WO2015181756A1 - Chloride salt of tat-nr2b9c - Google Patents

Chloride salt of tat-nr2b9c Download PDF

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Publication number
WO2015181756A1
WO2015181756A1 PCT/IB2015/053995 IB2015053995W WO2015181756A1 WO 2015181756 A1 WO2015181756 A1 WO 2015181756A1 IB 2015053995 W IB2015053995 W IB 2015053995W WO 2015181756 A1 WO2015181756 A1 WO 2015181756A1
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WO
WIPO (PCT)
Prior art keywords
formulation
tat
nr2b9c
concentration
trehalose
Prior art date
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PCT/IB2015/053995
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English (en)
French (fr)
Inventor
Jonathan David Garman
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NoNO Inc
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NoNO Inc
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Publication date
Priority to RU2016150678A priority Critical patent/RU2707885C2/ru
Priority to AU2015265487A priority patent/AU2015265487B2/en
Priority to CN201580028269.3A priority patent/CN106661126B/zh
Priority to KR1020217031792A priority patent/KR102407856B1/ko
Priority to HRP20200462TT priority patent/HRP20200462T1/hr
Priority to DK15799583.8T priority patent/DK3149048T3/da
Priority to EP20152821.3A priority patent/EP3757134B1/en
Priority to BR112016027814A priority patent/BR112016027814A2/pt
Priority to US15/313,940 priority patent/US10206973B2/en
Priority to MEP-2020-120A priority patent/ME03790B/me
Priority to MX2016015457A priority patent/MX374958B/es
Priority to EP15799583.8A priority patent/EP3149048B1/en
Priority to KR1020227019189A priority patent/KR102605540B1/ko
Priority to JP2016569655A priority patent/JP6622222B2/ja
Priority to SI201531132T priority patent/SI3149048T1/sl
Priority to PL15799583T priority patent/PL3149048T3/pl
Priority to CA2950395A priority patent/CA2950395C/en
Application filed by NoNO Inc filed Critical NoNO Inc
Priority to LTEP15799583.8T priority patent/LT3149048T/lt
Priority to ES15799583T priority patent/ES2784500T3/es
Priority to UAA201613470A priority patent/UA123820C2/uk
Priority to SM20200307T priority patent/SMT202000307T1/it
Priority to KR1020167036752A priority patent/KR102311038B1/ko
Priority to RS20200367A priority patent/RS60625B1/sr
Priority to SG11201609911YA priority patent/SG11201609911YA/en
Publication of WO2015181756A1 publication Critical patent/WO2015181756A1/en
Priority to IL249252A priority patent/IL249252B/en
Anticipated expiration legal-status Critical
Priority to ZA2016/08843A priority patent/ZA201608843B/en
Priority to US16/128,423 priority patent/US10758589B2/en
Priority to CY20201100251T priority patent/CY1122793T1/el
Priority to US16/904,230 priority patent/US11266714B2/en
Priority to AU2020267304A priority patent/AU2020267304C1/en
Priority to US17/545,995 priority patent/US11911438B2/en
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • 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
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    • 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
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    • A61P9/00—Drugs for disorders of the cardiovascular system
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus human T-cell leukaemia-lymphoma virus
    • C07K14/155—Lentiviridae, e.g. human immunodeficiency virus [HIV], visna-maedi virus or equine infectious anaemia virus
    • C07K14/16—HIV-1 ; HIV-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus human T-cell leukaemia-lymphoma virus
    • C07K14/155—Lentiviridae, e.g. human immunodeficiency virus [HIV], visna-maedi virus or equine infectious anaemia virus
    • C07K14/16—HIV-1 ; HIV-2
    • C07K14/163—Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Tins application is a non-pro visional of US 62/004,142, filed May 28, 2014, incorporated by reference in its entirety for all purposes.
  • Tat-NR2B9c (also known as NA-1) is an agent that inhibits PSD-95, thus disrupting binding to N-methyl-D-aspartate receptors (NMDARs) and neuronal nitric oxide synthases (riNOS) and reducing excitoxicity induced by cerebral ischemia. Treatment reduces infarction size and functional deficits.
  • TAT-NR2B9c has undergone a successful phase II trial (see WO 2010144721 and Aarts et al., Science 298, 846-850 (2002), Hill et al, Lancet Neurol, 11 :942 - 950 (2012)).
  • TAT-NR2B9c is free of serious side effects, it can be administered when stroke or other ischemic conditions or hemorrhagic conditions is suspected without a diagnosis according to art-recognized criteria having been made to confirm that no hemorrhage is present.
  • TAT-NR2B9c can be administered at the location where the stroke or neuro trauma has occurred (e.g., in the patients' home) or in an ambulance transporting a subject to a hospital.
  • TAT-NR2B9c has previously been described as a liquid composition of normal saline or phosphate buffered saline or a lyophilized composition from normal saline (WO2010144721 ).
  • the invention provides a chloride salt of a peptide which is TAT-NR2B9c (SEQ ID NO: 6) or differs from TAT-NR2B9c by up to 5 amino acid substitutions, insertions or deletions, or any other peptide disclosed as an active agent herein.
  • the chloride salt can be prepared by exchanging tnfluoroacetate for chloride in a tnfluoroacetate salt of TAT-NR2B9c.
  • the chloride salt can also be prepared by exchanging tnfluoroacetate for acetate and then acetate for chloride staring from a trifluoroacetate salt of TAT-NR2B9c.
  • greater than 99% of anions in the salt are chloride.
  • the invention further provides a prelyophilized formulation comprising a chloride salt as described above a buffer and a sugar.
  • the chloride salt is a chloride salt of TAT- NR2B9c.
