers e.g., PEO
• aldehydes e.g., Poloxamer 407
• the lyophilized pharmaceutical composition is in the fomi of a lyophilized cake.
• lyophilization of the pharmaceutical composition of the present disclosure may substantially remove ail volatile components from the composition.
• water may he substantially removed by lyophilization.
• DMSQ may be substantially removed by lyophilization.
• the lyophilized composition is substantially free from water and/or DMSO.
• the lyophilized composition contains less than about 5% by weight of water and/or DMSO In some embodiments, the lyophilized composition contains less than about 4% by weight of water anchor DMSO. In seme embodiments, the lyophilized composition contains less than about 3% by weight of water and/or DMSO. In some embodiments, the lyophilized composition contains less than about 2% by weight of water and/or DMSO. In some embodiments, the lyophilized composition contains less than about 1% by weight of water andtor DMSO.
• the lyophilized pharmaceutical composition has a higher stability to oxygen and/or light as compared to a comparable pharmaceutical composition comprising one or more solvents.
• the comparative composition is an otherwise identical composition.
• the paragraph above can be read as: the lyophilized pharmaceutical composition has a higher stability to oxygen and/or light as compared to an otherwise identical pharmaceutical composition comprising one or more solvents.
• the lyophilized composition comprises at least about 1% by weight of
• the lyophilized composition comprises about 1 % by weight to about 2 % by weight of CHIR99021. In some embodiments, the lyophilized composition comprises at least about 30% by weight of valproic add or a pharmaceutically acceptable salt thereof In some embodiments, the lyophilized composition comprises at least about 40% by weight of valproic add or a pharmaceutically acceptable salt thereof. In some embodiments, the lyophilized composition comprises about 30% by weight to about 50% by weight of valproic add or a pharmaceutically acceptable salt thereof In some embodiments, the lyophilized composition comprises at least about 50% by weight of po!oxamer.
• the lyophilized composi lion comprises at least about 60% by weight of poloxamer. In some embodiments, the lyophilized composition comprises about 50% by weight to about 70% by weight of poloxamer. In some embodiments, the lyophilized composition comprises about l .5% to about 2% by weight of CHIR99021 , about 42 5% by weight to about 47.5% by weight of sodium valproate, and the remaining percentage is Poloxamer 407.
• the level of an impurity present in the lyophilized pharmaceutical composition is less than about 10000 parts per million (ppm), less than about 1000 ppm, less than about 100 ppm, less than about 10 ppm, less than about 1 ppm, or less than about 0.1 ppm.
• the total level of all the impurities present in the iyophi!ized pharmaceutical composition is less than about 10000 parts per million (ppm), less than about 1000 ppm, less than about 100 ppm, less than about 10 ppm, less than about 1 ppm, or less than about 0.1 ppm.
• the impurity is a residual solvent.
• the impurity is selected from the group consisting of l-acetate-2-fonnate-l,2-propanedioi, acetic add, formic acid, formaldehyde, acetaldehyde, andpropionaldehyde.
• the level of polyethylene oxide presented in the lyophilized pharmaceutical composition is below about 3 %, below about 2 %, below about 1 %, below' about 0.5 %, or below about 0.1 %, as measured by high-performance liquid chromatography (HPLC).
• the total level of one or more impurities with cLog P of about 1 or less presented in the lyophilized pharmaceutical composition is from about 30 % to about 35 %, from about 25 % to about 29 %, from about 20 % to about 25 %, from about 15 % to about 19 %, from about 10 % to about 14 %, from about 5 % to about 9 %, or from about 0 % to about 4 %, as measured by high-performance liquid
• the total level of one or more im puriti es having a boiling point of about 220 °C or less presented in the lyophilized pharmaceutical composition is from about 35 % to about 40 %, from abort 30 % to about 34 %, from about 25 % to abort 29 %, from about 20 % to about 25 %, from about 15 % to about 19 %, from about 10 % to about 14 %, from about 5 % to about 9 %, or from about 0 % to about 4 %, as measured by high-performance liquid chromatography (HPLC).
• HPLC high-performance liquid chromatography
• the lyophilized pharmaceutical composition comprises memeified poloxamer (e.g., purified Poloxamer 407), and wherein the level of the one or more otic therapeutic agents (e.g., hearing loss treatment agents) presented in the lyophilized pharmaceutical composition is about 1.5 fold or higher, about 1.8 fold or higher, about 2 fold or higher, about 25 fold or higher, about 3 fold or higher, about 5 fold or higher, or about 10 fold or higher as compared to a comparable lyophilized pharmaceutical composition without purified poloxamer (e.g., purified Poloxamer 407).
• the comparable lyophilized pharmaceutical composition comprises unpurified poloxamer (e.g., unpurified Poloxamer 407).
• the lyophilized pharmaceutical composition comprises purified poloxamer (e.g., purified Poloxamer 407), and wherein the dissolved concentration of the one or more otic therapeutic agents (e.g., hearing loss treatment agents) presented in the lyophilized pharmaceutical composition is about 1.5 fold or higher, about 1.8 fold or higher, about 2 fold or higher, about 2.5 fiid or higher, about 3 fold or higher, about 5 fold or higher, or about 10 fold or higher as compared to an otherwise i denticai lyophilized pharmaceutical composition without promisified poloxamer (e.g., purified Poloxamer 407).
• the otherwise identical lyophilized pharmaceutical composition comprises unpurified poloxamer (e.g., unpurified Poloxamer 407).
• the lyophilized pharmaceutical composition comprises purified poloxamer (e.g., purified Poloxamer 407), and wherein the lyophilized pharmaceutical composition has lower batch-to-batch variability of one or more gelation properties (e.g., gelation temperature, viscosity, and/or stability') as compared to a comparable !yophilized pharmaceutical composition without purified poloxamer (e.g., purified Poloxamer 407).
• the comparable lyophilized pharmaceutical composition comprises unpurified poloxamer (e.g., unpurified Poloxamer 407).
• the lyophilized pharmaceutical composition compri ses purified poloxamer (e.g., purified Poloxamer 407), and wherein the lyophilized pharmaceutical composition has a lower gelation temperature, a narrower temperature range for gelation, and/or a higher viscosity as compared to a comparable lyophilized pharmaceutical composition withou purified poloxamer (e.g, purified Poloxamer 407).
• the comparable lyophilized pharmaceutical composition comprises unpurified poloxamer (e.g, unpurified Poloxamer 407).
• the lyophilized pharmaceutical composition comprises purified poloxamer (e.g, purified Poloxamer 407), and wherein the lyophilized pharmaceutical composition has a reduced degradation rate as compared to a comparable lyophilized pharmaceutical composition without purified poloxamer (e.g, purified Poloxamer 407).
• the comparable lyophilized pharmaceutical composition comprises unputified poloxamer (e.g, unpurified Poloxamer 407).
• the lyophilized pharmaceutical composition comprises one or more of a bulking agent (e.g, purifi ed Poloxamer 407); a stabilizing agent, a tonicity-adjusting agent, and a soothing agent
• the lyophilized pharmaceutical composition is prepared by iyophilizing the pharmaceutical composition of the present disclosure.
• the lyophilized pharmaceutical composition is prepared by the method of the present disclosure.
• the lyophilized pharmaceutical composition is suitable for preparing a reconstituted solution by a reconstitution process.
• the reconstitution process is less than about 1 hour. In some embodiments, the reconstitution process is less than about 30 minutes.
• the reconstituted solution is suitable for injection (e.g, intratympanic injection).
• the reconstituted solution maintains one or more rheometric properties of a pre- iyophi!ized solution which is used for preparing the lyophilized pharmaceutical composition.
• the reconstituted solution has a reduced degradation rate as compared to a reconstituted solution prepared from a comparable lyophilized pharmaceutical composition without purified poloxamer (e.g, purified Poloxamer 407).
• the comparable lyophilized pharmaceutical composition comprises unpurified poloxamer (e.g, unpurified Poloxamer 407)
• the reconstituted solution maintains one or more rheometric properties of a pre-!yophilized solution which is used for preparing the lyophilized pharmaceutical composition, when the reconstituted solution is prepared at the same solids content as the pre-lyophilized solution.
• the pharmaceutical composition is a pre-iyophi!ized pharmaceutical composition.
• the pharmaceutical composition may be famed by reconstituting the lyophilized compositions disclosed herein, for example to form an aqueous composition, for example a thermoreversible gel.
• aqueous composition for example a thermoreversible gel.
• components of the composition will have a certain concentration when the composition is aqueous (e.g. prior to lyophilization) which will change when the composition is lyophilized since, for example, w'ater is removed.
• the composition comprises a gelling agent and a compound of formula (1)
• the pharmaceutical composition comprises a gelling agent, valproic add or a pharmaceutically acceptable salt thereof at a concentration of greater than about 70 mg/ml, and one or more otic therapeutic agents.
• the pharmaceutical composition comprising a poloxamer, wherein at least 85% by weight of the poloxamer has an average molecular weight of greater than about 7250 Da, and valproic add or a pharmaceutically acceptable salt thereof at greater than 70 mg/mL.
• the pharmaceutical composition comprises a poloxamer, wherein less than 20% by wt.% of the poloxamer has an average molecular weight less about 7250 Da, and valproic acid or a pharmaceutically acceptable salt thereof at greater than 70 mgmL.
• the composition is suitable for iniratympanic injection.
• the gelling agent is a poloxamer (as described above and in the numbered embodiments) in some embodiments, the poloxamer comprises purified poloxamer.
• the poloxamer comprises purified poloxamer the poloxamer is purified poloxamer.
• the poloxamer is defined as above (as defined above and in the numbered embodiments).
• the compositions comprises one or more otic therapeutic agents (as defined above and in the numbered embodiments).
• composition gelling agent comprises a hyaluronic acid.
• composition gelling agent comprises a ceilulosic derivative.
• the one or more otic therapeutic agents include a GSK3 inhibitor.
• the one or more otic therapeutic agents include an HDAC inhibitor.
• the one or more otic therapeutic agents are selected from the tables above
• the one or more otic therapeutic agents include CHIR9902I or a pharmaceutically acceptable salt thereof.
• the concentration of CHIR99021 or a pharmaceutically acceptable salt thereof is less than about 10 mg/mL.
• the concentration of QfflR9902i or a pharmaceutically acceptable salt thereof is less than about 7.5 mg/mL.
• the concentration of CHIR99021 or a pharmaceutically acceptable salt thereof is about 3 to about 7 mg/mL. In some embodiments, the concentration of CHIR99G21 or a pharmaceutically acceptable salt thereof is about 4 to about 6 mg/mL In some embodiments, the concentration of CHIR99021 or a pharmaceutically acceptable saltthereof is about 1 to about 5 mg/mL In some embodiments, the concentration of CHIR99021 or a pharmaceutically acceptable salt thereof is about 2 to about 4 mg/mL. In some embodiments the one or more otic therapeutic agents are one or more hearing loss treatment agents.
• the one or more otic therapeutic agents include valproic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the one or more otic therapeutic agents include valproic acid or a pharmaceutically acceptable salt thereof and CHIR99021 or a pharmaceutically acceptable salt thereof
• the compositi on comprises a compound of formula (! (as described above and in the numbered embodiments).
• the compound of formula (I) and/or the one or more otic therapeutic agents are valproic acid or a pharmaceutically acceptable salt thereof.
• the pharmaceutically acceptable salt of valproic acid is sodium valproate.
• the concentration of valproic add or a phamoaceutically acceptable saltthereof is greater than about 100 mg/ml.
• the concentration of valproic add or a pharmaceutically acceptable saltthereof is about 100 to about 500 mg/mL.
• the concentration of valproic acid or a pharmaceutically acceptable salt thereof is about 100 to abort 350 mg/mL.
• the concentration of valproic acid or a pharmaceutically acceptable salt thereof is about 110 to about 160 mg/ml.
• the concentration of valproic acid or a pharmaceutically acceptable salt thereof is about 130 to about 140 mg/ml. In some embodiments, the concentration of valproic acid or a pharmaceutically acceptable salt thereof is about 125 to about 145 mg/ml. In some embodiments, the concentration of valproic add or a pharmaceutically acceptable salt thereof is abaft 128 to about 138 mg/ml. In some embodiments, the concentration of valproic acid or a pharmaceutically acceptable salt thereof is abort 133 mg/ml.
• the compound of formula (I) and/or the one or more otic therapeutic agents is not valproic add or a pharmaceutically acceptable salt thereof hr some embodiments, the compound of formula (I) and/or tire one or more otic therapeutic agents includes 2-(prop-2-yn-l-yl)-octanoic acid or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) and/or the one or more otic therapeutic agents includes phenylbutyric add or a pharmaceutically acceptable salt thereof hr some embodiments, the compound of formula (I) and/or the one or more otic therapeutic agents includes finoldc acid or a pharmaceutically acceptable salt thereof
• the one or more otic therapeutic agents can be different. In other embodiments, the one or more otic therapeutic agents do not include CHIR99021 or a pharmaceutically acceptable salt thereof In some embodiments the one or more otic therapeutic agents includes LY2090314 or a pharmaceutically acceptable salt thereof. In some embodiments tire one or more otic therapeutic agents includes AZD1080 or a pharmaceutically acceptable salt thereof In some embodiments the one or more otic therapeutic agents includes GSK3 XXH or a phannaceutieally acceptable salt thereof. In some embodiments the one or more otic therapeutic agents includes Compound 1-7 or a pharmaceutically acceptable salt thereof In some embodiments the one or more otic therapeutic agents includes Compound 1-1 or a phannaceutieally acceptable salt thereof
• the composition may comprise a poloxamer. While the poloxamer may vary'
• the poloxamer may comprise the following weight percentage of the composition.
• the concentration of poloxamer is about 2% to about 50% w/v.
• the concentration of poloxamer is about 2% to about 40% w/v.
• the concentration of poloxamer is about 2% to about 30% w/v.
• the concentration of poloxamer is about 2% to about 20% w/v.
• the concentration of poloxamer is about 10% to about 20% w/v.
• the concentration of poloxamer is about 12,5% to about 17.5% w/v.
• the concentration of poloxamer is about 13 % to about 17 5% w/v. In some embodiments, the concentration of poloxamer is about 13 % to about 17 5% w/v. In some
• the concentration of poloxamer is about 13 % to about 17% w/v. In some embodiments, the concentration of poloxamer is about 13.5 % to about 17% w/v. In some embodiments, the concentration of poloxamer is about 13.5 %to about 16.5% w/v. In some embodiments, the concentration of poloxamer is about 14% to about 16.5% w/v. In some embodiments, the concentration of poloxamer is about 14% to about 16% w/v. In some embodiments, the concentration of poloxamer is about 15% to about 17.5% w/v.
• the disclosure relates to a method for preparing a pharmaceutical compositi on (for example the compositions described above or by the numbered embodiments) comprising the steps of: (a) having an aqueous solution comprising a gelling agent; and (b) adding a solution of one or more otic therapeutic agents or a pharmaceutically acceptable salt thereof
• the aqueous solution further comprises valproic add or a pharmaceutically acceptable salt thereof to the first solution.
• the one or more otic therapeutic agents is CHK99021 or a pharmaceutically acceptable salt thereof.
• the one or more otic therapeutic agents is LY2090314 or a pharmaceutically acceptable salt thereof.
• the solution comprises a polar aprotic solvent.
• the polar aptotic solvent comprises DMSO.
• the polar aprotic solvent is DMSO.
• the polar aprotic solvent comprises dmethylforrnamide.
• the polar aprotic solvent comprises dimethylacetamide. In some embodiments, in step (b), the polar aprotic solvent comprises /V-methyl-2-pynolidone. The method of any preceding embodiment wherein the gelling agent comprises a poloxamer.
• the pharmaceutical composition is suitable for preparing the lyophilized pharmaceutical composition of the present disclosure (e.g., by a lyophiliza!ion process disclosed herein).
• the pre-lyophilized pharmaceutical composition comprises:
• CHIR99021 or a phannaceutieally acceptable salt thereof being present at a concentration ranging from 0.025 mg/ml to about 25 mg'rnl;
• valproic add or a pharmaceutically acceptable salt thereof being present at a concentration ranging from 0.5 mg/ml to about 500 mg/ml;
• poloxamer 407 being present at a concentration ranging from 1 wt% to about 25 wt%; and iv) dimethyl sulfoxide (DMSO) being present at a concentration below 7.5 wfrA
• the pre-lyophilized pharmaceutical composition comprises:
• CHIR99021 or a pharmaceutically acceptable salt thereof bang present at a concentration ranging from 0.025 mg / ml to about 25 mg/ml;
• valproic add or a pharmaceutically acceptable salt thereof being present at a concentration ranging from 0.5 mg/ml to about 500 mg/ml;
• poloxamer 407 being present at a concentration ranging from 1 wfr/o to about 25 wi%; and iv) dimethyl sulfoxide (DMSO) being present at a concentration below 25 wt%.
• DMSO dimethyl sulfoxide
• the pharmaceutically acceptable salt of valproic add is a sodium salt (e.g., sodium valproate).
• the phannaceutically acceptable salt of valproic add is a sodium salt (e.g., sodium valproate).
• Concentration can have the units of percent weight per volume (w/v) which can also be expressed as g/mL.