  • the buffer is histidine and the sugar is trehalose and the pH is 6-7.
  • acetate and trifluoroacetate each comprise less than 1% of anions by weight in the formulation.
  • acetate and trifluoroacetate each comprise less than 0.1% by weight of anions in the formuiation.
  • the chloride salt of the peptide is at a concentration of 70- 120 mg/ml
  • the histidine is at a concentration of 15-100 mM
  • the trehalose is at a
  • the chloride salt of the peptide is at a concentration of 70-120 mg/ml
  • the histidine is at a concentration of 20-100 mM
  • the trehalose is at a concentration of 100-140 mM.
  • the Tat-N 2B9c is at a concentration of 70-120 mg/ml
  • the concentration of trehalose is 100-140 mM.
  • the concentration of histidine is 20 mM and the concentration of trehalose is 100-200 mM, preferably 120 mM and the concentration of TAT-N 2B9c is 90 mg/ml.
  • the invention further provides a lyophilized formulation prepared by lyophilizing any of the prelyophilized formulations described above.
  • acetate and trifluoroacetate each comprise less than 1 % by weight of anions in the formulation.
  • acetate and trifluoroacetate each comprise less than 0.1% by weight of anions in the formulation.
  • the invention further provides a reconstituted formulation prepared by combining any of the lyophilized formulations described above with an aqueous solution.
  • the aqueous solution is water or normal saline.
  • the volume of the reconstituted formulation is 3-6 times the volume of the prelyophilized formulation.
  • the invention further provides a reconstituted formulation comprising TAT-NR2B9c or other active agent described herein at concentration of 15-25 mg/ml, a buffer and a sugar.
  • the buffer is histidine at a concentration of 4-20 mM and the sugar is trehalose at a concentration of 20-30 mM and the pH is 6-7.
  • acetate and trifluoroacetate each comprise less than 0.1% by weight of anions in the formulation.
  • the invention further provides a method of preparing a formulation, comprising storing a Ivophilized formulation sample as described above for at least a week at a temperature of at least 20°C; and reconstituting the lyophilized formulation.
  • the Ivophilized formulation is reconstituted in water or saline.
  • the method also includes
  • the formulation is stored for at least a year.
  • the storage is at ambient temperature.
  • the storage includes periods in which the temperature exceeds 37°C.
  • the patient has stroke or traumatic injury to the CNS.
  • the lyophilized sample is stored in an ambulance.
  • the patient has a
  • the patient is undergoing endovascular repair for an aneurysm.
  • Figure 1 Graph shows the infarct area of the rat brain after 3PVO stroke following treatment with different formulations of TAT-NR2B9c.
  • Figures 2A, B A) Bar graph demonstrating the stability of different TAT-NR2B9c formulations at -20°C and 40°C.
  • Y axis represents purity of the TAT-NR2B9c after I week at the storage temperature as measured by % total area using RP-HPLC.
  • Figure 3 Bar graph demonstrating the stability (by HPLC) of 20 rng/ml TAT-NR2B9c in Histidine buffer, pH 6.5, in the presence of different bulking agents and salt at -20oC and 40oC.
  • Figures 4 A, B Differential scanning calorimetry graphs of 20 mg/rnl TAT-NR2B9c in histidine buffer pH 6.5 in the presence of Mannitol (A) or Mannitol and NaCl (B).
  • Figures 5 A, B Differential scanning calorimetry graphs of 20 mg/ml TAT-NR2B9c in histidine buffer pH 6.5 in the presence of Trehalose (A) or Trehalose and NaCl (B).
  • Figures 6 A, B Differential scanning calorimetry graph of 20 mg/rnl TAT-NR2B9e in histidine buffer pH 6.5 in the presence of Dextran-40 (A) or Dextran-40 and NaCl (B).
  • Figures 7A, B A) Cake appearance following lyophilization of 3 mL of 90 mg/ml TAT- N 2B9c in 100 mM Histidine pH 6.5 with 120 mM Trehalose.
  • B Cake appearance of alternative TAT-NR2B9c formulations with different amounts of histidine and trehalose.
  • lyophilized formulations can include one or more of the following classes of components.
  • the classes are not mutually exclusive: in other words the same agent can component can fall within multiple classes.
  • a "bulking agent” provides structure to a freeze-dried peptide.
  • Bulking agents include, mannitol, trehalose, dextran-40, glycine, lactose, sorbitol, and sucrose among others.
  • bulking agents may also impart useful qualities in regard to modifying the collapse temperature, providing freeze-thaw protection, glass transition temperature and enhancing the protein stability over long-term storage. These agents can also serve as tonicity modifiers.
  • a buffer is an agent that maintains the solution pH in an acceptable range prior to lyophilization.
  • a preferred buffer is histidine.
  • Other buffers include succinate (sodium or potassium), histidine, citrate (sodium), gluconate, acetate, phosphate, Tris and the like.
  • Preferred buffers are effective in a pH range from about 5.5 to about 7 or about 6 to about 7; preferably a pH of about 6.5.
  • Examples of buffers that control the pH in this range include succinate (such as sodium succinate), gluconate, histidine, citrate and other organic acid buffers.
  • a "cryoprotectant” provides stability to a peptide against freezing-induced stresses, presumably by being preferentially excluded from the protein surface. It may also offer protection during primary and secondary drying, and long-term product storage.
  • examples are polymers such as dextran and polyethylene glycol; sugars (including sugar alcohols) such as sucrose, glucose, trehalose, and lactose; and surfactants such as polysorbates; and amino acids such as glycine, argmine, and serine.