• the concentration of CHIR99021 or the phannaceutically acceptable salt thereof in the pre-lyophiiized pharmaceutical composition ranges from about 0.05 mg/ml to about 5 mg/ml, from about 0.25 mg/ml to about 2.5 mg/ml, from about 0.5 mg/ml to about 1.75 mg/ml, or from about 1.45 mg/ml to about 1.65 mg/ml. In some embodiments, the concentration of CHIR99021 or the pharmaceutically acceptable salt thereof is about 1.55 mg/ml.
• the concentration of valproic add or the pharmaceutically acceptable salt thereof in the pre-lyophilized pharmaceutical composition ranges from about 2.5 mg/ml to about 200 mg/ml, from about 5 mg / ml to about 100 mg/ml, from about 15 mg/ml to about 50 mg/ml, or from about 43 mg/ml to about 46 mg/ml. In some embodiments, the concentration of valproic add or the pharmaceutically acceptable salt thereof is about 44.5 mg/ml.
• the concentration of poloxamer 407 in the pre-lyophilized pharmaceutical composition ranges from about 2.5 wfr/o to about 12.5 wt%, from about 5 wt% to about 11 wf/o, from about 6 wt to about 10 wt%, or from about 7 wt% to about 8 5 wt%. In some embodiments, the concentration of poloxamer 407 is about 8 wt%.
• the concentration of DMSO in the pre-lyophilized pharmaceutical composition ranges from about 0.5 wt% to about 5 wt%, from about 1 wt% to abort 4 wt%, from about 1.5 wl% to about 3.5 wt%, or from about 2 wt% to about 3 wl%. In some embodiments, the concentration of DMSO is about 2.5 wf/o. [0346] In some embodiments, the concentration of DMSO in the composition is about less than 5 wf/o, as described above. However, in other embodiments, it will be appreciated that the concentration of DMSO may be less than about 25 wt%.
• the concentration of DMSO is about less than 25 wt%. In some embodiments, the concentration of DMSO is about less than 20 wf/o. In some embodiments, the concentration of DMSO is about less than 15 wt%. In some embodiments, the concentration of DMSO is about less than 10 wt%.
• the concentration of DMSO is about less than 5 wt%. in some embodiments, wherein the concentration of DMSO is about 25 to about 15 wt°4 In some embodiments, wherein the concentration of DMSO is about 20 to about 10 wt°4 hi some embodiments, wherein the concentration of DMSO is about 15 to about 5 wt%. In some embodiments, wherein the concentration of DMSO is about 10 to about 5 wt%.
• the weight ratio between CHIR99021 or the pharmaceutically acceptable salt thereof and valproic acid or the pharmaceutically acceptable salt thereof in the pre-lyophilized pharmaceutical composition ranges from about 1 : 5 to about 1 :10, from about 1:10 to about 1:50, from about 1 :2Q to about 1:35, from about 1 :25 to about 1 :31, or from about 1 :27 to about 1 :29
• the weight ratio of CHI 99012 and valproic acid (or pharmaceutically acceptable sal ts thereof) will be substantially unchanged in the lyophilized and reconstituted pharmaceutical composition.
• the weight ratio between poloxamer 407 and the DMSO in the pre-lyophilized pharmaceutical composition ranges from about 1 :5 to about 40: 1 , from about 12 to about 15:1, from about 1 : 1 to about 8:1, from about 2:1 to about 4:1, orfrom about 2.5:1 to abcut 3.5:l. In some embodiments, the weight ratio between poloxamer 407 and the DMSO i s about 3 : 1.
• the weight ratio between CHIR99021 and poloxamer 407 in the pre-lyophilized pharmaceutical composition is about 0.02: 1 , the weight ratio between CHIR99021 and the DMSO i s about 0.06: 1 ; the weight ratio between valproic add sodium salt and poloxamer 407 is about 0.54: i; and/or the weight ratio between valproic add sodium salt and the DMSO is about 3.2:1.
• the concentration of CHIR99021 or the pharmaceutically acceptable salt thereof in the pre-lyophilized pharmaceutical composition ranges from about 1.45 mg/ml to about 1.65 mg/ml; the concentration of valproic add or the pharmaceutically acceptable salt thereof ranges from about 43 mg'ml to about 46 mg/ml; the concentration of poloxamer 407 ranges from about 7 wt% to about 8.5 wt%; and the concentration of DMSO ranges from about 2 wt% to about 3 wf/o.
• the concentration of CH1R99021 or the pharmaceutically acceptable salt thereof in the pre-lyophilized pharmaceutical composition is about. J .55 mg/ml; the concentration of valproic acid or the pharmaceutically acceptable salt thereof is about 44.5 mg/ml; the concentration of poloxamer 407 is about 8 wt%; and the concentration of DMSO is about 2.5 wf 4
• the pre-lyophilized pharmaceutical composition comprises:
• CHIR99021 or a pharmaceutically acceptable salt thereof being present at a concentration ranging from 0.025 mg/ml to about 25 mg'ml;
• valproic add or a pharmaceutically acceptable salt thereof being present at a concentration ranging from 1 mg/ml to about 500 mg/ml;
• poloxamer 407 being present at a concentration ranging from 1 wt% to about 25 wi%; and iv) dimethyl sulfoxide (DMSO) being present at a concentration below 7.5 wt%.
• DMSO dimethyl sulfoxide
• the pharmaceutically acceptable salt of valproic add is a sodium salt (eg., sodium valproate).
• the concentration of CHIR99Q21 or the pharmaceutically acceptable salt in the pre-lyophiiized pharmaceutical composition thereof ranges from about 0.05 mg/ml to about 10 mgrtrl, from about 0.25 mg/ml to about 2.5 mg/ml, from about 0.5 mg/ml to about 1.75 mg'inl, from about 0.85 mg'inl to about 1.15 mg/ml.
• tire concentration of CHER99021 or the pharmaceutically acceptable salt thereof is about 1.05 mg'inl.
• the concentration of valproic add or tire pharmaceutically acceptable salt thereof in the pre-lyophilized pharmaceutical composition ranges from about 25 mg/ml to about 200 mg/ml, from about 5 mg/ml to about 100 mg/ml, from about 15 mg/ml to about 50 mg/ml, from about 28 mg/ml to about 31 mg/ml. In some embodiments, the concentration of valproic acid or the pharmaceutically acceptable salt thereof is about 29.5 mg/ml.
• the concentration of poloxamer 407 in the pre-lyophilized pharmaceutical composition ranges from about 2.5 wt% to about 12.5 wt%, from about 5 wf% to about 11 wi%, from about 11 wf% to about 10 wt%, from about 7 wt% to about 8.5 wt%. In some embodiments, the concentration of poloxamer 407 is abort 7.5 wf%.
• the concentration of DMSO in the pre-lyophilized pharmaceutical composition ranges from about 0.5 wt% to about 5 wt%, from about 1 wt% to about 4 wi%, from about 1.5 wt% to about 3.5 wt%, from about 2 wt% to about 3 wt%.
• the concentration of DMSO is about 2.5 wt%
• tire weight ratio between CHIR99021 or the pharmaceutically acceptable salt thereof and valproic add or the pharmaceutically acceptable salt thereof in the pre-lyophilized pharmaceutical composition ranges from about 1 :5 to abort 1 : 10, from about 1 : 10 to about 1 :50, from about 1 :20 to about 1 :35, from about 1 :25 to about 1 :31 , or from about 1 :27 to about 1 :29.
• the weight ratio of CHIR99012 and valproic add (or pharmaceutically acceptable salts thereof) will be substantially unchanged in the lyophilized and reconstituted pharmaceutical composition.
• the weight ratio between poloxamer 407 and the DMSO in the pre-lyophilized pharmaceutical composition ranges from about 1 :5 to about 40: 1, from about 1 :2 to about 15:1, from about 1 : 1 to about 8: 1, from about 2: 1 to about 4: 1, from about.2.5:1 to about 3.5:1. In some embodiments, the weight ratio between poloxamer 407 and the DM SO is about 3:1.
• the weight ratio between CHI 99021 and poloxamer 407 in the pre-lyophilized pharmaceutical composition is about 0.016: 1; the weight ratio between the CH1R99021 and the DMSO is about 0.06:1; the weight ratio between valproic add sodium salt and poloxamer 407 is about 0.42: 1 ; and/or the weight ratio between valproic add sodium salt and the DMSO is about 1.5:1.
• the concentration of CHIR99021 or the pharmaceutically acceptable salt thereof ranges from about 0.95 mg'fnl to about 1.15 mg/ml in the pre-lyophilized pharmaceutical composition; the concentration of val proic acid or the pharmaceutically acceptable salt thereof ranges from about 28 mg/ml to about 31 mg/ml; the concentration of poloxamer 407 ranges from about 7 wt% to about.8.5 wt%; and the concentration of DMSO ranges from about 2 wl% to about 3 wt%.
• the concentration of CHIR99Q21 or the pharmaceutically acceptable salt thereof in the pre-lyophilized pharmaceutical composition is about 1.05 mg/ml; the concentration of valproic add or the pharmaceutically acceptable salt thereof is about 29.5 mg/ml; the concentration of poloxamer 407 is about 7.5 wt%; and the concentration of DMSO is about 2,5 wt°4
• the pre-lyophilized pharmaceutical composition comprises:
• CHIR99021 or a pharmaceutically acceptable salt thereof bang present at a concentration ranging from 0.025 mgml to about 25 mg/ml;
• valproic acid or a pharmaceutically acceptable salt thereof being present at a concentration ranging from 0.5 mg/ml to about 500 mg/ml
• poloxamer 407 being present at a concentration ranging from 1 wl% to about 25 wt%; and iv) dimethyl sulfoxide (DMSO) bdng present at a concentration below 7.5 wt%.
• DMSO dimethyl sulfoxide
• the pharmaceutically acceptable salt of valproic add is a sodium salt (e.g., sodium valproate).
• the concentration of CHIR99021 or the pharmaceutically acceptable salt thereof in the pre-lyophilized pharmaceutical composition ranges from about 0.05 mg/ml to about 5 mg/ml, from about 0.25 mg/ml to about 2 5 mg/ml, from about 0.5 mgml to about 1.75 mg'fnl, or from about 0.6 mg/ml to about 0.75 mgml. In some embodiments, the concentration of CHIR99021 or the pharmaceutically acceptable salt thereof ranges is about 0.7 mg'fnl.
• tire concentration of valproic add or the pharmaceutically acceptable salt thereof in the pre-lyophilized pharmaceutical composition ranges from about 2.5 mg'fnl to about 200 mg/ml, from about 5 mg'ml to abort 100 mg/ml, from about 15 mg/ml to about 50 mg'fnl, or from about 18 mg'fnl to about 21 mg'fnl. In some embodiments, the concentration of valproic add or the pharmaceutically acceptable salt thereof is about 19.5 mg/ml.
• the concentration of poloxamer 407 in the pre-lyophilized pharmaceuti cal composition ranges from about 2.5 wt% to about 125 wt%, from about 5 wt% to about 11 wt%, from about 6 wt to about 10 wt%, or from about 7 wt% to about 8 5 wt%. In some embodiments, the concentration of poloxamer 407 is about 7.5 wt%.
• the concentration of DMSO in the pre-lyophilized pharmaceutical composition ranges from about 0.5 wfr/o to about 5 wfr/o, from about 1 wfr/o to abort 4 wt%, from about 1.5 wt% to about 3.5 wfr/ ⁇ , or from about 2 wf/o to about 3 wt°4 in some embodiments, the concentration of DMSO is about 5 wt%.
• the weight ratio between CHIR99021 or the pharmaceutically acceptable salt thereof and valproic add or the pharmaceutically acceptable salt thereof in the pre-lyophilized pharmaceutical composition ranges from about 1 :5 to about 1:10, from about 1 : 10 to about 1 :50, from about 1 ;20 to about 1 :35, from about 1 :25 to about 1:31, or from about 1 :27 to about 1 :29.
• the weight ratio of CBIR99012 and valproic add (or pharmaceutically acceptable salts thereof) will be substantially unchanged in the lyophilized and reconstituted pharmaceutical composition.
• the weight ratio between poloxamer 407 and the DMSO in the pre-lyophilized phannaceutical composition ranges from about 1 :5 to about 40: 1, from about 1 :2 to about 15:1, from about 1 : 1 to about 8: 1, from about 2: 1 to about 4: 1, from about 2.5:1 to about 3.5:1.
• the weight ratio between poloxamer 407 and the DMSO in the pre-lyophilized pharmaceutical composition is about 3:1 ; the weight ratio between the CHIR99Q21 and poloxamer 4071 s about 0013: 1, the weight ratio between CHER99Q21 and the DMSO is about 006: 1; the weight ratio between valproic add sodium salt and poloxamer 407 i s about 0.23 : J ; and/or the weight ratio between valproic a d sodium salt arid the DMSO is about 1.8: 1.
• the concentration of CHIR99021 or the pharmaceutically acceptable salt thereof in the pre-lyophilized pharmaceutical composition ranges from about 0.6 mg/ml to about 0.75 mg/fnl; the concentration of valproic add or the pharmaceutically acceptable salt thereof ranges from about 18 mg/fnl to about 21 mg/ml; the concentration of poloxamer 407 ranges from about 7 wt% to about 8.5 wf/o; and the concentration of DMSO ranges from about 2 wfr/ o to about 3 wfr4
• the concentration of CHIR99021 or the pharmaceutically acceptable salt thereof in the pre-lyophilized pharmaceutical composition is about 0.7 nig/ml
• the concentration of valproic add or the pharmaceutically acceptable salt thereof is about 19.5 mgfnl
• the concentration of poloxamer 407 is about 7.5 wfr/o
• the concentration of DMSO is about 2.5 wfr/o.
• the pre-lyophilized pharmaceutical composition comprises one or more of water or a buffering agent; a bulking agent; a stabilizing agent (e.g., purified Poloxamer 407); a tonicity-adjusting agent; and a soothing agent.
• a stabilizing agent e.g., purified Poloxamer 407
• a tonicity-adjusting agent e.g., a tonicity-adjusting agent
• the present disclosure provides a method of preparing a lyophilized pharmaceutical composition of the present disclosure.
• the present disclosure provid es a method of processing the pharmaceuti cal composition of the present disclosure to form a lyophilized phannaceutical composition (e.g., the
• the method involves a lyophiiization process.
• the disclosure relates to a method of lyophilizing a pharmaceutical composition as described by the pharmaceutical composition above and the numbered embodiments, wherein the method comprises: (a) providing a pharmaceutical composition; (b) lyophilizing the composition by: (i) reducing the temperature in the fyophilizer to 45 °C at a rate of 0.5 °C per minute, and then holding it at 45 °C for 3 hours, (ii) applying a vacuum of 80 mTorr, (iii) increasing the temperature to -30 °C (at a rate of 0.5 °C per minute) and holding it at -30 °C for 15 hours under a vacuum of 80 mTorr, (iv) increasing tire temperature to 15 °C (at a rate of 0.5 °C per minute); and/or (v) holding the temperature at 15 °C for 20 hours under a vacuum of 80 mTorr, and (d) obtaining a lyophilized pharmaceutical composition.
• the composition comprises: (a) providing a pharmaceutical composition
• the disclosure relates to a method of lyophilizing a pharmaceutical composition as described by the pharmaceutical composition above and the numbered embodiments, wherein the method comprises: (a) providing a pharmaceutical composition, (b) lyophilizing the composition by: (i) reducing the temperature in the lyophilizer to about 45 °C at a rate of about 0.5 °C per minute, and then hoi ding i t at about - 45 °C for about 3 hours; (ii) applying a vacuum of about 80 mTorr, (iii) increasing the temperature to about -30 °C (at a rate of about 05 °C per minute) and holding it at about -30 °C for about 15 hours under a vacuum of about 80 mTorr, (iv) increasing the temperature to about 15 C' C (at a rate of about 0.5 °C per minute); and/or (v) holding the temperature at about 15 °C for about 20 hours under a vacuum of about 80 mTon;
• the pharmaceutical composition is sterilized prior to the lyophiiization process.
• the pharmaceutical co position is sterilized through filtration (e.g., a sterile filtration) using a filter, for example a microporous membrane.
• the filter comprises a nylon, polycarbonate, cellulose acetate, po!yvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polyetiiersulfone (PES), or any combination thereof.
• PVDF po!yvinylidene fluoride
• PTFE polytetrafluoroethylene
• PES polyetiiersulfone
• the filter is a polyethersulfone (PES) membrane filter or a
• the filter has a pore size of about 0.01 pm, about 0.02 pm, about 0.05 p , about 0.08 pm, about 0.1 pm, about 0.2 pm, about 0.3 pm, about 0.4 pm, about 0.5 pm, or about 1 pm.
• microorgani sms e.g., bacteria, mold, or yeast
• particle are substantially removed from the pharmaceutical composition by the filtration.
• the method comprises the steps of:
• step (i) catling the pharmaceutical composition at a first temperature below 0 °C for a first period of time; ii) removing one or more solvents from the resulting mixture of step (i) at a second temperature below 0 C' C, and at a reduced pressure below' 760 Tear, for a second period of time.
• the method comprises one or more st ps selected from:
• the pharmaceutical composi tion comprises the one or more otic therapeutic agents (e.g., hearing loss treatmen t agents) and the poloxamer.
• the pharmaceutical composition comprises the one or more otic therapeutic agents (e.g., hearing loss treatment agents) and poloxamer 407.
• the pharmaceutical composition comprises the one or more otic therapeutic agents (e.g., hearing loss treatment agents) and purified poloxamer 407.
• the pharmaceutical composition comprises CHIR99021 , valproic add sodium salt, the poloxamer, DMSO, and water. In some embodiments, the pharmaceutical composition comprises CHIR99Q21, valproic add sodium salt, poloxamer 407, DMSO, and water. In some embodiments, the pharmaceutical composition comprises CH1R99021 , valproic acid sodium salt, purified poloxamer 407,
• the method comprises one or more steps selected from:
• iib holding the pharmaceutical composition at the second temperature and under the reduced pressure for the second period of time ranging from about 10 hours to about 15 hours; iic) warming the pharmaceutical composition at a rate of about 0.5 °C per minute to 20 °C;
• the reconstituted soluti on is prepared by adding a diluent to the lyophilized pharmaceutical composition of the present disclosure.
• the disclosure relates to a method for reconstituting a lyophilized pharmaceutical composition (as described above or in the numbered embodiments), the method comprising: (a) providing the lyophilized pharmaceutical composition of any preceding embodiment; (h) reconstituting the lyophilized pharmaceutical composition with a pharmaceutically acceptable diluent; and (c) obtaining a reconsti tuted pharmaceutical composition.
• reconstituting the lyophilized pharmaceutical composition comprises dissolving the lyophilized pharmaceutical composition in the pharmaceutically acceptable diluent. In some embodiments, dissolving the lyophilized pharmaceutical composition in the pharmaceutically acceptable diluent takes less than about 1 hour. In some embodiments, dissolving the lyophilized pharmaceutical composition in the pharmaceutically acceptable diluent takes less than about 45 minutes. In some embodiments, dissolving the lyophilized pharmaceutical composition in the pharmaceutically acceptable diluent takes less than about 30 minutes. In some embodiments, dissolving the lyophilized pharmaceutical composition in the pharmaceutically acceptable diluent takes less than about 15 minutes. In some embodiments, dissolving the lyophilized pharmaceutical composition in the pharmaceutically acceptable diluent takes less than about 10 minutes.
• a reconstituted pharmaceutical composition can be obtained by the method for reconstituting a lyophilized pharmaceutical composition.
• a reconstituted pharmaceutical composition comprises the lyophilized composition of the present disclosure and a diluent
• the composition reconstitutes in less abort 1 hour. In some embodiments, the composition reconstitutes in less than about 45 minutes. In some embodiments, the composition reconstitutes in less than about 30 minutes. In some embodiments, the composition reconstitutes in less than about 15 minutes. In some embodiments, the composition reconstitutes in less than about 10 minutes.
• the lyophilized pharmaceutical composition is prepared by lyophilizing the pharmaceutical composition of the present disclosure.
• the lyophilized pharmaceutical composition is prepared by the method of the present disclosure.
• the lyophilized pharmaceutical composition comprises one or more otic therapeutic agents (e.g., hearing loss treatment agents) and a gelling agent.
• the diluent comprises water and dimethyl sulfoxide (DMSO).
• the concentration of DMSO in the diluent ranges from about 1% w/w to about 15% w/w, from about 2% w/w to about 12% w/w, from about 3% w/w to about 10% w/w, from about 4% w/w to about 9% w/w, from about 5% w/w to about 8% w/w, from about 5.5% w/w to about 7.5% w/w, from about 5.8% w/w to about 7% w/w, from about 6% w/w to about 6.8% w/w, or from about 6.2% w/w to about 6.6% w/w.
• the concentration of DMSO in the diluent is about 6.4% w/w.
• the diluent is 64 w/w% DMSO
• the amount of the diluent added during the reconstitution ranges from about 1 m ⁇ , to about 6 m ⁇ ,, from about 2 m ⁇ to about 5 m ⁇ , from about 2.5 pL to about 45 pL, from about 2.8 m ⁇ to about 4 pL, from about 3 pL to about 3.8 pL, or from about 3.2 pL to about 3.6 pL pa mg of tire lyophilized pharmaceutical composition.
• the amount of the diluent added during the reconstitution is about 3.4 pL per mg of the lyophilized pharmaceutical composition.
• the amount of the diluent added during the reconstitution is about 20 grams, about 30 grams, about 40 grams, about 50 grams, about 60 grams, about 70 grams, about 80 grams, about 90 grams, about 100 grams, about 120 grams, about 150 grams, about 200 grams, about 300 grams, about 500 grams, about 800 grams, or about 1000 grams
• the amount of the diluent added during the reconstitution is about 0. lmL - about 1 5mL, about 0.3 ml - about 1.3 mL, about 0.5 ml - about 1.1 mL or about 0.7 mL - about 0.9 mL. In some embodiments, the amount of the diluent added during the reconstitution is about 0.85mL.
• the diluent is sparged with nitrogen for about 10 seconds to about 30 minutes, from about 20 seconds to about 20 minutes, from about 30 seconds, to about 10 minutes, from about 40 seconds to about 5 minutes, from about 50 seconds to about 3 minutes, or from about 1 minute to about 2 minutes prior to being added to the lyophilized pharmaceutical composition.
• the diluent is sterile filtered (e.g., using aPES 0.2 pm filter and/or a 10 mL syringe) prior to being added to the lyophilized pharmaceutical composition.
• the mixture of the lyophilized pharmaceutical composition and the dil uent is held at temperature lower than ambient temperature for a period time, thereby forming the reconstituted solution.
• the reconstitution process is conducted without any agitation of the mixture of the lyophilized pharmaceutical composition and the diluent (e.g., shaking, scnication, or vortexing).
• the reconstitution process comprises gently rotating the container (e.g., the vial) to mix the lyophilized pharmaceutical composition and the diluent, and/or gently tapping the container (e.g., the vial) until the lyophilized pharmaceutical composition and the diluent form a homogeneous solution.
• the mixture of the lyophilized pharmaceutical composition and the diluent is held at a temperature ranging from about -10 °C to about 20 °C, from about -5 °C to about 15 °C, from about 0 °C to about 10 °C, from about 1 °C to about 9 °C, or from about 2 °C to about 8 °C. In some embodiments, the mixture of the lyophilized pharmaceutical composition and the diluent is held at a temperature ranging from about 5-8 °C.
• the mixture of the lyophilized pharmaceutical composition and the diluent is held for a period of time (e.g., reconstitution time) being about 6 hours or less, about 3 hours or less, about 2 hairs or less, about 1 hours or less, about 50 minutes or less, about 40 minutes or less, about 30 minutes or less, about 20 minutes or less, or about 10 minutes or less.
• the mixture of the lyophilized pharmaceutical «imposition and the diluent is held for 20 minutes.
• the reconstitution process comprises addition of the diluent to the lyophilized pharmaceutical composition and storing the 'vial at 2-8 °C. In some embodiments, the reconstitution process comprises addition of the diluent to the lyophilized pharmaceutical composition and storing the vial at 2-8 °C and gently tapping the container (e.g., the vial) until the lyophilized pharmaceutical composition and the diluent form a homogeneous solution.
• the reconstitution process comprises addition of the diluent to the lyophilized pharmaceutical composition and storing the vial at 2-8 °C and gently tapping the container (e.g., the vial) until the lyophilized pharmaceutical composition and the diluent form a homogeneous solution without scnication or vortexing (for exampl e in order to avoid po!oxamer degradation or drug precipitation).
• the reconstitution process comprises addition of about 0.85 ml of diluent to the lyophilized pharmaceutical composition and storing the vial at 2-8 °C and gently tapping the container (e.g., the vial) until the lyophilized pharmaceutical composition and the diluent fonn a homogeneous solution without sonication or vortexing.
• the reconstitution process comprises addition of about 0.85 mL of diluent to the lyophilized pharmaceutical composition and storing the vial at 2-8 °C and gently tapping the container (e.g., the vial) until the lyophilized pharmaceutical composition and the diluent fonn a homogeneous solution without sonication or vortexing where the diluent is 6.4 w/w% DMSO in water.
• any of the reconsisiu!ion processes can be used to measure improved reconstitution time, for example the improvements discussal herein e.g. relative to non-lyophilized solid forms in certain
• the improvement in reconstitution time disclosed herein is specifically measured using a reconstitution process in which about 0.85 mL of diluent is added to the lyophilized pharmaceutical composition, the vial is stored at 2-8 °C and gently tapped until the lyophilized pharmaceutical composition and the diluent fonn a homogeneous solution without sonication or vortexing, where the diluent i s 6.4 w/w%
• the reconstituted solution is a clear- solution at ambient temperature (e.g., between 20 °C and 26 °C).
• the reconstituted solution is suitable for injection at ambient temperature (e.g., between 20 °C and 26 °C).
• the reconstituted solution has a gelation temperature being higher than ambient temperature (e.g., between 20 °C and 26 °C; preferably 25 °C) and being lower than the temperature of human body (e.g., between 36 °C and 39 °C; preferably 37 °C).
• the reconstituted solution has a gelation temperature range of about 2 °C or about 3 °C.
• the reconstituted solution is stable upon storage of at a temperature ranging from about -10 °C to about 20 °C, from about -5 °C to about 15 °C, from about 0 °C to about 10 °C, from about 1 °C to about 9 °C, or from about 2 °C to about 8 °C.
• the reconstituted solution is stored for about 10 minutes or longer, about 20 minutes or longer, about 30 minutes or longer, about 40 minutes or longer, about 50 minutes or longer, about 1 hour or longer, about 2 hours or longer, about 3 hours or longer, about 4 hours or longer, about 5 hours or longer, or about 6 hours or longer prior to use.
• 0.1 % or less about 0.09% or less, about 0.08% or less, about 0.07% or less, about 0.06% or less, about 0.05% or less, about 0.04% or less, abort 0.03% or less, about 0.02% or less, or about 0.01% or less of one or mere otic therapeutic agents (e.g., CHIR99021 and/or sodium valproate) degrades during the storage.
• one or mere otic therapeutic agents e.g., CHIR99021 and/or sodium valproate
• the reconstituted solution has a pH value ranging from about 4 to about 13, from about 5 to about 12, from about 6 to about 1 1 , from about 65 to about 10.5, or from about 7 to about 10.
• the reconstituted solution is suitable for injection at ambient temperature (e.g., between 20 °C and 26 °C) through a needle (e.g., a needle having an inner diameter of about 3 81 mm or less, abaft 3.43 m or less, about 3.00 m or less, about 269 mm or less, about 2.39 mm or less, about 2 16 mm or less, about 1.80 mm or less, about 160 m or less, about 1.37 mm or less, about 1.19 mm or less, about 1.07 mm or less, about 0.84 mm or less, about 0.69 mm or less, about 0.60 mm or less, about 0.51 mm or less, about 0.41 mm or less, about 0.34 mm or less, about 0.31 mm or less, or about 0.26 mm or less).
• a needle e.g., a needle having an inner diameter of about 3 81 mm or less, abaft 3.43 m or less, about 3.00 m or less, about 269
• the reconstituted solution is formulated for in] ection in a volume of about 1 ml or less, about 900 m ⁇ or less, about 800 m ⁇ or less, about 700 m ⁇ or less, about 600 m ⁇ or less, about 500 m ⁇ or less, about 400 m ⁇ or less, about 300 m ⁇ or less, about 200 m ⁇ or less, or about 100 or less.
• the reconstituted solution comprises:
• CHIR99021 or a pharmaceutically acceptable salt thereof being present at a concentration ranging from 0.05 mg/ml to about 50 mg/ml;
• valproic acid or a phannaceutically acceptable salt thereof being present at a concentration ranging from 1 mg/ml to about 1000 mg/ml;
• poloxamer 407 being present at a concentration ranging from 2 wt% to about 50 wt%; and iv) dimethyl sulfoxide (DMSO) being present at a concentration below 15 wt%.
• DMSO dimethyl sulfoxide
• the phannaceutically acceptable salt of valproic acid is a sodium salt. In some embodiments, the phannaceutically acceptable salt of valproic acid is sodium valproate
• the concentration of CH1R99021 or the pharmaceutically acceptable salt thereof in tiie reconstituted solution ranges from about 0.1 mg/ml to about 10 mg/ml, from about 0.5 mgtinl to about 5 g/ml, from about J mg/ml to about 3.5 mg/ml, or from about 2.9 g/ml to about 3.3 mg/ml.
• the concentration of CHIR99021 or the phannaceutically acceptable salt thereof is about 3.1 mg/fnl.
• the concentration of valproic add or the pharmaceutically acceptable salt thereof in the reconstituted solution ranges from about 5 mg/ml to about 400 mg'ml, from about 10 mg/ml to about 200 mg/ml, from about 30 mg'rnl to about 100 mg'rnl, or from about 86 mg'rnl to about 92 mg'rnl. In some embodiments, the concentration of valproic acid or the pharmaceutically acceptable salt thereof i s about 89 mg/ml.
• the concentration of pofoxamer 407 in the reconstituted solution ranges from about 5 wi% to about 25 wt%, from about 10 wt% to about 22 wt%, from about 12 wt% to about 20 wt%, or from about 14 wt% to about 17 wt%.
• tire concentration of poloxam er 407 is about 16 wi%.
• the concentration of DMSO in the reconstituted solution ranges from about 1 wt% to about 10 wt%, from about 2 wt% to about 8 wt%, from about 3 wt% to about 7 wi%, or from about 4 wt% to about 6 wt%. In some embodiments, the concentration of DMSO is about 5 wt%.
• the weight ratio between CH1R99021 or the pharmaceutically acceptable salt thereof and valproic acid or the pharmaceutically acceptable salt thereof in the reconstituted solution ranges from about 1 :5 to about 1 : 10, from about 1 : 10 to about 1 :50, from about 1 ;20 to about 1 ;35, from about. 1 :25 to about 1 :31, or from about 1 :27 to about 1 :29.
• the weight ratio between poloxamer 407 and the DMSO ranges in the reconstituted solution from abort 1 : 5 to about 40:1, from about 1 :2 to about 15:1, from about 1 : 1 to about 8:1, from about 2: 1 to about 4: 1, or from about 2.5:1 to about 3.5:1. In some embodiments, the weight ratio between poloxamer 407 and the DMSO is about 3:1.
• the wdght ratio between CHIR99021 and poloxamer 407 in the reconstituted solution is about 0.02: 1 ; the weight ratio between CHIR99021 and the DMSO is about 0.06: 1; the weight ratio between valproic add sodium salt and poloxamer 407 is about 054: 1; and/or the wdght ratio between valproic a d sodium sal t and the DMSO is about 3.2:1.
• the concentration of CHIR99Q21 or the pharmaceutically acceptable salt thereof in the reconstituted solution ranges from about 2.9 mg'ml to about 3.3 mg'ml; the concentration of valproic add or the pharmaceutically acceptable salt thereof ranges from about 86 mg'ml to about 92 mg/ml; the concentration of poloxamer 407 ranges from about 14 wt% to about 17 wt%; and the concentration of DMSO ranges from abort 4 wt% to about 6 wf/o.
• the concentration of CHIR99021 or the pharmaceutically acceptable salt thereof in the reconstituted solution range; from about 3.2 mg/ml to about 3.3 mg/ml; the concentration of valproic a d or the pharmaceutically acceptable salt thereof ranges from about 87 mg'ml to about 90 mg'ml; the concentration of poloxamer 407 ranges from about 14 wt% to about 16 wt%; and the concentration of DMSO ranges from about 4 wi% to about 5 wt%
• the concentration of CHI 99021 or the pharmaceutically acceptable salt thereof in the reconstituted solution is about 3.1 mg/ml; the concentration of valproic acid or the pharmaceutically acceptable salt thereof is about 89 mg'ml; the concentration of poloxamer 407 is about 16 wt%; and the concentration of DMSQ is about 5 wt%.
• die reconstituted solution comprises:
• CHIR99G21 or a pharmaceutically acceptable salt thereof being present at a concentration ranging from 0.05 mg'ml to about 50 mg/ml;
• valproic acid or a pharmaceutically acceptable salt thereof being present at a concentration ranging from 1 mg'ml to about 1000 mg'ml;
• poloxamer 407 being present at a concentration ranging from 2 wt% to about 50 wi%; and iv) dimethyl sulfoxide (DMSO) being present at a concentration below 15 wt%.
• DMSO dimethyl sulfoxide
• the pharmaceutically acceptable salt of valproic add is a sodium salt. In some embodiments, the pharmaceutically acceptable salt of valproic «id is sodium valproate.
• the concentration of CHIR99021 or the pharmaceutically acceptable salt thereof in the reconstituted solution ranges from about 0.1 mg/ml to about 10 mg/ml, from about 0.5 mg'ml to about 5 mg'ml, from about 1 mg'ml to about 3.5 mg'ml, from about 1.9 mg’ml to about 2.3 mg'ml. In some embodiments, the concentration of CHIR99021 or the pharmaceutically acceptable salt thereof is about 2 1 mg'ml.
• the concentration of valproic acid or the pharmaceutically acceptable salt thereof in the reconstituted solution ranges from about 5 mg/ml to about 400 mg'ml, from about 10 mg'ml to about 200 mg'ml, from about 30 mg'ml to about 100 mg'ml, from about 56 mg'ml to about 62 mg'ml. In some embodiments, the concentration of valproic acid or the pharmaceutically acceptable salt thereof is about 59 mg'ml.