  • a lyoprotectant provides stability to the peptide during the drying or " dehydration " process (primary and secondary drying cycles), presumably by providing an amorphous glassy matrix and by binding with the protein through hydrogen bonding, replacing the water molecules that are removed during the drying process. This helps to maintain the peptide conformation, minimize peptide degradation during the lyophi zation cycle and improve the long-term product stability.
  • Examples include polyols or sugars such as sucrose and trehalose.
  • deamidation inhibitors include fatty acid esters of sorbitan polyethoxylates (e.g., polysorbate 20 or poiysorbate 80), poioxamer 188, and detergents.
  • surfactants some common ones are fatty acid esters of sorbitan polyethoxylates (e.g., polysorbate 20 or poiysorbate 80), poioxamer 188, and detergents.
  • lyophilization refers to a process by which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment.
  • a "pharmaceutical formulation” or composition is a preparation that permits an active agent to be effective, and lacks additional components which are toxic to the subjects to which the formulation would be administered.
  • Reconstitution time is the time that is required to rehydrate a Iyophilized formulation with a solution to solution which is free of particles to the naked eye.
  • a "stable" Iyophilized peptide formulation is one with no significant changes observed at 20° C for at least one week, month, or more preferably at least three months, at least six months or a year. Changes are considered insignificant if no more than 10%, preferably 5%, of peptide is degraded as measured by SEC-HPLC.
  • the rehvdrated solution is colorless, or clear to slightly opalescent by visual analysis.
  • the concentration, pH and osmolality of the formulation have no more than +/-10% change after storage. Potency is within 70-130%, preferably 80-120% or sometimes 80-100% of a freshly prepared control sample. No more than 0%, preferably 5% of clipping is observed.
  • Stability can be measured by various methods reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N. Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993).
  • the term "isotonic" means that the formulation of interest has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 270-328 mOsm. Slightly hypotonic pressure is 250-269 and slightly hypertonic pressure is 328-350 mOsm. Osmotic pressure can be measured, for example, using a vapor pressure or ice-freezing type osmometer. [0030] Tonicity Modifiers: Salts (NaCl, KC1, MgCl 2 , CaCl 2 ) can be used as tonicity modifiers to control osmotic pressure. In addition, cryprotecants/lyoprotectants and/ or bulking agents such as sucrose, mannitol, or glycine can serve as tonicity modifiers.
  • Peptides synthesized by solid state methods are typically produced as trifluoroacetate salts because trifluoroacetic acid (TFA) is used for deprotection of peptides and/or removal of peptides from resins.
  • TFA trifluoroacetic acid
  • the trifluoroacetate is usually replaced with acetate as a counterion because acetate is nontoxic and the replacement of trifluoroacetate by acetate is straightforward.
  • TAT-NR2B9c synthesized to- date as described in WO2010144721 or PCT/US2013/071755 and elsewhere.
  • Acetate is often a preferred counterion for pharmaceutically peptides because trifluoroacetate can be exchanged for acetate with a minor change in the typical purification process of a peptide resulting from solid phase synthesis in which the final wash is performed with acetic acid instead of trifluoroacetic acid followed by elution with acetonitrile.
  • the present invention provides lyophilized formulations of active agents, particularly of TAT-NR2B9c as a chloride salt. Such formulations are stable at ambient temperature (e.g., at least 20°C) thus facilitating maintenance of supplies of such a formulation in ambulances or the like or with emergency personnel for administration at the scene of illness or accident or between such scene and a medical facility.
  • Lyophilized formulations are prepared from a prelyophilized formulation comprising an active agent, a buffer, a bulking agent and water. Other components, such as cryo or
  • lyopreservatives a tonicity agent pharmaceutically acceptable carriers and the like may or may ⁇ be present.
  • a preferred active agent is a chloride salt of TAT-NR2B9c.
  • a preferred buffer is histidine.
  • a preferred bulking agent is trehalose. Trehalose also serves as a cryo and lyo- preservative.
  • An exemplary prelyophilized formulation comprises the active agent (e.g., chloride salt of TAT-NR2B9c), histidine (10-100 mM, 1 5-100 rnM 15-80 mM, 40-60 raM or 15- 60 mM, for example, 20 mM or optionally 50 mM, or 20-50mM)) and trehalose (50-200 mM, preferably 80-160 mM, 100-140 mM, more preferably 120 mM).
  • the pH is 5.5 to 7.5, more preferably, 6-7, more preferably 6.5.
  • the concentration of active agent is 20-200 mg/'ml, preferably 50-150 rng/ml, more preferably 70-120 rng/ml or 90 mg/'ml.
  • an exemplary prelyophilized formulation is 20 mM histidine, 120 raM trehalose, and 90 mg/ ' ml chloride salt of TAT-NR2B9c.
  • an acetylation scavenger such as lysine can be included, as described in co-pending application 057769-446850, to further reduce any residual acetate or trifluoroacetate in the formulation
  • lyophilized formulations After lyophilization, lyophilized formulations have a low-water content, preferably from about 0%-5% water, more preferably below 2.5% water by weight. Lyophilized formulations can be stored in a freezer (e.g., -20 or -70° C), in a refrigerator (0-4° C) or at room temperature (20-25° C).
  • Active agents are reconstituted in an aqueous solution, preferably water for injection or optionally normal saline (0.8-1.0% saline and preferably 0.9% saline). Reconstitution can be to the same or a smaller or larger volume than the prelyophilized formulation. Preferably, the volume is larger post- reconstitution than before (e.g., 3-6 times larger).
  • a prelyophilization volume of 3-5 ml can be reconstituted as a volume of 10 mL, 12 mL, 13.5 ml, 15 mL or 20 mL or 10-20 mL among others.