• the concentration of poloxamer 407 in the reconstituted solution ranges from about 5 wt% to about 25 wl%, from about 10 wt% to about 22 wt%, from about 12 wt% to about 20 wt%, from about. 14 wt% to about 17 wt%. In some embodiments, the concentration of poloxamer 407 i s about 15 wt%.
• the concentration of DMSO in the reconstituted sol ution ranges from about J wt% to about 10 wt%, from about 2 wt% to about 8 wt%, from about 3 wt% to about 7 wi%, from about 4 wt% to about 6 wt%. In some embodiments, the concentration of DMSO is about 5 wt%.
• the weight ratio between CHIR99021 or the pharmaceutically acceptable salt thereof and valproic acid or the pharmaceutically acceptable salt thereof in the reconstituted solution ranges from about 1 :5 to about 1:10, from about 1 : 10 to about 1 :50, from about 1 :20 to about 1 :35, from about 1 :25 to about 1 :31, or from about 1 :27 to about 1 :29.
• the weight ratio between poloxamer 407 and the DMSO in the reconsti tuted solution ranges from about 1 :5 to about 40: 1 , from about 1 :2 to about 15:1, from about 1 : 1 to about 8:1, from about 2: 1 to about 4:1, from about 2.5 : 1 to about 3.5:1. in some embodiments, the weight ratio between poloxamer 407 and the DMSO is about 3:1.
• the weight ratio between CHIR99021 and poloxamer 407 in the reconstituted solution is about 0.016: 1; the weight ratio between the CHIR99Q21 and the DMSO is about 0.06: 1; the weight ratio between valproic acid sodium salt and poloxamer 407 is about 0.42: 1 ; and/or the weight ratio between valproic acid sodium salt and the DMSO is about 1.5:1.
• the concentration of CHIR99021 or the pharmaceutically acceptable salt thereof in the reconstituted solution ranges from about 1 9 mg/ml to about 2.3 mg'ml ; the concentration of valproic add t ite pharmaceutically acceptable salt thereof ranges from about.56 mg/ml to about 62 mg/ml, the concentration of poloxamer 407 ranges from about 14 wt% to about 17 wt%; and the concentration of DMSO ranges from abort 4 wt% to about 6 wt .
• the concentration of CHIR99021 or the pharmaceutically acceptable salt thereof in the reconstitute! solution is about 2.1 mg/ml; the concentration of valproic acid or tire pharmaceutically acceptable salt thereof is about 59 mg/ml; the concentration of poloxamer 407 is about 15 wi%; and the concentration of DMSO is about 5 wt%.
• the reconstituted solution comprises:
• CHIR99021 or a pharmaceutically acceptable salt thereof being present at a concentration ranging from 0.05 mg/ml to about 50 mg/ml
• valproic add or a pharmaceutically acceptable salt thereof being present at a concentration ranging from 1 fflg'inl to about 1000 mg/ml;
• poloxamer 407 being present at a concentration ranging from 2 wt% to abort 50 wt ; and iv) dimethyl sulfoxide (DMSO) being present at a concentration below 15 wt%.
• DMSO dimethyl sulfoxide
• the pharmaceutically acceptable salt of valproic add is a sodium salt (e.g., sodium valproate).
• the concentration of CHIR99021 or the pharmaceutically acceptable salt thereof in the reconstituted solution ranges from about 0.1 mg/ml to about 10 mg/ml, from about 0.5 mg/ml to about 5 mgtinl, from about 1 mg'ml to about 3.5 mg/ml, or from about. 1.2 mgtinl to about 1.5 mg/ml. In some embodiments, the concentration of CHIR99021 or the pharmaceutically acceptable salt thereof ranges is abort 1.4 mg/ml.
• the concentration of valproic add or the pharmaceutically acceptable salt thereof in the reconstitute! solution ranges from about 5 mg/ml to about 400 mg/ml, from abort 10 mg/ml to about 200 mgtirrl, from about 30 m ml to abort 100 mg/ml, or from about 36 mgml to about 42 rng/ml. In some embodiments, the concentration of valproic add or the pharmaceutically acceptable salt thereof is about 39 mg'ml.
• the concentration of poloxamer 407 in the reconstituted solution ranges from about 5 wt% to about 25 wt%, from about 10 wt% to about 22 wt%, from about 12 wt% to about.20 wt%, or from about 14 wt% to about 17 wt%. In some embodiments, the concentration of poloxamer 407 is about 15 wt .
• the concentration of DMSO in the reconstituted solution ranges from about 1 wt% to about 10 wt%, from about 2 wt to about 8 wt%, from about 3 wt% to about 7 wt%, or from about 4 wt% to about 6 wi%. In some embodiments, the concentration of DMSO is about 5 wt%.
• the weight ratio between CHIR99021 or the pharmaceutically acceptable salt thereof and valproic add or the pharmaceutically acceptable salt thereof in the reconstituted solution ranges from about 1 :5 to about 1 :10, from about 1 : 10 to about 1 :50, from about 1 :20 to about 1 :35, from about 1 :25 to about 1 :31, or from about 1 :27 to about 1 :29.
• the weight ratio between poloxamer 407 and the DMSO in the reconstituted solution ranges from about 1 :5 to about 40: 1, from about 1 :2 to about 15:1, from about 1 : 1 to about 8: 1, from about 2: 1 to about 4: 1, from about 2.5: 1 to about 3.5: 1.
• the weight ratio between poloxamer 407 and the DMSO in the reconstituted solution is about 3 : 1; the weight ratio between the CHIR99021 and poloxamer 407 is about 0.013 : 1 ; the weight ratio between CHIR99021 and the DMSO is about 006: 1; the weight ratio between valproic acid sodium salt and poloxamer 407 is about 0.23: 1 ; and/or the weight ratio between valproic acid sodium salt and the DMSO is about 1.8: 1.
• the concentration of CHIR99021 or the pharmaceutically acceptable sal t thereof in the reconstituted solution ranges from about 1.2 mtyml to about 1.5 mg/ml; the concentration of valproic add or the pharmaceutically acceptable salt thereof ranges from about 36 mg/ml to about 42 mg/ml; the concentration of poloxamer 407 ranges from about 14 wt to about 17 wt%; and the concentration of DMSO ranges from about 4 wt% to about 6 wi%.
• the concentration of CHIR99021 or the pharmaceutically acceptable salt thereof in the reconstituted solution is about 1.4 mg/ml; the concentration of valproic acid or the pharmaceutically acceptable salt thereof is about 39 mg/ml; the concentration of poloxamer 407 is about 15 wt%; and the concentration of DM SO is about 5 wt%.
• the reconstituted solution compri ses, in additi on to the acti ve agents, one or more of water or a buffering agent; a bulking agent (e.g., purified Poloxamer 407); a stabilizing agent, a tonicityadjusting agent and a soothing agent
• the reconstituted solution comprises, in addition to the active agents, purified poloxamer (e.g., purified Poloxamer 407), and wherein the reconstituted solution has a higher stability to oxygen and/or light as compared to a comparable reconstituted solution without (e.g., purified Poloxamer 407).
• the comparable reconstituted solution comprises unpurified Poloxamer (e.g., unpurified
• the level of an impurity present in the reconstituted solution is less than about 10000 parts per million (ppm), less than abort 1000 ppm, less than abort 100 ppm, less than about 10 ppm, less than about 1 ppm, or less tha about.0. J ppm.
• the impurity is selected from the group consisting of 1 -acetate-2 ⁇ fomiate- 1 ,2- propanediol, acetic add, formic add, formaldehyde, acetaldehyde, and propianaldehyde.
• the level of polyethylene oxide present in the reconstitute! solution is below about 3 %, below about 2 %, below about 1 %, below about 0.5 %, or below about 0.1 %, as measured by high- performance liquid chromatography (HPLC).
• the total 1 evei of one or more impurities with cLqg P of about 1 or less present in the reconstituted solution is from about 30 % to about 35 %, from about 25 % to about 29 %, from about 20 % to about 25 %, from about 15 % to about 19 %, from about 10 % to about 14 %, from about 5 % to about 9 %, or from about 0 % to about 4 %, as measured by high-performance liquid chromatography (HPLC).
• HPLC high-performance liquid chromatography
• the total level of one or more impurities having a boiling point of about 220 °C or less present in the reconstitute! solution is from about 35 % to about 40 %, from about 30 % to about 34 %, from about 25 % to about 29 %, from about 20 % to about 25 %, from about 15 % to about 19 %, from about 10 % to about 14 %, from about 5 % to about 9 %, or from about 0 % to about 4 %, as measured by high- performance liquid chromatography (HPLC).
• HPLC high- performance liquid chromatography
• the reconstituted solution comprises purified poloxamer (e.g., purified Poloxamer 407), and wherein the level of the one or more otic therapeutic agents (e.g., hearing loss treatment agents) present in the reconstituted solution is about 1.5 fold or higher, abort 1.8 fold or higher, about 2 fold or higher, about 2.5 fold or higher, about 3 fold or higher, about 5 fold or higher, or about 10 fold or higher as compared to a comparable reconstituted solution without purified poloxamer (e.g., purified Poloxamer 407).
• the comparable reconstituted solution comprises unpurified poloxamer (e.g., unpurified Poloxamer 407).
• ih e reconstituted solution comprises purified poloxamer (e.g., purified Poloxamer 407), and wherein the reconstituted solution has lower batch-to-batch variability of one or more gelation properties (e.g., gelation temperature, viscosity, and/or stability) as compared to a comparable reconstituted solution without purified poloxamer (e.g., purified Poloxamer 407).
• the comparable reconstituted solution comprises unpurified poloxamer (e.g., unpurified Poloxamer 407).
• the reconstituted solution comprises purified poloxamer (e.g., purified Poloxamer 407), and wherein the reconstituted solution has a lower gelation temperature, a narrower gelation temperature range, a more sustained release of the hearing loss treatment agent, and/or a higher viscosity 7 as compared to a reconstituted solution without purified poloxamer (e.g., purified Poloxamer 407).
• the comparable reconstituted solution comprises unpurified poloxamer (e.g , unpurified Poloxamer 407).
• the reconstituted solution comprises purified poloxamer (e.g., purified Poloxamer 407), and wherein the reconstituted solution has a lower gelation temperature than the gelation temperature of an otherwise identical compositi on with unpurified poloxamer rather than purified poloxamer, wherein the temperature is about 1 °C lower, about 2 °C lower, about 3 °C lower, about 4 °C lower, about 5 °C lower, about 6 °C lower, about 7 °C lower, about 8 °C lower, about 9 °C lower, about 10 °C lower, about 11 °C lower, about 12 °C lower, or about 13 °C lower than the gelation temperature of an otherwise identical reconstituted solution with unpurified poloxamer (e.g., unpurified Poloxamer 407) rather than purified poloxamer as described herein.
• unpurified Poloxamer 407 rather than purified poloxamer as described herein.
• the recoastituted solution comprises purified poloxamer (e.g., purified Poloxamer 407), and wherein the reconstituted solution has a narrower gelation temperature range compared to the gelation temperature range of an otherwise identical composition with unpurified poloxamer rather than purified poloxamer.
• the gelation temperature range is the range of temperatures over which the formulation transitions from being a fluid to being a gel .
• composition with unpurified poloxamer generally transition from a fluid to a gel over a range of about 10 °C, whereas otherwise identical compositions with purified poloxamer (e.g., purified Poloxamer 407) transition firm a fluid to a gel over a range of about 2 °C to about 3 X:.
• purified poloxamer e.g., purified Poloxamer 407
• the reconstituted solution comprises purified poloxamer (e.g., purified
• the reconstituted solution has a reduced degradation rate as compared to a comparable reconstituted solution without purified poloxamer (e.g., purified Poloxamer 407).
• the comparable reconstituted solution comprises unpurified poloxamer (e.g , unpurified
• the reconstituted solution is suitable for injection (e.g., intratympanic injection).
• the reconstituted solution maintains one or more rheometric properties of a pharmaceutical composition which is used for preparing the !yophilized pharmaceutical composition.
• the reconstituted solution has a reduced degradation rate as compared to a reconstituted solution prepared from a comparable lyophilized pharmaceutical composition without purified poloxamer (e.g., purified Poloxamer 407).
• the comparable lyophilized pharmaceutical composition comprises unpurified poloxamer (e.g , unpurified Poloxamer 407).
• the reconstituted solution comprises one or more of water or a buffering agent a bulking agent (e.g., purified Poloxamer 407); a stabilizing agent; a tonicity-adjusting agent; and a soothing agent.
• a buffering agent e.g., purified Poloxamer 407
• the pharmaceutical composition or reconstituted solution of the present disclosure comprises water.
• the pharmaceutical composition or reconstituted solution of the present disclosure comprises a buffering agent.
• Hie buffer controls the pH of the reconstituted solution to a range of from about 4 to about 13, from about 5 to about 12, from about 6 to about 11, from about 6.5 to about 10.5, or from about 7 to about 10.
• buffering agent examples include, but are not limited to, citrate buffering agents, acetate buffering agents, phosphate buffering agents, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gfuconic add, cakium glycerophosphate, calcium lactate, calcium lactobionate, propanoic add, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, caldum hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium dirate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate
• Lubricating agents may be selected fro the non-limiting group consisting of magnesium stearate* caldum stearate, stearic add, silica, talc, malt glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauiyi sulfate, and combinations thereof.
• the buffering agent comprises phosphate buffered saline, THIS, Iris acetate, tris HC1-65, sodium dtrate, histidine, arginine, sodium phosphate, iris base-65, hydroxyethyl starch, or any combination thereof
• a poloxamer can be used in certain embodiments as a gelling agent.
• An aldehyde is a compound containing a functional group with the structure CHO, consisting of a carbon double- bonded to oxygen with the carbon atom also bonded to a hydrogen atom.
• Aldehydes including formaldehyde, acetaldehyde, and propionaldehyde, are potential impurities and degradation products of poloxamers and may be formed e.g. when the poloxamer is present in a gel. Lyophilization beneficially removes aldehydes present in the test composition. Lyophilized compositions disclosed herein can also be more stable than the gel form, for example in relation to the levels of aldehyde present overtime.
• lyophilization removes aldehydes from the compositions of the present disclosure.
• preservatives such as antioxidants are not required in the lyophilized compositions of the present disclosure, for example because of the low levels of aldehydes present
• the concentration of aldehydes is less than about 1, about 2, about 3, about 4, about 5 or about 10 ppm (ug/g). In some embodiments of the lyophilized pharmaceutical composition, the concentration of aldehydes is less than about 10 ppm (pg/g). In some embodiments of the lyophilized pharmaceutical composition, the concentration of aldehydes is less than about 5 ppm (pg/g). In some embodiments of the lyophilized pharmaceutical composition, the concentration of aldehydes is less than about 4 ppm (ug/g). In some embodiments of the lyophilized pharmaceutical composition, the concentration of aldehydes is less than about 3 ppm (pg/g).
• the concentration of aldehydes is less than about 2 ppm (pg/g). In some embodiments of the lyophilized pharmaceutical composition, the concentration of aldehydes is less tha about 1 ppm (pg/g).
• the aldehydes are volatile aldehydes. [0478] !n some embodiments, the aldehydes comprise molecules where each individual molecule has a molecular weight of less than 300 Da. hi some embodiments, the aldehydes comprise molecules where each individual molecule has a molecular weight of less than 200 Da. In some embodiments, the aldehydes comprise molecules where each individual molecule has a molecular weight of less than 100 Da.
• the aldehydes comprise formaldehyde, acetaldehyde, and/or propionaldehyde.
• antioxidants include, but are not limited to, RRR-Alpha-Tocopherol, d-Alpha tocopherol; d-alpha tocopheryl acetate; dl-alpha tocopheryl acetate; d-alpha tocophayl acid succinate; dl-alpha tocopheryl acid succinate; beta tocopherol; delta tocopherol; gamma tocopherol; tocopherols excipient, Ascorbic Add; Ascorbyl palmitate; erythorbic add; sodium ascorbate; sodium eiythorbate; butylated hydroxytoluene; Butylated Hydroxyanisole; Anhydrous citric add; fumaric add; malic acid; sodium dtrate; dihydrate; tartaric acid; Citric Add Monohydrate; Edetic Add; Dipotassium edetate; disodium edetate; edetate caldum disodium; sodium edetate, trisodium
• the pharmaceutical composition of the present disclosure does not comprise an antioxidant.
• the lyophilized pharmaceutical composition of the present disclosure does not comprise an antioxidant.
• the reconstituted lyophilized pharmaceutical composition of the present disclosure does not comprise an antioxidant.
• the pharmaceutical composition of the present disclosure does not comprise an antioxidant and has a concentration of aldehydes which is less than about 1, about 2, about 3, about 4, about 5 or about 10 ppm (pg/g) .
• the lyophilized pharmaceutical composition of the present di sclosure does not comprise an antioxidant and has a concentration of aldehydes which is less than about 1, about 2, about 3, about 4, about 5 or about 10 ppm (pg/g) .
• the reconstituted pharmaceutical composition of the present disclosure does not comprise an antioxidant and has a concentration of aldehydes which is less than about 1, about 2, about 3, about 4, about 5 or about 10 ppm (pg/g) .