  • the concentration of histidine is preferably 2-20 mM, e.g., 2-7 mM, 4.0-6.5 mM, 4.5mM or 6 mM;
  • the concentration of trehalose is preferably 15-45 mM or 20-40 mM or 25-27 mM or 35-37 mM.
  • the concentration of lysine is preferably 100-300 mM, e.g., 150-250mM, 150-170 mM or 210-220 mM.
  • the active agent is preferably at a concentration of 10-30 mg/ml, for example 15-30, 18-20, 20 mg/ml of active agent (e.g., TAT-NR2B9c) or 25-30, 26-28 or 27 mg/mL active agent.
  • active agent e.g., TAT-NR2B9c
  • An exemplary formulation after reconstitution has 4-5 mM histidine, 26-27 mM trehalose, 150-170 mM lysine and 20 mg/ml TAT-NR2B9c (with concentrations rounded to the nearest integer).
  • a second exemplary formulation after reconstitution has 5-7 mM histidine, 35-37 mM trehalose, 210-220 mM lysine and 26-28 mg/ml TAT-NR2B9c (with concentrations rounded to the nearest integer).
  • the reconstituted formulation can be further diluted before administration such as by adding into a fluid bag containing normal saline for intravenous infusion.
  • TAT-NR2B9c is preferably lyophiiized in the same vial as that in winch it will be reconstituted for use.
  • An aqueous solution of TAT-NR2B9c is added to the vial optionally after filtering through a sterilizing filtration system, such as a 0.22 micron filter standardly used for peptides.
  • Formulations can be lyophiiized in a controlled cycle, such as described in the Examples.
  • a prelyophilized formulation can be placed in a vial, and lyophiiized at reduced temperature and pressure. After lyophilization, vials can be sealed. For use, the lyophilizate is reconstituted with water for injection, normal saline or other pharmaceutically acceptable carrier or diluent.
  • a variety of containers are suitable for lyophilization.
  • a container should be able to withstand the outside pressure when the container is sealed and stored under partial vacuum.
  • the container should be made of a material that allows a reasonable transfer of heat from outside to inside.
  • the size of the container should be such that the solution to be lyophilized occupies not more than 20% of the useful volume or may be overfilled with an excess, in accord with then- prevailing USP recommendations for the volume in a container. For example, a 0.5 ml solution may be filled in a 3 ml vial.
  • the vials may be made of glass e.g. borosilicate, or plastic, e.g. polypropylene.
  • Glass bottles commonly used for lyophilizmg biological materials can be used.
  • Another suitable container is a two-compartment syringe wherein one compartment contains the lyophilized TAT-NR2B9c peptide cake and the other compartment contains the aqueous diluent.
  • the vacuum within the vials or ampules may be released by filling the system with an inert gas, stoppered in place using standard equipment and then crimp sealed. Such a method will ensure a sterile final product.
  • Other two-part solutions such as a bag with a breakable seal between the lyophilized drug compartment and the diluent can be used as well.
  • TAT-NR2B9c active agent
  • other active agents as described below can be prepared as chloride salts or formulated according to the principles described for TAT-NR2B9c. Specific concentrations given for TAT-NR2B9c can be used as is for other agents or converted to give equirnolar concentrations of the other agent and TAT-NR2B9c.
  • Active agents inhibit interaction between PSD-95 and one or more NMDARs (e.g., 2A, 2B, 2C or 2D) or nNOS (e.g., Swiss-Prot P29475) by binding to PSD-95.
  • NMDARs e.g., 2A, 2B, 2C or 2D
  • nNOS e.g., Swiss-Prot P29475
  • Such agents are useful for reducing one or more damaging effects of stroke and other neurological conditions mediated at least in part by NMDAR excitotoxicity.
  • Such agents include peptides having an amino acid sequence including or based on the PL motif of a NMD A Receptor or PDZ domain of PSD-95.
  • Such peptides can also inhibit interactions between PSD-95 and nNOS and other glutamate receptors (e.g., kainite receptors or AMPA receptors), such as KV1.4 and GluR6.
  • Preferred peptides inhibit interaction between PDZ domains 1 and 2 of postsynaptic density-95 protein (PSD-95)(human ammo acid sequence provided by Stathakism, Genomics 44(l):71-82 (1997)) and the C-terminal PL sequence of one or more NMD A Receptor 2 subunits including the NR2B subunit of the neuronal N-methyl-D-aspartate receptor (Mandich et al, Genomics 22, 216-8 (1994)).
  • NMDAR2B has GenBank ID 4099612, a C-terminal 20 ammo acids
  • FNGSSNGHVYEKLSSIESDV (SEQ I D NO: 11) and a PL motif ESDV (SEQ ID NO: 12).
  • Preferred peptides inhibit the human forms of PSD-95 and human NMDAR receptors. However, inhibition can also be shown from species variants of the proteins. A list of NMD A and giutaniate receptors that can be used appears below:
  • Peptides can include or be based on a PL motif from the C-terminus of any of the above subunits and have an amino acid sequence comprising [S/'T]-X-[V'L.]. This sequence preferably occurs at the C-terminus of the peptides of the invention.
  • Preferred peptides have an amino acid sequence comprising i L D N Q j-i S Tj-i D I. Q N j ⁇ i V L
  • Exemplary peptides comprise: ESDV (SEQ ID NO: 12), ESEV (SEQ ID NO:29), ETDV (SEQ ID NO: 9).
  • ETEV SEQ ID NO:40
  • DTDV SEQ ID NO:41
  • DTEV SEQ ID NO:42
  • DTEV SEQ ID NO:42
  • Two particularly preferred peptides are KLSSIESDV (SEQ ID NO: 5), and KLSSIETDV (SEQ ID NO:43).
  • Such peptides usually have 3-25 amino acids (without an internalization peptide), peptide lengths of 5-10 ammo acids, and particularly 9 annno acids (also without an internalization peptide) are preferred. In some such peptides, all amino acids are from the C -terminus of an NMD A receptor (not including amino acids from an internalization peptide).
  • Peptides and peptidomimetics of the invention can contain modified ammo acid residues for example, residues that are N-alkylated.
  • N- terminal alkyl modifications can include e.g., N- Methyl, N-Ethyi, N-Propyl, N-Butyl, N-Cyeiohexyimethyi, N-Cyclyhexylethyl, N-Benzyl, N- Phenylethyl, N-phenylpropyl, N-(3, 4-Dichlorophenyl)propyl, N-(3,4-Difluorophenyl)propyl, and N-(Naphthalene-2-yl)ethyl).
  • NR2B9c SEQ ID NO: 6
  • PDZ-binding activity is exhibited by peptides having only three C-terminal amino acids (SDV).
  • Bach also reports analogs having an amino acid sequence comprising or consisting of XitSX 2 V (SEQ ID NO: 68), wherein t and S are alternative amino acids, Xj is selected from among E, Q, and A, or an analogue thereof, X 2 is selected from among A, Q, D, N, N-Me-A, N-Me-Q, N-Me-D, and N-Me-N or an analog thereof.
  • the peptide is N-alkylated in the P3 position (third amino acid from C-terminus, i.e. position occupied by tS).
  • the peptide can be N-alkylated with a cyclohexane or aromatic substituent, and further comprises a spacer group between the substituent and the terminal amino group of the peptide or peptide analogue, wherein the spacer is an alkyl group, preferably selected from among methylene, ethylene, propylene and butylene.
  • the aromatic substituent can be a naphthal en-2-yl moiety or an aromatic ring substituted with one or two halogen and/or alky] group.
  • Peptidoniimetics also include retro peptides.
  • a retro peptide has a reverse amino acid sequence.
  • Peptidoniimetics also include retro inverse peptides in which the order of ammo acids is reversed from so the originally C-terminal amino acid appears at the N-terminus and D-amino acids are used in place of L-amino acids.
  • WO 2008/014917 describes a retro-inverso analog of Tat-NR2B9c having the amino acid sequence vdseisslk-rrrqrrkkrgyin (SEQ ID NO: 69) (lower case letters indicating D amino acids), and reports it to be effective inhibiting cerebral ischemia.
  • Another effective peptide described herein is Rv-Tat-NR2B9c (RRRQRRKKRGYKLSSIESDV; SEQ ID NO:70).
  • a linker e.g., a polyethylene glycol linker
  • a linker can be used to dimerize the active moiety of the peptide or the peptidomimetic to enhance its affinity and selectivity towards proteins containing tandem PDZ domains. See e.g., Bach et al., (2009) Angew. Chem. Int. Ed. 48:9685- 9689 and WO 2010/004003.
  • a PL motif-containing peptide is preferably dimerized via joining the N-termini of two such molecules, leaving the C -termini free.
  • a pentamer peptide IESDV (SEQ ID NO: 71 ) from the C-terminus of NMDAR 2B was effective in inhibiting binding of NMDAR 2B to PSD-95.
  • IETDV (SEQ ID NO: 73) can also be used instead of IESDV.
  • about 2-10 copies of a PEG can be joined in tandem as a linker.
  • the linker can also be attached to an internalization peptide or lipi dated to enhance cellular uptake. Examples of illustrative dimeric inhibitors are shown below (see Bach et al, PNAS 109 (2012) 3317-3322). Any of the PSD-95 inhibitors disclosed herein can be used instead of IETDV, and any internalization peptide or lip j dating moiety can be used instead of tat. Other linkers to that shown can also be used.
  • IETAV is assigned SEQ ID N():26, YGRKKRRQRRR SEQ ID N0:2, and rrrqrrkkr, SEQ ID NO: 10, lower case letters indicated D-amino acids.
  • peptides, peptidomimetics or other agent can be confirmed if desired, using previously described rat models of stroke before testing in the primate and clinical trials described in the present application.
  • Peptides or peptidomimetics can also be screened for capacity to inhibit interactions between PSD-95 and NMDAR 2B using assays described in e.g., US 20050059597, which is incorporated by reference.
  • Useful peptides typically have IC50 values of less than 50 ⁇ , 25 ⁇ , 10 ⁇ , 0.1 ⁇ or 0.01 ⁇ in such an assay.
  • Preferred peptides typically have an IC50 value of between 0.001-1 ⁇ , and more preferably 0.001-0.05, 0,05-0.5 or 0.05 to 0. ⁇ .
  • Peptides such as those just described can optionally be derivatized (e.g., acetylated, phosphorylated, myristoylated, geranylated, pegylated and/or glycosylated) to improve the binding affinity of the inhibitor, to improve the ability of the inhibitor to be transported across a cell membrane or to improve stability.
  • derivatized e.g., acetylated, phosphorylated, myristoylated, geranylated, pegylated and/or glycosylated
  • this residue can be phosphorylated before use of the peptide.
  • a pharmacological agent can be linked to an internalization peptide to facilitate uptake into cells and/or across the blood brain barrier.
  • Internalization peptides are a well-known class of relatively short peptides that allow many cellular or viral proteins to traverse membranes.
  • Internalization peptides also known as cell membrane transduction peptides or cell penetrating peptides can have e.g., 5-30 ammo acids.
  • Such peptides typically have a cationic charge from an above normal representation (relative to proteins in general) of arginine and/or lysine residues that is believed to facilitate their passage across membranes.