• the phaimaceuti cal composition or reconstituted sol ution of the present disclosure comprises a bulking agent
• the bulking agent comprises poloxamer (e.g., poloxamer 407), mannitol, sucrose, maltose, trehalose, dextrose, sorbitol, glucose, raf!inose, glycine, histidine, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K12 or polyvinylpyrrolidone K17), lactose, or any combination thereof.
• the poloxamer e.g., poloxamer 407 is the gelling agent and/or the bulking agent.
• the poloxamer e.g., poloxamer 407) is the gelling agent and the bulking agent.
• the composition comprises a gelling agent (such as poloxamer, e.g. Poloxamer 407)
• the composition does not comprise an additional bulking agent (such as mannitol, sucrose, maltose, trehalose, dextrose, sorbitol, glucose, raffinose, glycine, histidine, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone Kl 2 or polyvinylpyrrolidone K17), lactose, or any combination thereof).
• an additional bulking agent such as mannitol, sucrose, maltose, trehalose, dextrose, sorbitol, glucose, raffinose, glycine, histidine, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone Kl 2 or polyvinylpyrrolidone K17), lactose, or any combination thereof).
• a bulking agent can positively enhance the lyophi!izatian process, leading to an improved dried/lyophilized product in terms of appearance and characteristics.
• a solution of poloxamer 407 can be lyophilized in the absence of a bulking agent to form a porous cake of substantial volume (e.g. see Figure 9) and not a flat sheet of dried mass (e.g. see Figure 10).
• a molecule such as sodium valproate (NaVPA) was added to poloxamer 407 solution.
• a polymeric lyophilized cake mass produced in this way reconstituted well and retained the rheological properties similar to the pre-lyophilized sol ution.
• the pharmaceutical composite on of the present disclosure does not comprise bulking agent in addition to the gelling agent
• the lyophilized pharmaceutical composition of the present disclosure does not comprise a bulking agent in addition to the gelling agent
• the reconstituted lyophilized pharmaceutical composition of the present disclosure does not comprise a bulking agent in addition to the gelling agent.
• the pharmaceutical composition or reconstituted solution of the present disclosure comprises a stabilizing agent.
• the stabilizing agent comprises Polyethylene Glycol, saccharides, ascorbic add, acetylcysteine, bisulfite, metabi sulfite, mmothioglyercol, inositol, oleic acid, or any combination thereof.
• the stabilizing agent comprises a cr oprotectant
• the cryoprotectant is a polyol (e.g., a did or a trio! such as propylene glycol (i.e , 1 ,2-propanediol), 1 ,3-propanediol, glycerol, (+/-)-2-methyl-2,4-pentanediol, l,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethyleneglycol, or diethylene glycol), a ncndetergent sulfobetaine (e.g., M3SB-2Q1 (3-(l-pyridino)-l-propane sulfonate), an osmolyte (e.g., L-proline or trimethylamineN-oxide dihydrate), a polymer (e.g., polyethylene
• the salt is selected from the group consisting of lithium salts (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, or any hydrate thereof), magnesium salts (e.g., magnesium acetate or a hydrate thereof), and sodium salts (e.g., sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate, or any hydrate thereof).
• the formulation comprises one or more sodium salts.
• the formulation comprises sodium chloride
• the stabilizing agent comprises a surfactant.
• the surfactant compri ses one or more anionic surfactants (e.g., 2-acrylamido-2-methylpropane sulfonic add, ammonium lauiyl sulfate, ammonium perfluorononanoate, docusate, disodium cocoamphodiacetate, magnesium laureth sulfate, perfluorobutanesulfonic add, peifluorononancic add, perfluorooctanesulfbnic add, perfluorooctanoic acid, potassium lauiyl sulfate, sodium alkyl sulfate, sodium dodecyl sulfate, sodium
• anionic surfactants e.g., 2-acrylamido-2-methylpropane sulfonic add, ammonium lauiyl sulfate, ammonium perfluorononanoate, docusate, disodium cocoa
• dodecylbenzenesulfonate sodium laurate, sodium laureth sulfate, sodium lauroyl sarcosinate, sodium myreth sulfate, sodium nonanoyloxybenzenesulfonate, sodium pareth sulfate, sodium stearate, or sulfolipid
• one or more cationic surfactants e.g., behentrimonium chloride, benzalkonium chloride, benzethonium chloride, benzododednium bromide, bronidox, carbethopendednium bromide, cetalkonium chloride, cetrimonium bromide, cetrimonium chloride, cetylpyridinium chloride, didecyldimethylammorrium chloride,
• dmethyldioctadecylammonium bromide dimethyldioctadecylammonium chloride, domiphen bromide, lauiyl methyl gluceth-10 hydroxypropyl imonium chloride, octenidine dihydrochloiide, olaflur, n-deyl-1,3- propanediamine, paliutoxin, stearalkonium chloride, tetramethylammonium hydroxide, or thonzenium bromide), one or more zwiterionic surfactants (e.g., cocamidopropyi betaine, cocamidopropyi hydroxysultaine, dipalmitoylphosphaticfrlcholine, egg lecithin, hydroxysultaine, lecithin, myristamine oxide, peptitergents, or sodium lauroamphoacetate), and/or one or more non-ionic surfactants (e.g.
• the pharmaceutical composition or reconstituted solution of the present disclosure comprises a tonidty-adjusting agent
• the tonkity-adj usiing agent comprises NaCl, dextrose, dextran, ficoll, gelatin, mannitol, sucrose, glydne, glycerol, or any combination thereof.
• the pharmaceutical composition or reconstituted solution of the present disclosure comprises a soothing agent.
• the soothing agent comprises Jidocaine [0503]
• the pharmaceutical composition or reconstituted solution of the present disclosure may include any substance useful in pharmaceutical compositions.
• the pharmaceutical composition or reconstituted solution of the present disclosure may include one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrants, fillers, g!idamts, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, and other species. Excipien ts such as waxes, butters, coloring agents, coating agen ts, flavorings, and perfuming agents may also be included. Pharmaceutically acceptable excipients are well known in tire art (see for example Remington’ s The Science and Practice ofPharmacy, 21 a Edition, A. R Gennaro; Lippincott, Williams & Wilkins, Baltimore, AID, 2006).
• diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microaystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and/or combinations thereof
• Granulating and dispersing agents may be selected from the non-limiting list consisting of potato starch, com starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, ben tonite, cellulose and wxxxl products, natural sponge, cati on- exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyfyinyl-pyrrolidone)
• crospQvidane sodium carboxymeihyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and/or combinations thereof.
• Surface active agents and/or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic add, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and ledthin), colloidal clays (e.g., bentonite [aluminum silicate] and
• VEEGUM® [magnesium aluminum silicate]
• long chain amino add derivatives high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triaeetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyaciylie a d, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, ceMosie derivatives (e.g., cafooxymethyicellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan tatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monoo
• ether [BKU® 30]), poly( ⁇ inyl- pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauiyl siilfate, PLURQNIC®F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, henzalkonium chloride, docusate sodium, and/or combinations thereof.
• a binding agent may be starch (e.g., cornstarch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylceliui ose,
• starch e.g., cornstarch and starch paste
• gelatin e.g., cornstarch and starch paste
• sugars e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol
• natural and synthetic gums e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum,
• preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, addic preservatives, and/or other- preservatives.
• antioxidants include, but are not limited to, alpha tocopherol, ascorbic add, ascorbyl palmitale, butylated hydroxyanisole, butylated hydroxytoluene, monothioglyceroi, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, anchor sodium sulfite.
• chelating agents include ethylenediaminetetraacetic add (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic add, fumaric acid malic add phosphoric add, sodium edetate, tartaric acid and/or trisodium edetate.
• EDTA ethylenediaminetetraacetic add
• citric acid monohydrate disodium edetate
• dipotassium edetate dipotassium edetate
• edetic add fumaric acid malic add
• fumaric acid malic add phosphoric add
• sodium edetate tartaric acid and/or trisodium edetate.
• antimicrobial preservatives include, but are not limited to, benzaikonium chloride, benzethcnium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresoi, chloroxylenol, cresol, ethyl alcohol, glycerin, hexeticline, imidurea, phenol, phenoxy ethanol, phenylethyl alcohol, phenylmercuiic nitrate, propylene glycol, and/or thimerosal.
• antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic add, hydraxybenzoie add potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic add.
• alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol.
• acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric add acetic acid, dehydroascorbic add, ascorbic add, sorbic add and/or phytic add.
• preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SEES), sodium bisulfite, sodium metabi sulfite, potassium sulfite, potassium metabi sulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, CERMABEN®ISL NEOLONETM, KATHONTM, and/or EUXYL®.
• buffering agents include, but. are not limited to, dtrate buffering agents, achate buffering agents, phosphate buffering agents, ammonium chloride, calcium carbonate, calcium chloride, calcium dtrate, calcium glubionate, calcium giuceptate, caldum gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic add calcium levulinate, pentanoic add, dibasic calcium phosphate, phosphoric add, tribasic calcium phosphate, caldurn hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfon
• Lubricating agents may be selected from the non-limiting group consisting of magnesium stearate, calcium stearate, stearic add, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium laiayi sulfate, sodium lauryl sulfate, and combinations thereof
• oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, camauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seal, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademianut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandal wood, sasquana,
• octyldodecanol oleyl alcohol, silicone oil, and/or
• the term "pharmaceutically acceptable salt” takes its normal meaning in the ait. In certain embodiments it refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergi c response and the like, and are commensurate with a reasonable benefit/risk ratio.
• Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describes pharmaceutically acceptable salts in detail in I Pharmaceutical Sciences (1977) 66: 1-19.
• Pharmaceutically acceptable salts of the compounds provided herein include those derived from suitable inorganic and organic acids and bases.
• Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic adds such as hydrochloric acid, hydrobromic acid, phosphoric add, sulfuric add and perchloric acid or with organic adds such as acetic add, oxalic acid, maleic add, tartaric acid, citric acid, succinic add or maionic acid or by using other methods used in the ait such as ion exchange.
• Other pharmaceutically acceptable sal ts include adipate, alginate, ascorbate, aspartate,
• benzenesulfonate besylate, benzoate, bisulfate, borate, butyrate, camphorate, eamphorsulfonate, dtrate, cydopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydraxy-ethanesulfonate, iactcbionate, lactate, laurate, lauiyl sulfate, malate, maleate, malonate, methanesulfcnate, 2- naphthalenesulfonate, nicotmate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulf
• organic acids from which salts can be derived include, for example, acetic add, propionic acid, glycolic add, pyruvic acid, oxalic add, lactic add, trifluoraceiic acid, maleic add, malonic add, sucdnic acid, fumaric acid, tartaric acid, citric add, benzoic add, cinnamic acid, mandelic acid, methanesulfonic add, ethanesulfbnic acid, p-toluenesulfonic add, salicylic ad d, and the like.
• the salts can be prepared in situ during the isolation and purification of the disclosed compounds, or separately, such as by reacting the free base or free add of a parent compound with a suitable base or add, respectively.
• Pharmaceutically acceptable salts derived from appropriate bases include alkali metal and alkaline earth metal.
• Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like.
• Further pharmaceutically acceptable salts include, when appropriate, potassium, sodium, calcium, and magnesium salts.
• Alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to ten carbon atoms (e.g., Ci-io alkyl).
• a numerical range such as “ 1 to 10” refers to each integer in the given range, e.g., " 1 to 10 carbon atoms” means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although the present definition also covers the occurrence of the term "alky! where no numerical range is designated.
• “alkyl” c an be a C , alkyl group.
• alkyl groups have 1 to 10, 1 to 8, 1 to 6, or 1 to 3 carbon atoms.
• Representative saturate! straight chain alkyls include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; while saturated branched alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tart-butyl, -isopentyl, 2-methy!butyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-rnetliylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl,
• alkyl is attached to the parent molecule by a single bond.
• an alkyl group is optionally substituted by one or more of substituents which independently include: alkyl, alkenyl, alkynyl, alkoxy, cydoalkyl, aryl, or halo.
• substituents which independently include: alkyl, alkenyl, alkynyl, alkoxy, cydoalkyl, aryl, or halo.
• a substituted alkyl can be selected from fluoromethyi, difluoromethyl, trifluoromethyl, 2- fluoroehyl, 3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl, andphenethyl.
• alkenyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, and having from two to ten carbon atoms (i.e., C2-10 alkenyl). Whenever it appears herein, a numerical range such as “2 to 10" refers to each integer in the given range; e.g , "2 to 10 carbon atoms” means that the alkenyl group can consist of 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms. In certain embodiments, an alkenyl comprises two to eight carbon atoms.
• an alkenyl comprises two to six carbon atoms (e.g., C2-6 alkenyl).
• Tire alkenyl is attached to the parent molecular structure by a single bond, for example, ethenyl (i.e., vinyl), prop-l-enyl (i.e., allyl), but- 1-enyl, pent-l-enyi, penta-l,4-dienyl, and the like.
• the me or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl).
• Examples of C2-4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (Cr), 2-butenyl (Cs), 2-methylprop-2-enyl (Cr), butadienyl (&) and the like.
• Examples of C2-6 alkenyl groups include the aforementioned C24 alkenyl groups as well aspentenyl (Cs), pentadienyl (Cs), hexenyl ((3 ⁇ 4 2,3-dimethyl-2-butenyl (Ce) and the like.
• alkenyl examples include heptenyl ( €7), octenyl (Cs), octatrienyl (Cs) and the like. Unless stated otherwise in the specification, an alkenyl group can be optionally substituted by me or more substituents which
• alkyl independently include: alkyl, alkenyl, alkynyi, alkoxy, eydoalkyl, aryl, or halo.
• Alkynyi refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond, having from two to ten carbon atoms (i.e., C2-10 alkynyi). Whenever it appears herein, a numerical range such as “2 to 10" refers to each integer in the given range; e.g., "2 to 10 carbon atoms” means that the alkynyi group can consist of 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms. In certain embodiments, an alkynyi comprises two to eight carbon atoms.
• an alkynyi has two to six carbon atoms (e.g., C2-0 alkynyi)
• Tire alkynyi is attached to the parent molecular structure by a single bond, for example, ethynyl, propynyi, butynyl, pentynyl, 3-methyl- 4-pentenyl, hexynyl, and the like.
• an alkynyi group can be optionally substituted by one or more substituents which independently include: alkyl, alkenyl, alkynyi, alkoxy, cycloalkyl, aryl, and halo.
• Alkoxy' refers to the group -O-alkyl, including from 1 to 10 carbon atoms of a straight, branched, saturated cyclic configuration and combinations thereof, atached to the parent molecular structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, pentoxy, eyclopropyloxy, cydohexyloxy and tire like. "Lower alkoxy " refers to alkoxy groups containing one to six carbons. In some embodiments, CM alkoxy i s an alkoxy group which encompasses both straight and branched chain alkyls of from 1 to 4 carbon atoms.
• an alkoxy group can be optionally substituted by one or mere substituents which independen tly include: alley!, alkenyl, alkynyi, alkoxy', eydoalkyl, aryl, and halo.
• Aryl refers to a radical with 6 to 14 ring atoms (e.g, G I 4 aromatic or AA aryl) which has at least one ring having a conjugated pi electron system which is carbocydic (e.g, phenyl, fluorenyl, and naphthyl).
• the aryl is a Guo aryl group.
• bivalent radicals formed from substituted benzene derivatives and having the free valences at ring atoms are named as substitute! phenylene radicals.
• bivalent radicals derived from univalent polycyclic hydrocarbon radicals whose names end in"- yl" by removal of one hydrogen atom from the carbon atom with the free valence are named by adding "-idene" to the name of the corresponding univalent radical, e.g, a naphthyl group with two points of atachment is tamed naphthylidene.
• a numerical range such as "6 to 14 aryl” refers to each integer in the given range; e.g, "6 to 14 ring atoms” means that the ary! group can consist of 6 ring atoms, 7 ring atoms, etc, up to and including 14 ring atoms.
• the term includes monocyclic or fused-ring polycyclic (i.e, rings which share adjacent pairs of ring atoms) groups.
• Polycyclic aryl groups include bicycles, tricycles, tetracycies, and the like. In a multi-ring group, only me ring is required to be aromatic, so groups such as indanyl are encompassed by the aryl definition.
• Non-limiting examples of aryl groups include phenyl, phenalenyl, naphthalenyl, tetrahydronaphthyl, phenanthrenyl, anthracenyl, fiuorenyl, indolyl, indanyl, and the like.
• an aryl moiety can be optionally substi tuted by one or more substituents which independently include: alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, aryl, and halo.
• substituents which independently include: alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, aryl, and halo.
• this term includes any of o-tolyl, m-tolyl, and p-tolyl groups.
• “tolyi” includes any of the three isomeric univalent aromatic radicals derived from toluene.
• Cydoalkyl and “carbocyclyl” each refer to a monocyclic or polycyclic radical that contains only carbon and hydrogen, and can be saturated or partially unsaturated. Partially unsaturated cydoalkyl groups can be termed “cydoalkenyl” if the carbocycle contains at least one double bond, or "cycloalkynyl” if the carbocycle contains at least one triple bond. Cydoalkyl groups include groups having from 3 to 13 ring atoms (i.e , C3-13 cydoalkyl).