  • Some such peptides have at least 5, 6, 7 or 8 arginine and/or lysine residues. Examples include the antennapedia protein (Bonfanti, Cancer Res. 57, 1442-6 (1997)) (and variants thereof), the tat protein of human
  • immunodeficiency virus the protein VP22, the product of the UL49 gene of herpes simplex virus type 1, Penetratin, SynBl and 3, Transportan, Amphipathic, gp41NLS, polyArg, and several plant and bacterial protein toxins, such as ncm, abrin, modeccin, diphtheria toxin, cholera toxin, anthrax toxin, heat labile toxins, and Pseudomonas aeruginosa exotoxin A (ETA).
  • ncm abrin
  • modeccin diphtheria toxin
  • cholera toxin diphtheria toxin
  • anthrax toxin anthrax toxin
  • heat labile toxins heat labile toxins
  • Pseudomonas aeruginosa exotoxin A ETA
  • a preferred internalization peptide is tat from the HIV virus.
  • a tat peptide reported in previous work comprises or consists of the standard ammo acid sequence YGRKKRRQRRR (SEQ ID NO: 2) found in HIV Tat protein. If additional residues flanking such a tat motif are present (beside the pharmacological agent) the residues can be for example natural amino acids flanking this segment from a tat protein, spacer or linker amino acids of a kind typically used to join two peptide domains, e.g., gly (ser) 4 (SEQ ID ⁇ 0: -! -! ⁇ . TGEKP (SEQ ID NO:45),
  • GGRRGGGS SEQ ID NO:46
  • LRQRDGERP SEQ ID NO:47
  • the number of flanking amino acids other than an active peptide does not exceed ten on either side of YGRKKRRQRRR (SEQ ID NO:2).
  • tat peptide comprising additional amino acid residues flanking the C-terminus of YGRKKRRQRRR (SEQ ID NO: 2) is YGRKKRRQRRRPQ (SEQ ID NO:48), However, preferably, no flanking amino acids are present.
  • Other tat peptides that can be used include GRKKRRQRRRPQ (SEQ ID NO: 4) and GRKKRRQRRRP (SEQ ID NO:72).
  • variants of the above tat peptide having reduced capacity to bind to N-type calcium channels are described by WO/2008/109010.
  • Such variants can comprise or consist of an amino acid sequence XGRKKRRQRRR (SEQ ID NO:49), in which X is an amino acid other than Y or nothing (in which case G is a free N-terminal residue).
  • a preferred tat peptide has the N- terminal Y residue substituted with F.
  • a tat peptide comprising or consisting of
  • FGRKKRRQRRR (SEQ ID NO: 3) is preferred.
  • Another preferred variant tat peptide consists of GRKKRRQRRR (SEQ ID NO: 1).
  • Another preferred tat peptide comprises or consists of RRRQRRKKRG or RRRQRRKKRGY (amino acidsl-10 or 1-11 of SEQ ID NO:70).
  • Other tat derived peptides that facilitate uptake of a pharmacological agent without inhibiting N-type calcium channels include those presented below.
  • X can represent a free ammo terminus, one or more amino acids, or a conjugated moiety.
  • Internalization peptides can be used in inverse or retro or inverso retro form with or without the linked peptide or peptidomimetic being in such form.
  • a preferred chimeric peptide has an amino acid sequence comprising or consisting of YGRKKRRQRRR-KLSSIESDV (SEQ ID NO:6, also known as TAT-NR2B9c or Tat-NR2B9c), or YGRKKRRQRRR-KLS SIETD V (SEQ ID NO:7).
  • chimeric peptides differ from SEQ ID NO:6 or NO:7 by up to 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions (internal or at the ends).
  • Other preferred peptides include RRRQRRKKRGY-KLSSIESDV (SEQ ID NO: 70, also known, as RvTat-NR2B9c or having an amino acid sequence comprising or consisting of
  • Internalization peptides can be attached to pharmacological agents by conventional methods.
  • the agents can be joined to internalization peptides by chemical linkage, for instance via a coupling or conjugating agent.
  • a coupling or conjugating agent Numerous such agents are commercially available and are reviewed by S. S. Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press (1991).
  • cross-linking reagents include J-succimmidyl 3 -(2- pyndyldithio) propionate (SPDP) or N,N'-(l,3-phenylene) bismaleimide; ⁇ , ⁇ '-ethylene-bis- (iodoacetamide) or other such reagent having 6 to 11 carbon methylene bridges (which relatively specific for sulfhydryl groups); and l,5-difluoro-2,4-dinitrobenzene (which forms irreversible linkages with amino and tyrosine groups).
  • SPDP J-succimmidyl 3 -(2- pyndyldithio) propionate
  • N,N'-(l,3-phenylene) bismaleimide N,N'-(l,3-phenylene) bismaleimide
  • cross-linking reagents include p,p'-difluoro-m, m'-dinitrodiphenylsulfone (which forms irreversible cross-linkages with amino and phenolic groups); dimethyl adipimidate (which is specific for amino groups); phenol-1,4- disulfonylchloride (which reacts principally with amino groups); hexamethylenediisoc anate or diisothiocyanate, or azophenyl-p-diisocyanate (which reacts principally with ammo groups); glutaraldehyde (which reacts with several different side chains) and disdiazobenzidme (which reacts primarily with tyrosine and histidine).
  • pharmacological agents that are peptides attachment to an internalization peptide can be achieved by generating a fusion protein comprising the peptide sequence fused, preferably at its N-terminus, to an internalization peptide.