• a numerical range such as “3 to 10" refers to each integer in the given range; e.g., "3 to 13 carbon atoms” means that the cydoalkyl grasp can consist of 3 carbon atoms, 4 carbon atans, 5 carbon atoms, etc., up to and including 13 carbon atoms.
• the term "cydoalkyl” also includes bridged and spiro-fused cyclic structures containing no heteroatoms.
• the term also includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of ring atoms) grasps.
• Polycyclic aryl groups include bicycles, tricycles, tetracycies, and the like.
• “cydoalkyl” can be a C3-8 cydoalkyl radical. In some embodiments,“cydoalkyl” can be a C3-5 cydoalkyl radical .
• Illustrative examples of cydoalkyl groups include, but are not limited to the following moieties: C3-6 carbocyclyl groups include, without limitation, cyclopropyl ((3 ⁇ 4), cyclobutyl (Ci), cyclopentyl ((3 ⁇ 4), cyelopentenyl (Cs), cyclohexyl ( €3 ⁇ 4), cydohexenyl (( cydohexadienyl ((3 ⁇ 4 and the like.
• C3-7 carbocyclyl groups include norbomyl (C7).
• Examples of C3-8 carbocyclyl groups include the aforementioned C3-7 carbocydyl groups as well as cydoheptyl(C7), cydoheptadienyl (C 7), cydoheptatrienyl (C?), cyclooctyl (Cs), bicyclo[2.2.1 Jheptanyl, bicydo[2.2.2]octanyl, and the like.
• C3-13 carbocydyl groups include the aforementioned C -s carbocydyl groups as well as octahydro-lH indenyl, decahydronaphthalenyl, spiro[4.5]decanyl and the like.
• a cydoalkyl group can be optionally substitute! by one or more substituents which independently include: alkyl, alkenyl, alkynyl, alkoxy, cydoalkyl, aryl, and halo.
• a cydoalkenyl group can have 3 to 13 ring atoms, such as 5 to 8 ring atoms. In some embodiments, a cycloalkynyl group can have 5 to 13 ring atoms.
• a “covalent bond” or “direct bond” refers to a single bond joining two groups.
• Halo means fluoro, chioro, bromo or iodo.
• haloalkyl include alkyl, alkenyl, alkynyl and alkoxy' structures that are substituted with one or more halo groups or with combinations thereof
• fluoroalkyl and fluoroalkoxy include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine, such as, but not limited to, tifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, l-fluoromethyl-2- fluoroethyl, and the like.
• alkyl, alkenyl, alkynyl and alkoxy groups are as defined herein and can be optionally further substituted as defined herein.
• the presen t disclosure relates to inducing, promoting, or enhancing the growth, proliferation or regeneration of inner ear tissue, particularly inner ear supporting cells and hair cells by using the composition disclosed herein.
• Some embodiments relate to method s for controlled proliferation of stem cells comprising an initial phase of inducing sternness while inhibiting differentiation and a subsequent phase of differentiation of the stem cells into tissue cells.
• cochlear supporting cell or vestibular supporting cell populations are treated with a hair cell regeneration agent in accordance to the methods of the disclosure, whether the population is in vivo or in vitro, the treated supporting cells exhibit stern-like behavior in that the treated supporting cells have the capacity to proliferate and differentiate and, more specifically, differentiate into cochlear hair cell s or vestibular hair cells.
• an agent induces and maintains the supporting cells to produce daughter stem cells that can divide fix many generations and maintain the ability to have a high proportion of the resulting cells differentiate into hair cells.
• the proliferating stem cells express stem cell marker(s) selected from one or more ofLgrS, Sox2, Opeml, Phex, lin28, Lgr6, cyclinDl, Msxl, Myb, Kit Gdnt3, Zic3, Dppa3, Dppad, DppaS, Nanqg, Esnb, Rexl, DnmtSa, DnmOb, DnmOl, IJtfl, Tell, Oct4, Klf4, Pax6, Six2, Zicl, Zic2, Otx2, Bmil, CDX2, STAT3, Smadl, Smad2, smad2/3, smad4, smadS, and smad7.
• the proliferating stem cells express stem cell markers) selected from one or more ofLgrS, the
• the methods may be used to maintain, or even transiently increase sternness (i.e , self-renewal) of a pre-existing supporting cell population prior to significant hair cell formation in some embodimen ts
• the pre-existing supporting cell population comprises inner pillar cells, outer pillar cells, inner phalangeal ceils, Deiter cells, Hensen cells, Boetcher ceils, and/or Claudius cells. Morphological analyses with immunostaining (including cell counts) and lineage tracing across a Representative Microscopy Samples may be used to confirm expansion of one or more of these cell-types.
• the pre-existing supporting cells comprise Lgr5+ cells. Morphological analyses with immunostaining (including cell counts) and qPCR and RNA hybridization may be used to confirm Lgr5 upregulation amongst the cell population.
• the therapy preferably involves the administration of a small molecule, peptide, antibody, or other non-nucleic add molecule or nucleic add delivery' vector unaccompanied by gene therapy.
• the therapy involves the administration of a small organic molecule.
• hearing protection or restoration is achieved through the use of a (non-genetic) therapeutic that is injected in the middle ear and diffuses into the cochlea.
• the cochlea relies heavily on all present cell types, and the organization of these ceils is important to their function. Supporting ceils play an important role in neurotransmitter cycling and cochlear mechanics. Thus, maintaining a rosette patterning within the organ of Cord may be important for function.
• Cochlear mechanics of the basilar membrane activate hair cell transduction. Due to the high sensitivity of cochlear mechanics, it is also desirable to avoid masses of cells. I all, maintaining proper distribution and relation of hair cells and supporting cells along the basilar membrane, even after proliferation, is likely a desired feature lor hearing as supporting cell function and proper mechanics is necessary for normal hearing.
• the hearing loss treated by using a composition as disclosed herein is senorineural healing loss or hidden healing loss.
• Sensorineural healing loss accounts for approximately 90% of hearing loss and it often arises from damage or loss of hair cells in the cochlea.
• There are numerous causes of hair cell damage and loss and the agents and treatments described herein may be used in the context of sensorineural hearing loss arising from any cause of hair cell damage or loss.
• hair cells may be damage and loss may be induced by noise exposure, leading to noise-induced sensorineural hearing loss.
• sensorineural healing loss is noise-induced sensorineural hearing loss.
• Noise-induced sensorineural hearing loss can be a result of chronic noise exposure or acute noise exposure.
• Ototoxic drugs for example cisplatin and its analogs, aminoglycoside antibiotics, salicylate and its analogs, or loop diuretics, can also cause sensorineural hearing loss.
• sensorineural hearing loss is drug-induced sensorineural hearing loss. Infection may damage cochlear hair ceils, and may he a cause of sudden sensorineural hearing loss.
• sensorineural hearing loss is sudden sensorineural hearing loss (SSNHL). Sudden sensorineural hearing can also be idiopathic. Hair cells can also be lost or damaged over time as part of the ageing process in humans.
• sensorineural hearing loss is age-related sensorineural hearing loss (also known as presbycusis).
• the present disclosure provides a method of facilitating the regeneration of a tissue and/or a cell, comprising delivering a pharmaceutically effective amount of the pharmaceutical composition or the reconstituted solution of the present disclosure to the tissue and/or the cell.
• the present disclosure provides a method of treating a subject who has, or is at risk of developing a disease associated with absence or a lack of a tissue and/or a cell, comprising administering to the
• the present disclosure provides a method of increasing a population of vestibular cells in a vestibular tissue, comprising deliveiying a pharmaceutically effective amount of the pharmaceutical composition or the reconstituted solution of the present disclosure to the population.
• the present disclosure provides a method of treating a subject who has, or is at risk of developing a vestibular condition, comprising administering to the subject a pharmaceutically effective amount of the pharmaceutical composition or the reconstituted solution of the present disclosure.
• the present disclosure provides a method of increasing a population of cochlear cells in a cochlear tissue, comprising delivering a pharmaceutically effective amount of the pharmaceutical composition or the reconstituted solution of the present di selosure to the population.
• the present disclosure provides a method of treating a subject who has, or is at risk of developing a cochlear condition, comprising administering to the subject a pharmaceutically effective amount of the pharmaceutical composition or the reconstituted solution of the present disclosure.
• the present disclosure provides a method of increasing a population of cells found in the Organ of Corti, comprising deliverying a pharmaceutically effective amount of the pharmaceutical composition or the reconstitute! solution of the present disclosure to the population.
• the present disclosure provides a method of increasing a population of hair cells found in the Organ of Corti, comprising deliverying a pharmaceutically effective amount of the pharmaceutical composition or the reconsti tuted solution of the present disclosure to the population.
• the present disclosure provides a method of increasing a population of inner hair cell s found in the Organ of Corti, comprising deliverying a pharmaceutically effective amount of the pharmaceutical composition or the reconstituted solution of the present disclosure to the population.
• the present disclosure provides a method of increasing a population of outer hair cells found in the Organ of Corti, comprising delivetying a pharmaceutically effective amount of the pharmaceutical composition or the reconstituted solution of the present disclosure to the population.
• the present disclosure provides a method of increasing a population of neuronal cells found in the Organ of Corti, comprising deliverying a pharmaceutically effective amount of the pharmaceutical composition or the reconstituted solution of the present disclosure to the population.
• the present disclosure provides a method of treating a subject who has, or is at risk of developing a hearing condition, comprising administering to the subject a pharmaceutically effective amount of the pharmaceutical composition or the reconstituted solution of the present disclosure.
• the present disclosure provides the pharmaceutical composition or the reconstituted solution of the present disclosure for use in facilitating the generation of a tissue and/or a cell.
• the present disclosure provides the pharmaceutical composition or the reconstituted solution of the present disclosure for use in treating a subject who has, or is at risk of developing, a disease associated with absence or a lack of a tissue and/or a cell.
• the present disclosure provides the pharmaceutical composition or the reconstituted solution of the present disclosure for use in increasing a population of vestibular cells in a vestibular tissue.
• the present disclosure provides the pharmaceutical composition or the reconstituted solution of tiie present disclosure for use in treating a subj t who has, or is at risk of developing a vestibular condition.
• the present disclosure provides the pharmaceutical composition or the reconstituted solution of the present disclosure for use in increasing a population of cochlear cells in a cochlear tissue
• the present disclosure provides the pharmaceutical composition or the reconstituted solution of the present disclosure for use in treating a subject who has, or is at risk of developing a cochlear condition.
• the present disclosure provides the pharmaceutical composition or the reconstituted solution of the present disclosure for use in increasing a population of cells found in the Organ of Corti.
• the present disclosure provides the pharmaceutical composition or the reconstituted solution of the present disclosure for use in increasing a population of hail cells found in the Organ of Corti .
• the present disclosure provides the pharmaceutical composition or the reconstituted solution of the present disclosure for use in increasing a population of inner hair cells found in the Organ of Corti.
• the present disclosure provides the pharmaceutical composition or the reconstituted solution of the present disclosure for use in increasing a population of outer hair cells found in the Organ of Corti .
• the present disclosure provides the pharmaceutical composition or the reconstituted solution of tiie present disclosure for use in increasing a population of neuronal cells found in the Organ of Corti.
• tiie present disclosure provides the pharmaceutical composition or tiie reconstituted solution of tiie present disclosure for use in treating a subj ect who has, or is at risk of developing a hearing condition.
• the present disclosure provides for use of the pharmaceutical composition or the reconstituted solution of the present disclosure in the manufacture of a medicament for facilitating the generation of a ti ssue and/or a cell .
• the present disclosure provides for use of the pharmaceutical composition or the reconstituted solution of the present disclosure in the manufacture of a medicament for in treating a subject who has, or is at risk of developing, a disease associated with absence or a lack of a tissue and/or a cell.
• the present disclosure provides for use of the pharmaceutical composition or the reconstituted solution of the present disclosure in the manufacture of a medicament for increasing a population of vestibular cells in a vestibular tissue.
• the present disclosure provides for use of the pharmaceutical composition or the reconstituted solution of the present disclosure in the manufacture of a medicament for treating a subject who has, or is at. risk of developing a vestibular condition
• the present disclosure provides for use of the pharmaceutical composition or the reconstituted solution of the present disclosure in the manufacture of a medicament for increasing a population of cochlear cells in a cochlear tissue.
• the present disclosure provides for use of the pharmaceutical composition or the reconstituted solution of the present disclosure in the manufacture of a medicament for heating a subj ect who has, or is at risk of developing a cochlear condition.
• the present disclosure provides for use of the pharmaceutical composition or the reconstituted solution of the present disclosure in the manufacture of a medicament for increasing a population of cells found in the Organ of Corti
• the presort disclosure provides for use of the pharmaceutical composition or the reconstituted solution of the present disclosure in the manufacture of a medicament for increasing a population of Mr cells found in the Organ of Corti.
• the present disclosure provides for use of the pharmaceutical composition or the reconstituted solution of the present disclosure in the manufacture of a medicament for increasing a population of inner hair cells found in the Organ of Corti.
• the present disclosure provides for use of the pharmaceutical composition or the reconstituted solution of the present disclosure in the manufacture of a medicament for increasing a population of outer hair cells found in the Organ of Corti
• the present disclosure provides for use of the pharmaceutical composition or the reconstituted solution of the present disclosure in the manufacture of a medicament for increasing a population of neuronal cells found in the Organ of Corti.
• the pharmaceutical composition or reconstituted solution of the present disclosure is delivered extratympaniealiy (i.e., onto the eardrum).
• the pharmaceutical composition or reconstituted solution of the present disclosure is delivered intrafympanieal!y (i.e., into the middle ear).
• the pharmaceutical composition or reconstituted sol uticn of the present disclosxe is delivered continuously.
• the pharmaceutical composition or reconstituted solution of the present disclosxe is delivered as a bolus injection.
• about about 1 ml or less, about 900 m ⁇ or less, about 800 m ⁇ or less, about 700 m ⁇ or less, about 600 m ⁇ or less, about 500 m ⁇ or less, about 400 m ⁇ or less, about 300 m ⁇ or less, about 200 m! less, or about 100 or less of the pharmaceutical composition or reconstituted solution is injected
• the pharmaceutical composition or reconstituted solution of the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 10 g'kg from about 0.001 mg'kg to about 10 mg/kg, from about 0.005 mg/kg to about 10 g/kg, from about 0.01 mg'kg to about 10 mg'kg, from about 0.05 mg/kg to about 10 mg'kg, from about 0.1 mg/kg to about 10 mg/kg from about 1 mg/kg to about 10 mg/kg, from abort 2 mg/kg to about 10 mg/kg from about 5 mgkg to about 10 mg/kg from about 00001 mg/kg to about 5 mg/kg, from about 0.001 mg/kg to about 5 mg/kg from about 0.005 mg/kg to about 5 mg/kg from about 0.01 mgkg to about 5 mg/kg from about 0.05 mgkg to about 5 mg/kg from about 0.1 mg/kg to about 5 mgkg from about 1 mgkg to about 5 mgkg from about 2 mgkg to about 5 mg/kg from
• a dose of about 0001 mgkg to about 10 mgkg of a therapeutic and/or prophyl actic (e.g , mRNA) of aLNP may be administered.
• a dose of about 0.005 mgkg to about 2.5 mg/kg of a therapeutic and/or prophylactic may be administered.
• a dose of about 0.1 mg/kg to about 1 mgkg may be administered hi some embodiments, a dose of about 0.05 mgkg to about 0.25 mg/kg may be administered.
• a dose may be administered one or more times per day, in the same or a different amount, to obtain a desired level of mRNA expression and/or therapeutic, diagnostic, prophylactic, or imaging effect
• desired dosage may be delivered, for example, three times a day, two times a day, once a day, every oilier day, every third day, every week, every two weeks, every three weeks, or every four weeks.
• the desired dosage may be delivered using multiple administrations (e.g: , two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
• a single dose may be administered, for example, prior to or after a surgical procedure or in the instance of an acute di sease, disorder, or condi tion.
• the administration of the pharmaceutical composition or reconstituted solution results in a plasma concentration for the one or more otic therapeutic agents (e.g., CHIR9902I and sodium valproate) having a maximum plasma concentration at at time ranging from 10 minutes to about 3 hours, from about 20 minutes to about 2 hours, or form about 30 minutes to about 1 hour.
• the one or more otic therapeutic agents e.g., CHIR9902I and sodium valproate
• Articles such as“a,”“an,” and“the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include“or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
• the disclosure includes embodiments in which exactly one member of the grasp is present in, employed in, or otherwise relevant to a given product or process.
• the disclosure includes embodiments in which more than one, or all, of the group members are present in, employed in, or otherwise relevant to a given product or process.
• the terms“approximately” and“about,” as applied to one or more values of interest, refer to a value that is similar to a stated reference value.
• the term“approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%o, 14%, 13%, 12%,
• the term“approximately” or“about’ refers to +/- 10% of the recited value.
• “about’ when used in the context of an amount of a given compound in a lipid component of aLNP,“about’ may mean +/- 10% of the recited value.
• the expressions“one or more of A, B, or C,”“one or more A, B, or C,”“one or more of A, B, and C,”“one or more A, B, and C,”“selected from the group consisting of A, B, and C”,“selected from A, B, and C”, and the like are used interchangeably and ail refer to a selection from a group consisting of A, B, and/or C, i.e , one or more As, one or more Bs, one or more Cs, or any combination thereof, unless indicated otherwise.
• the term“bulking agenfr’ refers to an agent that adds bulk to a pharmaceutical composition and/or modifies one or more the properties of the pharmaceutical composition (e.g., the appearance of the cake, the porosity, drug stability, and/or the reconstitution time).
• compositions are described as having including, or comprising specific components, it is contemplated that compositions also consist essentially of, cr consist of the recited components. Similarly, where methods or processes are described as having, including or comprising specific process steps, the processes also consist essentially of, or consist of ’ the recited processing steps. Further, it shoid be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions can be conducted simultaneously.
• the term“comparable pharmaceutical composition” refers to a pharmaceutical composition with comparable parameters, as of the pharmaceutical composition being compared (e.g, the one or more otic therapeutic agents (e.g., hearing loss treatment agents) and gelling agents therein, and/or the concentration of the one or more otic therapeutic agents (e.g , hearing loss treatment agents) and gelling agents).
• the“comparable pharmaceutical composition” comprises a poloxamer (e.g, Poloxamer 407) with lower purity as compared to pharmaceutical composition being compared.
• the “comparable pharmaceutical composition” does not comprise a purified poloxamer (e.g., purified Poloxamer 407).
• the“comparable pharmaceutical composition” comprise a unpurified poloxamer (e.g., unpurified Poloxamer 407).
• the term“comparable reconstituted solution” refers to a reconstituted solution with comparable parameters as of the reconstituted solution being compared (e.g., the one or more otic therapeutic agents (e.g., hearing loss treatment agents) and gelling agents therein, and/or the concentration of the one or more otic therapeutic agents (e.g., hearing loss treatment agents) and gelling agents).
• the “comparable reconstituted solution” comprises a poloxamer (e.g., Poloxamer 407) with lower purity as compared to reconstituted solution being compared.
• the“comparable reconstituted solution” does not comprise a purified poloxamer (e.g., purified Poloxamer 407). In some embodiments, the “comparable reconstituted solution” comprise a unpurified poloxamer (e.g., unpurified Poloxamer 407).
• the“comparable reconstituted solution” is pepared from a pharmaceutical composition comprising a poloxamer (e.g., Poloxamer 407) with lower purity as compared to pharmaceutical composition used for preparing the reconstituted solution being compared.
• the “comparable reconstituted solution” is pepared from a pharmaceutical composition not comprising a purified poloxamer (e.g., purified Poloxamer 407).
• the“comparable reconstituted solution” is pepared from a pharmaceutical composition comprising a unpurified poloxamer (e.g., unpurified Poloxamer 407).
• the term“impurity” refers to a compound that is underarable for the pharmaceutical composition.
• the impurity is selected from solvents, l-acetate-2-formate- 1,2- propanediol, acetic add, formic add, formaldehyde, acetaldehyde, propionaldehyde, low MW poloxamers, and degradants from CHIR99021 and valproic add.
• the term“soothing agenfr’ refers to an agent capable of mitigating the discomfort from administration of the formulation to patients.
• the terra“stabilizing agent” refers to an agent capable of maintaining the one or more desirable properties of the pharmaceutical composition (e.g., reduced suspetabiliiy to degradation by heat, light, or air).
• the term“unpurified poloxamer” refers to a poloxamer not bring purified (e.g., by the process disclosed herdn).
• the unpurified poloxamer e.g., unpurified Poloxamer 407
• the unpurified poloxamer e.g., unpurified Poloxamer 407 is not purified by any liquid-liquid extraction or size exclusion chromatography.
• purified poloxamer may in some embodiments refer to poloxamer that is at least 85% by weight poloxamer that has a molecular weight of at least 7250 Da.
• Purified poloxamer can in some embodiments be prepared by following the method of: A Fakhari, M Corcoran, A Schwarz,
• the present disclosure provides methods for preparing any of the pharmaceutical compositions and reconstituted solutions described herein.
• the present disclosure also provides detailed methods for preparing various pharmaceutical compositions and reconstituted solutions following the procedures described in the Examples.
• compositions ate described as having, including or compri sing specific components it is contemplated that compositions also consist essentially of, or consist of, the recited components.
• methods or processes are described as having including or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps.
• order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions can be conducted simultaneously.
• any description of a method of treatment includes use of the compounds to provide such treatment or prophy!axi s as i s described herein, as well as use of the compounds to prepare a medicament to treat or prevent such condi tion.
• the treatment includes treatment of human or non-human animals including rodents and other disease models.
• sterile refers to solutions, products, equipment, or glass ware that are treated and / or handled to be free from bacteria or other living microorganisms.
• the term“subject’ is interchangeable with the term“subject in need thereof’, both of which refer to a subject having a disease or having an increased risk of developing the disease.
• A“subject’ includes a mammal .
• the mammal can be e.g , a human or appropriate non-human mammal, such as primate, mouse, rat, dog, cat, cow horse, goat, camel, sheep or a pig.
• the subj ect can al so be a bird or fowl .
• the mammal is a human.
• a subject in need thereof can be one who has been previously diagnosed or identified as having an imprinting disorder.
• a subject in need thereof can also be one who has (e.g, is suffering from) an imprinting disorder.
• a subject in need thereof can be one who has an increased risk of developing such disorder relative to the population at large (i.e., a subject who is predisposed to developing such disorder relative to the population at large).
• a subject in need thereof can have a refractoiy or resistant imprinting disorder (i.e., an imprinting disorder that doesn't respond or hasn’t yet responded to treatment).
• the subject may be resistant at start of treatment or may become resistant during treatment
• the subject in need thereof received and failed all known effective therapies for an imprinting disorder.
• the subject in need thereof received at least one prior therapy.
• the subject has an imprinting disorder.
• the term“sterilization” refers to process for ensuring the removal of undesired contamination including bacteria, mold and yeast and particles using e.g., a 0.2-micron filter.
• Filter materials used in the sterilization of liquids include, but are not limited to, nylon, polycarbonate, cellulose acetate, pdyvinylidene fluoride (PVDF), and poiyethersulfone (PES).
• the term“tonicity” refers to a measured level of effective osmolarity.
• the tonicity refers to a measured level of the effective osmotic pressure gradient, as defined by the water potential of two solutions separated by a semipermeab!e membrane
• the term“tonicity-adjusting agent” refers to an agent capable of changing the tonicity of the pharmaceutical composition or solution to a desired level.
• the term“treating” or“treat” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes tire administration of a compound of the present disclosure, or a pharmaceutically acceptable salt, polymorph or solvate thereof to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder.
• the term “treat” can also include treatment of a cell in i ntro or an animal model.
• a compound of the present disclosure or a pharmaceutically acceptable salt, polymorph or solvate thereof can or may also be used to prevent a relevant disease, condition or disorder, or used to identify suitable candidates for such purposes.
• the term“preventing,”“prevent,” or“protecting against” describes reducing or eliminating the onset of the symptoms or amplications of such disease, condition or disorder.
• the term“pharmaceutical composition” is a formulation containing one or more otic therapeutic agents (e.g., hearing loss treatment agents) of the present disclosure in a form suitable for administration to a subject.
• the pharmaceutical composition is in bulk or in unit dosage form.
• the unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet a single pump on an aerosol inhales or a vial.
• the quantity of active ingredient (e.g. , a formulation of the disclosed compound or salt hydrate, solvate or isomer thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved.
• the dosage will also depend on the route of administration.
• routes of administration A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperiteneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like.
• Dosage forms for the topical or transdermal administration of a compound of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
• the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.
• the term“pharmaceutically acceptable” refers to those compounds, anions, cations, materials, compositions, earners, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or amplication, commensurate with a reasonabl e benefit/risk ratio.
• the term“pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use.
• a “pharmaceutically acceptable excipienf’ as used in the specification and claims includes both one and more than one such excipient
• a pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration.
• routes of administration include parenteral, e.g, intravenous, intradermal, subcutaneous, oral (e.g, inhalation), transdermal (topical), and transmucosal administration.
• Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or oilier synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• the pH can be adjusted with acids or bases, such as hydrochloric add or sodium hydroxide.
• the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose dais made of glass or plastic.
• a compound or pharmaceutical composition of the disclosure can be administered to a subject in many of the well-known methods currently used for chemotherapeutic treatment.
• a compound of the disclosure may be injected into the blood stream or body cavities or taken orally or applied through the skin with patches.
• the dose chosen should be sufficient to constitute effective treatment but not so high as to cause unacceptable side effects.
• the state of the disease condition (e.g., imprinting disorders, and the like) and the health of the patient should preferably be closely monitored during and for a reasonable period after treatment
• the term“therapeutically effective amount” refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified di sease or condi tion, or to exhibit a detectable therapeutic or inhibitoiy effect.
• the effect can be detected by any assay method known in the art.
• the precise effecti ve amount for a subject will depend upon the subject’s body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration.
• Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.
• the disease or condition to be treated is an imprinting disorder.
• the therapeutically effective amount can be estimated initially either in cell culture assays, e.g, of neoplastic cells, or in animal models, usually rats, mice, rabbits, dogs, or pigs.
• the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans
• Therapeutic/prophylactic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g, ED» (the dose therapeutically effective in 50% of the population) and LDso (the dose lethal to 50% of the population).
• the dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LDso/ED».
• Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage may vary within this range depending upon the
• Dosage and administration are adjusted to provide sufficient levels of the active agents) or to maintain the desired effect.
• Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combinations), reaction sensitivities, and tolerance/response to therapy.
• Long-acting pharmaceutical compositions may be administered every' 3 to 4 days, every week, or once every two weeks depending on half- life and clearance rate of the particular formulation.
• compositions containing active compounds of the present disclosure may be manufactured in a manner that is generally known, e.g. , by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• Pharmaceutical compositions may be fonnulated in a conventional manner using one or more pharmaceutically acceptable earners comprising excipients and/or auxiliaries that facilitate processing of the active compounds into preparations that can be rosed pharmaceutically.
• the appropriate formulation is dependent upon the route of administration chosen.
• the pharmaceutical composition or reconstituted solution of the present disclosure is refrigerated or frozen for storage and/or shipment (e.g, being stored at a temperature of 4 °C or lower, such as a temperature between about - 150 °C and about 0 °C or between about -80 °C and about -20 °C
• the present disclosure also relates to a method of increasing stability' of the pharmaceutical composition or reconstituted solution and by storing the pharmaceutical composition or reconstituted solution at a temperature of 4 °C or lower, such as a temperature between about - 150 °C and about 0 °C or between about -80 °C and about -20 °C, e.g, about -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C, 40 °C, -50 °C, -60 °C, -70 °C, -80 °C, -90 °C, -130 °C or -150 °C).
• a temperature of 4 °C or lower such as a temperature between about - 150 °C and about 0 °C or between about -80 °C and about -20 °C, e.g, about -5 °C, -10 °C, -15 °C, -20
• compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• suitable earners include physiological saline, bacteriostatic water, Ciemophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
• the composition must be steril e and should be fluid to the extent that easy syringeahili ty exi ts. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of
• the carrier can be a sol vent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof
• the proper fluidity' can be maintained, for example, by the use of a carting such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanoL phenol, ascorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride in the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
• Sterile inj ectab!e solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those
• sterile powders for the preparation of sterile injectable solutions methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• Oral compositions generally include an inert diluent or an edible pharmaceutically acceptable carrier. They ca be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the fomi of tablets* troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid earner is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
• the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragaeanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as algtnic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Stearates; a giidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
• a binder such as microcrystalline cellulose, gum tragaeanth or gelatin
• an excipient such as starch or lactose, a disintegrating agent such as algtnic acid, Primogel, or com starch
• a lubricant such as magnesium stearate or Stearates
• a giidant such as coll
• Systemic administration can also be by transmucosal or transdermal means.
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• Such penetrants are generally known in the ait, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid deri vati ves.
• Transmucosal administration can be accomplished through the use of nasal sprays or patches, thin films, tablets to be used for buccal or sublingual application or suppositories.
• the active compounds are formulated into ointments, salves, creams, gels, patches or microneedle delivery systems as generally known in the art.
• the active compounds can be prepared with pharmaceutically acceptable camera that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
• a controlled release formulation including implants and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, poiyg!yeolic add, collagen, polyorthoesters, polylacticglycolic add and poiyiactic add. Methods for preparation of such formulations will be apparent to those skilled in the ait.
• Hie materials can al so be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
• Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described inU.S. Pat. No.4,522,811.
• Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.
• the dosages of the pharmaceutical compositions used in accordance with the disclosure vary depending on the agent, the age, weight, and clinical condition of the recipient patient and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage.
• the dose should be sufficient to result in slowing and preferably regressing the symptoms of the imprinting disorder and also preferably causing complete regression of the imprinting disorder.
• Dosages can range from about 0.01 mg/kg per day to about 5000 mg/kg per day. In preferred aspects, dosages can range from about 1 mg/kg per day to about 1000 mg'kg per day.
• the dose will be in the range of about 0.1 mg/day to about 50 g/day ; about 0.1 mgiiay to about 25 g/day; about 0.1 mg day to about 10 gday; about 0.1 mg to about 3 g/day; or about 0.1 mg to about 1 g/day, in single, divided, or continuous doses (which dose may be adj usted for the patient s weight in kg body surface area in m 2 , and age in years).
• An effective amount of a phamiaceutieal agent is that which provides an objectively identifiable improvement as noted by the clinician or other qualified observer. Improvement in survival and growth indicates regression.
• the term“dosage effective manner” refers to amount of an active compound to produce the desired biological effect in a subject or ceil .
• compositions can be included in a container, pack, or dispenser together with instructions for administration.
• the term“pharmaceutically accep table salts” refer to derivati ves of the compounds of the present disclosure wherein the parent compound is modified by making acid or base salts thereof.
• pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic adds, and the like.
• pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
• such conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic adds selected from 2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulfbnic, 1,2-ethane sulfonic, fumaric, gfucoheptcnic, gluconic, glutamic, glycolic, glycollyarsanilic, hexylresordnic, hydrabarnic, hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxymphthoic, isethionic, lactic, lactobionic, lauiyl sulfonic, maleic,
• Oilier examples of pharmaceutically acceptable salts include hexanoic add, cyclopentane propionic add, pyruvic add, malonic acid, 3-(4-hydroxybenzoyl)benzoie add, dnnamie add, 4-chlorobenzenesulfonic add, 2-naphthalenesulfonic add, 4-toluenesulfonic add, eamphorsulfonic add, 4-methylbicyclo-[2.2.2]-oct-2- ene- 1 -carboxylic acid, 3-phenylpropionic add, trimethylacetic add, tertiary butylacetic add, muconic add, and the like.
• the present disclosure also encompasses salts famed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tramethamine, N- methylglucamine, and the like.
• a metal ion e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion
• an organic base such as ethanolamine, diethanolamine, triethanolamine, tramethamine, N- methylglucamine, and the like.
• the ratio of the compound to the cation or anion of the salt can be 1:1, or any ration other than 1 :1, e.g., 3:1, 2:1, 1:2, or 1 :3.
• the compounds of the present disci osure can also be prepared as esters, for example, phannaceu!ically acceptable esters.
• a carboxylic add function group in a conpound can be converted to its corresponding ester, e.g, a methyl, ethyl or other ester.
• an alcohol group in a compound can be converted to its corresponding ester, e.g. , acetate, propionate or other ester.
• the compounds of the present disclosure can be a prodrug (that may include an ester) of any compound disclosed herein.
• the compounds of the present disclosure can also be prepared as co-crystals with oilier compounds.
• the compounds, or pharmaceuti cally acceptable salts thereof, are administered orally, nasally, transdermally, pulmonary, inhalationally, buccaliy, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally and parenterally. I one embodiment the compound is administered orally.
• One skilled in the art will recognize the advantages of certain routes of administration.
• the dosage regimen utilizing the compounds is selected in accordance with a variety of factors including type, species, age, weight sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed.
• An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the dmg required to prevent, counter, or arrest the progress of the condition.
• Example 1 Preparation of composition of CHIR99021, valproic mid, poloxamer 407, andDMSO.
• CHIR99021 To prepare 1 ml of gel, 25 m ⁇ of CHIR99021 solution and 25 m ⁇ of DMSO w3 ⁇ 4re added to the poloxamer 407 solution that contains valproic add. CHIR99021 may come out of solution and the mixture may be incubated at 37 °C to re-dissolve CHIR99Q2L and then cooled to abort 4 °C to fonri a flowable mixture.
• Valproic add means sodium valproate in this example.
• Example 2 Preparation and stability analysis i composition ofCHlR99021, sodium valproate , pdoxamer 407, and DMSO for fyopUlization.
• Example 3 Lyophilization of composition of CHIR99021, sodium valproate ⁇ , poloxamer 407, and DMSO, and stability amdysis of the lyophilized conpositimi
• the temperature was then slowly increased to 15 °C (at a rate of 0.5 °C per minute).
• Tire temperature was held at 15 °C for 20 hours under a vacuum of 80 mTorr.
• the glass vial s were stoppered under nitrogen and vacuum, and then the vacuum was then released completely while backfilling the lyophilizer with nitrogen.
• the glass vials were removed from the lyophilizer, capped, and crimped in a sterile environment.
• the 5 ml glass vials containing individual doses of formulated cake may be stared at -20 °C until use.