  • a peptide (or other agent) inhibiting PSD-95 to an internalization peptide can be linked to a lipid (lipidation) to increase
  • Lipidation is preferably performed on the N-terminal amino acid but can also be performed on internal amino acids, provided the ability of the peptide to inhibit interaction between PSD-95 and NMDAR 2B is not reduced by more than 50%.
  • lipidation is performed on an ammo acid other than one of the four most C-terminal amino acids.
  • Lipids are organic molecules more soluble in ether than water and include fatty acids, glycerides and sterols.
  • Suitable forms of lipidation include myristoy!ation, palmitoylation or attachment of other fatty acids preferably with a chain length of 10-20 carbons, such as lauric acid and stearic acid, as well as geranylation, geranylgeranylation, and isoprenylation. Lipidations of a type occurring in posttranslational modification of natural proteins are preferred. Lipidation with a fatty acid via formation of an amide bond to the alpha- amino group of the N-terminal amino acid of the peptide is also preferred.
  • Lipidation can be by peptide synthesis including a prelipidated amino acid, be performed enzymatically in vitro or by recombinant expression, by chemical crossiinkmg or chemical denvatization of the peptide. Amino acids modified by myristoyiation and other lipid modifications are commercially available.
  • Lipidation preferably facilitates passage of a linked peptide (e.g., KLSS1ESDV (SEQ ID NO: 5), or KLSSIETDV (SEQ ID NO: 43)) across a cell membrane and/or the blood brain barrier without causing a transient reduction of blood pressure as has been found when a standard tat peptide is administered at high dosage ⁇ e.g., at or greater than 3 mg ' 'kg), or at least with smaller reduction that than the same peptide linked to a standard tat peptide.
  • a linked peptide e.g., KLSS1ESDV (SEQ ID NO: 5), or KLSSIETDV (SEQ ID NO: 43
  • Pharmacologic peptides can be synthesized by solid phase synthesis or recombinant methods.
  • Peptidomimetics can be synthesized using a variety of procedures and methodologies described in the scientific and patent literature, e.g.. Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley & Sons, Inc., NY, al ⁇ Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin, Chem. Biol, 1 : 114-119;
  • Peptides of the type described above are typically made by solid state synthesis.
  • peptides are typically initially produced as trifloroaeetate salts.
  • the tnfluoroacetate can be replaced with another anion by for exampie, binding the peptide to a solid support, such as a column, washing the column to remove the existing counterion, equilibrating the column with a solution containing the new counterion and then eluting the peptide, e.g., by- introducing a hydrophobic solvent such as acetonitrile into the column.
  • Replacement of tnfluoroacetate with acetate can be done with an acetate wash as the last step before peptide is eluted in an otherwise conventional solid state synthesis.
  • Replacing tnfluoroacetate or acetate with chloride can be done with a wash with ammonium chloride followed by elution.
  • Use of a hydrophobic support is preferred and preparative reverse phase HPLC is particularly preferred for the ion exchange.
  • Trifluoroacetate can be replaced with chloride directly or can first be replaced by acetate and then the acetate replaced by chloride.
  • Counterions whether trifluoroacetate, acetate or chloride, bind to positively charged atoms on TAT-NR2B9c, particularly the N -terminal amino group and ammo side chains arginine and lysine residues.
  • TAT-NR2B9c particularly the N -terminal amino group and ammo side chains arginine and lysine residues.
  • chloride salt of TAT-N 2B9c or other active agent means that in a preparation of the salt, chloride is the predominant anion by weight (or moles) over all other anions present in the aggregate in the salt. In other words, chloride constitutes greater than 50% and preferably greater than 75%, 95%, 99%, 99.5% or 99.9% by weight or moles of the all anions present in the salt. In such a salt or formulation prepared from the salt, acetate and trifluoroacetate in combination and individually constitutes less than 50%, 25%, 5%, 5% or 0.1 of the anions in the salt or formulation.
  • the lyophilized formulations are useful in treating a variety of diseases, particularly neurological diseases, and especially diseases mediated in part by excitotoxity.
  • diseases and conditions include stroke, epilepsy, hypoxia, subarachnoid hemorrhage, traumatic injury to the CNS not associated with stroke such as traumatic brain injury and spinal cord injury, other cerebral ischemia, Alzheimer's disease and Parkinson's disease.
  • Other neurological diseases treatable by agents of the invention not known to be associated with excitotoxicity include anxiety and pain.
  • a stroke is a condition resulting from impaired blood flow in the CNS regardless of cause.
  • Potential causes include embolism, hemorrhage and thrombosis.
  • Some neuronal cells die immediately as a result of impaired blood flow. These cells release their component molecules including glutamate, which in turn activates NMD A receptors, which raise intracellular calcium levels, and intracellular enzyme levels leading to further neuronal cell death (the excitotoxicity cascade).
  • the death of CNS tissue is referred to as infarction.
  • Infarction Volume i.e., the volume of dead neuronal cells resulting from stroke in the brain
  • the symptomatic effect depends both on the volume of an infarction and where in the brain it is located.
  • Disability index can be used as a measure of symptomatic damage, such as the Rankin Stroke Outcome Scale (Rankin, Scott Med J;2:200-15 (1957)) and the Barthel Index.
  • the Rankm Scale is based on assessing directly the global conditions of a patient as follows.
  • the Barthel Index is based on a series of questions about the patient's ability to carry out 10 basic activities of daily living resulting in a score between 0 and 00, a lower score indicating more disability (Mahoney et al, Maryland State Medical Journal 14:56-61 (1965)),
  • stroke severity/outcomes can be measured using the NTH stroke scale, available at world wide web ninds.nih.gov/doctors/NIH Stroke ScaleJBooklet.pdf,
  • the scale is based on the ability of a patient to carry out 11 groups of functions that include assessments of the patient's level of consciousness, motor, sensory and language functions.