• Example 4 Fonmdation andsfabitiiy analysis of injection dosages from lyopki!ized composition of CH1R9902I, sodiumvalproate, pohxamer 407, and DMSO.
• Formulation of Dosage 1 6.4 grams of DMSO was added to a beaker containing 93.6 grams of purified water. The mixture was stirred for 3-5 minutes until homogeneous.
• the solution was sparged with nitrogen for 1 -2 minutes, and was then sterile filtered into a clean container using PES 0.2 urn filter and 10-ml syringe 0.85 ml of the filtered solution was added to the lyophiiized composition (Example 3) in the 5 ml vial, and the mixture was held at 2-8 °C for 20 minutes or until a clear solution was formed.
• Formulation cf Dosage 4 6.47 grams of DMSO was added to a beaker containing 92.35 grams of purified water and 1.18 grams of benzyl alcohol. The mixture was stirred for 3-5 minutes until homogeneous. The solution was sparged with nitrogen for 1 -2 minutes, and was then sterile filtered into a clean container using poiyethersulfone (PES) 0.2 um filter and 10-ml syringe. 085 ml of the filtered solution was added to the lyophiiized compositi on (Example 3) in the 5 ml vial, and the mixture was hel d at 2-8 °C for 20 minutes or until a clear ⁇ solution was fanned.
• PES poiyethersulfone
• Dosage 1 are based on observed amounts from three batches of lyophiiized product and reconstituted product ready for dosing.
• the ranges for Dosages 2 and 3 are based on observed amounts from two batches of lyophiiized product and reconstituted product ready for dosing.
• mice with SNHL were deafened using an established method where mice were exposed to 8-16 kHz octave band noise for 2 hairs at>l 16 dB (Wang et a!, 2002). This model was shown to cause immediate and extensive hair cell loss, but also to cause damage to other structures, such as the lateral wall, supporting cells, and spiral ganglion, all of which could limit the extent of possible hearing recovery (Wang et al, 2002).
• ABRs Auditory brainstem responses
• DPO AEs Distortion product octoacoustic emissions
• CHIR99021 and VTA were delivered locally by intra-tympanic inj ecdon into the middle ear using a pulled glass pipette that mimics the standard clinical middle ear injection technique.
• Tire deli very' vehicle w3 ⁇ 4s adapted from previous work using thermo-reversible poloxamer gels to deliver drugs into the middle ear for diffusion into the cochlea (Salt et al,
• mice were administered 10 uL of a composition containing 87.6mg ml NaVPA (527mM) and 1 39mg / ml CHIR99021 (approximately 3mM). Using established techniques (ITirose et al, 2014), perilymph was sampled from 7 animals and analyzed using mass spectrometry to determine entry' of CHIR99021 and VPA into the cochlea.
• total hair cell total hair cell
• IHC inner hair cell
• OHC outer hair ceil
• Tlii s in vivo study showed that the components of the composition, CHIR99021 and VPA, improved auditory thresholds and restored hair cells in a mouse model of noise-induced hearing loss.
• Example 8 Treatment with reconstituted lyophHizedtest compositions containing CH1B99021, Na VPA and pokmimer leads to improvement in hearing in an animal model
• CHIR99021 in an animal efficacy model were evaluated.
• Tone bursts 5 ms in duration (0.5 ms cos 2 R/F) were presented 500-1000 times at 20/s in descending intensity' using a 5 dB minimum step size until wave I of the ABR could no longer be visually discerned. The stimulus level was then increased until the response re-appeared. Recording utilized Biosig32 and TDT hardware. ABR thresholds were obtained at 5, 10, 20, 283, and 40 kHz, 24h and 5 weeks after post-exposure by an operator blinded to experimental treatment. ABRs were plotted and reported using standard error. Comparisons from 24h to 5wks, both within and across groups, were compared using a two-tailed t-test.
• Intratympanic inj ections were performed after ABR testing, 24 hrs after noise exposure.
• the therapeutic composition was maintained at a cold temperature in order reduce viscosity and allow' loading of the syringe and injection through the pipette.
• 1 ml allotments of the composition 1 ml allotments of therapeutic agents dissolved in cold Poloxamer 407 plus Evans blue were drawn into a 1 ml disposable syringe to which 34 cm of polyethylene plastic tubing was attached.
• the tubing was sized to fit snuggly to a glass 1.5 mm OD mierocapillary tube (WPI), pulled to a fine point in a custom pipette puller and broken with forceps to a tip width of 40-60 pm.
• WPI glass 1.5 mm OD mierocapillary tube
• the assembly was kept in the dark at 4 °C until use.
• the pulled pipette was attached to a micro-positioner.
• Mice were injected with ketamine/xylazine (8Q/15 mg/kg) and positioned ventral!y in a custom head-holder.
• the cartilage ring at the base of the right pinna was slightly expanded by a bloodless cut on the ventral side and a retractor and forceps were used to reveal a full view of the pinna.
• the positioner-tubing assembly was then used to make two holes in the tympanic membrane just large enough to admit the pipette. The first hole served as a vent hole and was made just anterior to the umbo.
• the pipette was aimed at the posterior margin of the tympanic membrane, just ventral to the incus (pars flacida). Leaving the pipette in place, the middle ear was filled slowly by depressing the syringe, from postero-dorsai to antero-ventral until excess Poloxamer began to emerge from the vent hole. Topical Lidocaine was then applied, and mice were aliow3 ⁇ 4d to recover under a warming lamp. Mice show normal acti vity levels within 24 hrs and no middle ear infections or surgical complications.
• mice were deeply anesthetized using pentobarbital and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer. Cochleae were then removed and placed in the same fixative for 24 hrs, after which thev were transferred to 0.12 M sodium EDTA and stored at 4 °C for later processing.
• Figure s shows the results of a NaVP A + CH1R99021 solution with a low concentration of
• Figure 4 shows the results of a NaVPA + CH0199021 solution with an isotonic concentration of
• NaVPA e.g. see Example 6 however the poloxamer is omitted. As seal from figure 4, a significant improvement in thresholds is seen at all frequencies.
• Figure 3 shows the results of a reconstituted test composition (e.g. see Example 10). As seen from figure 3, a significant improvement in thresholds is seen at 20kHz and 28.3kHz. This validates that reconstituting lyophilized compositions comprising NaVPA CH1R99021 and Poloxamer 407 is a viable strategy for administering NaVPA and CHIR99Q21 since the effects on hearing improvement are similar to administering
• test compositions in this example contain P4Q7 at -15.5% w/v% (e.g. see Table 34) and can be made following the method of Example
• Example 10 Preparation and fyopMUz fi don of a composition of CHIR99021, sodium valproate, polm mer 407, and DMSQ for injection (test composition) (GMP).
• Step 1 6453 g of water for injection (WFI) was added to a 20 L jacketed formulation vessel. The temperature of the water was controlled between 24 °C using the jacketed vessel assembly. 1322 g of P407 was added to the chilled water in portions while stirring the solution at 300 rprn using an overhead stirrer for ⁇ initial mixing (speed was adjusted to ensure no frothing while mixing). The temperature of P407 in water was maintained cold to ensure that the solution was free flowing during the actual compounding step although at this step, temperature control was not critical. The stock solution of P407 was then used in the next steps of compounding the test composition.
• WFI water for injection
• Step 2 7G9g of solid sodium valproate was weighed and added in small portions, totheP407 aqueous solution from step 1 above, while maintaining temperature of the solution to 2-8 °C and continuously stirring the solution using an overhead stirrer. Tire mixing speed was adjusted to ensure no frothing while mixing. The required solution temperature was achieved by setting the temperature of the jacketed vessel to 15 °C. Mixing was completed in 60 minutes.
• Step 3 26.3 g ofCHIR99021 was weighed and added to pre-warmed (at 32-35 °C) 407 g of
• Step 4 The dear solution obtained in Step 4 was then diluted by addition of 8917.4 g of WFI at 2-8
• Step S The diluted solution was filtered using a polyethersulfone (PES) membrane based standard Sartopore® 2, 0.2 pm pore size, 1000 cm 2 capsule filter under aseptic conditions.
• Step 6 Filling glass vials with individual doses: A tray of sterile glass vials and sterile stoppers were transferred in a sterile environment. For each 5 mL glass vial, 2.2 grams of sterile poloxamer 407, sodium valproate, and CHIR99021 solution was dispensed as an individual dose. The dispense was performed using filling machine. The stoppers were then partially inserted into the necks of each vial aseptically. The composition forms a gel at about 37 °C.
• Steps 1 -6 take about 12 hours to complete.
• Step ? Lyophilization of poloxamer 407, sodium valproate, CHR99021 , and DMSO solution:
• the tray of filled glass vials was placed into a lyophilizer in a sterile environment Tire temperature in the lyophi!izer was slowly reduced to -45 °C (at a rate of 0.5 C' C per minute) and then held at -45 °C for 3 hours. A vacuum of 80 mTorr was applied to the lyophilizer. The temperature was then slowly increased to -30 °C (at a rate of 0.5 °C per minute) and then held at -30 °C fix 15 hours under a vacuum of 80 mTorr. The temperature was then slowly increased to 15 °C (at a rate of 0.5 °C per minute). The temperature was held at 15 °C for 20 hours under a vacuum of 80 mTorr.
• the glass vial s were stoppered under nitrogen and vacuum, and then the vacuum was then released completely write backfilling the lyophilizer with nitrogen.
• the glass vials were removal from the lyophilizer, capped, and crimped in a sterile environment.
• the 5 ml glass vials containing individual doses of the cake test composition may be stored at -20 °C until use.
• i t is important to use an appropriate lyophilization eyrie that consists of suitable temperature and suitable vacuum pressure used during the drying process (e.g. see step 7).
• the dried mass can result in a flat sheet e.g. see Figure 8.
• the flat sheet visible in Figure 9 was generated by freezing the “wet” test composition (i.e. the product from step 6) in a sample vial with liquid nitrcgen. The sample was then lyophilized by drying at room temperature raider a vacuum pressure at 400mTorr.
• the test composition produced using a suitable lyophilization cycle, such as outlined in Step 7 above, produces a lyophilized cake as shown in Figure 9.
• Table 26 shows the results when CHIR99021 is added at different stages in the manufacture of the test composition. Table 26: order of addition of components in the test composition
• a CHIR99021 stock solution was prepared at the given concentration in DMSO because the solubility of CFHR99021 is very limited in almost all organic solvents except polar solvents, such as DMSO.
• the first step in the sequence of compounding was to add pre-weighed solid sodium valproate to the stock P407 aqueous solution over ice. The temperature of P407 solution is important to keep the viscosity of this thennoreversible polymer in a liquid free-flowing state. After a clear solution was obtained, the CHIR99021 -DM SO stock solution was added slowly to the NaVPA-P4Q7-water solution.
• CT1IR99021-DMSO The slow addition of CT1IR99021-DMSO was necessary to avoid precipitation of CHIR99021ffom the solution.
• the Cl 11R99021 - DMSO stock solution is added after the NaVPA has dissolved to minimize the time that CH1R99021 is in solution. It has been established that for the test composition, compounding must be completed in under 12 hours otherwise CFHR9902I can begin to precipitate out of solution. CHIR99021 also begins to degrade after extended periods of time in solution.
• Step 1 To a 151, jacketed vessel, water for injection
• Step 2 The sparged solution was then sterile filtered using PES membrane based standard Sartopore® 2, 0.2 pm pore size, 1000 cm 2 capsule fil ter under aseptic conditions.
• Step 3 The filtered solution was filled aseptica!ly into 3 ⁇ rnL sterile glass vials, stoppered using sterile Teflon faced rubber stoppers and crimp sealed with aluminum seals to obtain sterile Diluent
• the Diluent can be used in the following reconstitution procedures.
• Diluent 1 0.85 mL + lyophilized cake. Using a syringe, add 0.85 mL to the lyophilized cake of the test composition, and rest in a fridge or ice hath (2-8 °C) fix 20 minutes or until clear solution is formed. Gentle tapping on the exterior of the vessel to help the cake dissolve in the Diluent may be required while keeping it in fridge (or ice bath). To avoid degradation of the composition, minimize stirring and/or vortexing.
• Placebo composition [0691] Placebo composition:
• Diluent 1, 0.95 mL + Placebo cake Using a syringe, add the Diluent to the Placebo cake, and rest in a fridge or ice bath (2 ⁇ 8°C) for 60 minutes or until clear solution is formed. Gentle tapping on the exterior of the vessel to help the cake dissolve in the Diluent may be required while keeping it in fridge (or ice bath). To avoid degradation of the composition, minimize stirring and/or vortexing.
• Example 13 Stability qfNon-Lyophiliml Compositions Versus Stability of lyophilized Compositions
• The“wet” test composition i e. non-lyophilized - see Example 10, steps 1-6
• tire lyophilized composition has been tested for stability for 6 months at refrigerated conditions and for 2 years at -20 C' C storage, and remains stable.
• The“wet” test composition will freeze at -20 °C which negatively impacts the test composition and so the stability of the frozen composition was not studied).
• test composition was compounded fresh (see steps 1-6 of Example 10), stored in the refrigerator and tested at time intervals of 0, 5, 24, 48, 54, 120 hours for NaVPA and CHIR99Q21 content and appearance. See Figures 10 and 11 for the results.
• the assay level of CHER99021 decreased if cm its initial level over time, while the assay level ofNaVPA remained constant over this time.
• the solution of the test composition developed precipitate and solution turned hazy somewhere between 24 and 48 hours and then developed precipitate after 120 hours of solution storage under refrigerated conditions.
• the“wet” test composition CH1R99021 and NaVPA is not stable during storage.
• the ly ophilized test composition was reconstituted with 0.85 mL of Diluent under refrigeration for approximately 30 minutes.
• the reconstituted solution was stored in the refrigerator and tested at time intervals of 0, 1, 2, 6, 8, and 24 hours for NaVPA and CHIR99021 drug content and appearance.
• NaVPA and CHIR99021 assay levels within the reconstituted test composition were stored inside polypropylene syringes and kept refrigerated remained stable with initial levels for 6 hours as shown in Figure 12. Sometime between 8 and 24 hours, the assay level of CHIR99021 decreased.37% from its initial level, while the assay level ofNaVPA remained constant over this time.
• the lyophilized test composition was studied for storage stability.
• the lyophilized composition was stored in a glass container with rubber closure and crimp seal under refrigeration for 6 months and at -20 °C for 24 months.
• the lyophilized test compositi on was tested for drug content. It is sear that the lyophilized test composition remains stable (without signs of decomposition) for at least 6 months under refrigeration and 24 months at -20 C' C as shown below.
• Table 27 the lyophilized composition stability data for GMP lot B 17030018.
• Example 14 Reduced Reconstitution Time qfLyophili l Poloxamer.
• a Lyophiiized test composition was prepared according to Examples 2 and 3.
• Lyophilized P407 was prepared according to Examples 2 and 3 ; however, the steps of adding NaVPA and CHIR99021 were omitted.
• Powder P407 i.e. non-lyophilized
• BASF BASF
• Table E shows the reconstitution time (or dissolution time in the case of powdered P407) using 850
• the lyophilized test composition reconstitutes within 20 minutes of the addition of Diluent.
• the test composition has a poloxamer (P407) concentration of 16.15 %w/v and a sodium valproate concentration of 533 25mM. Therefore, to probe the effect of salts other than sodium valproate on reconstitution time, a poloxamer solution at 16.15% concentration either al one or including a salt (e.g. an inorganic salt or organic add salt) at a concentration of 533.25mM, was lyophilized. Tire lyophilized material was then reconstituted using Diluent for reconstitution (6.4% DMSO by wt.% in water) or with water. The results are displayed in Table 28.
• a salt e.g. an inorganic salt or organic add salt
• the reconstitution time oflyophilized poloxamer is largely improved due to the addition of organic add salts such as Sodium valproate, Magnesi um divalproate (magnesi um valproate), Sodi um 2-(prcjp-2-yn- 1 - yl) octanoate (also refeired to as the sodium salt of 2-hexyl-4-pentyndc add) and is found to be 4-20 min.
• organic add salts such as Sodium valproate, Magnesi um divalproate (magnesi um valproate), Sodi um 2-(prcjp-2-yn- 1 - yl) octanoate (also refeired to as the sodium salt of 2-hexyl-4-pentyndc add) and is found to be 4-20 min.
• Purification of Poloxamer 407 refers to the removal of residual smaller chains of polymer, such as monomers and dirnens, therefore providing more consistent gelation properties.
• P407 was purified accordance with a published procedure: A. Fakhari, M Corcoran, A Schwarz, liiennoge!ling Properties of Purified Poloxamer 407, Heliyon (2017), 3(8), e00390.
• unpurified P407 means that no purification methods have been performed.
• purified means that some forni of purification method has been performed.
• unpurified P407 is material obtained directly from the supplier and used without any further manipulation.
• Poloxamer 407 was characterized using two HPLC methods: RPLC-CAD (reverse phase liquid chromatography with a corona charged aerosol detector) and SEC-CAD (size exclusion chromatography with the same corona CAD detector). Poloxamer 407 does not have a chromophore and so it is undetectable by traditional ultraviolet (LA' 1 ) detectors.
• the CAD detector is a mass sensitive detector that wcrics in a manner similar to a mass spectrometer detector.
• SEC Size exclusion chromatography

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