  • An ischemic stroke refers more specifically to a type of stroke that caused by blockage of blood flow to the brain.
  • the underlying condition for this type of blockage is most commonly the development of fatty deposits lining the vessel walls. This condition is called atherosclerosis. These fatty deposits can cause two types of obstruction.
  • Cerebral thrombosis refers to a thrombus (blood clot) that develops at the clogged part of the vessel
  • Cerebral embolism refers generally to a blood clot that forms at another location in the circulatory system, usually the heart and large arteries of the upper chest and neck.
  • a second important cause of embolism is an irregular heartbeat, known as arterial fibrillation. It creates conditions in which clots can form in the heart, dislodge and travel to the brain. Additional potential causes of ischemic stroke are hemorrhage, thrombosis, dissection of an artery or vein, a cardiac arrest, shock of any cause including hemorrhage, and iatrogenic causes such as direct surgical injury to brain blood vessels or vessels leading to the brain or cardiac surgery. Ischemic stroke accounts for about 83 percent of all cases of stroke.
  • Transient ischemic attacks are minor or warning strokes.
  • TIA Transient ischemic attacks
  • conditions indicative of an ischemic stroke are present and the typical stroke warning signs develop.
  • Hemorrhagic stroke accounts for about 17 percent of stroke cases. It results from a weakened vessel that ruptures and bleeds into the surrounding brain. The blood accumulates and compresses the surrounding brain tissue.
  • the two general types of hemorrhagic strokes are intracerebral hemorrhage and subarachnoid hemorrhage. Hemorrhagic stroke result from rupture of a weakened blood vessel ruptures.
  • Hemorrhagic strokes can also arise from a hemorrhagic transformation of an ischemic stroke which weakens the blood vessels in the infarct, or a hemorrhage from primary or metastatic tumors in the CNS which contain abnormally weak blood vessels. Hemorrhagic stroke can also arise from iatrogenic causes such as direct surgical injury to a bram blood vessel.
  • An aneurysm is a ballooning of a weakened region of a biood vessel. If left untreated, the aneurysm continues to weaken until it ruptures and bleeds into the brain.
  • An arteriovenous malformation (AVM) is a cluster of abnormally formed blood vessels.
  • a cavernous malformation is a venous abnormality that can cause a hemorrhage from weakened venous structures. Any one of these vessels can rupture, also causing bleeding into the bram.
  • Hemorrhagic stroke can also result from physical trauma. Hemorrhagic stroke in one part of the brain can lead to ischemic stroke in another through shortage of blood lost in the hemorrhagic stroke.
  • One patient class amenable to treatments are patients undergoing a surgical procedure that involves or may involve a blood vessel supplying the brain, or otherwise on the brain or CNS. Some examples are patients undergoing cardiopulmonary bypass, carotid stenting, diagnostic angiography of the brain or coronary arteries of the aortic arch, vascular surgical procedures and neurosurgical procedures. Additional examples of such patients are discussed in section IV above. Patients with a brain aneurysm are particularly suitable.
  • Such patients can be treated by a variety of surgical procedures including clipping the aneurysm to shut off blood, or performing endovascular surgery to block the aneurysm with small coils or introduce a stent into a blood vessel from which an aneurysm emerges, or inserting a microcatheter. Endovascular procedures are less invasive than clipping an aneurysm and are associated with a better patient outcome but the outcome still includes a high incidence of small infarctions.
  • Such patients can be treated with an inhibitor of PSD95 interaction with NMDAR 2B and particularly the agents described above including the peptide YGRKKRRQRRRKLS SIESD V (SEQ ID NO: 6, also known as Tat-NR2B9c).
  • the timing of administration relative to performing surgery can be as described above for the clinical trial.
  • Another class of patients amenable to treatment are patients having a subarachnoid hemorrhage with or without an aneurysm (see US 61/570,264).
  • a lyophilized formulation is administered such that the active agent (e.g., NR2B9c) is administered in an amount, frequency and route of administration effective to cure, reduce or inhibit further deterioration of at least one sign or symptom of a disease in a patient having the disease being treated.
  • a therapeutically effective amount means an amount of active agent sufficient significantly to cure, reduce or inhibit further deterioration of at least one sign or symptom of the disease or condition to be treated in a population of patients (or animal models) suffering from the disease treated with an agent of the invention relative to the damage in a control population of patients (or animal models) suffering from that disease or condition who are not treated with the agent.
  • the amount is also considered therapeutically effective if an individual treated patient achieves an outcome more favorable than the mean outcome in a control population of comparable patients not treated by methods of the invention.
  • a therapeutically effective regime involves the administration of a therapeutically effective dose at a frequency and route of administration needed to achieve the intended purpose.
  • the active agent is administered in a regime comprising an amount frequency and route of administration effective to reduce the damaging effects of stroke or other ischemic condition.
  • the outcome can be determined by infarction volume or disability index, and a dosage is considered therapeutically effective if an individual treated patient shows a disability of two or less on the Rankin scale and 75 or more on the Barthel scale, or if a population of treated patients shows a significantly improved (i.e., less disability) distribution of scores on a disability scale than a comparable untreated population, see Lees et at L, N Engl J Med 2006;354:588-600.
  • a single dose of agent is usually sufficient for treatment of stroke.
  • the invention also provides methods and formulations for the prophylaxis of a disorder in a subject at risk of that disorder.
  • a subject has an increased likelihood of developing the disorder (e.g., a condition, illness, disorder or disease) relative to a control population.
  • the control population for instance can comprise one or more individuals selected at random from the general population (e.g., matched by age, gender, race and/or ethnicity) who have not been diagnosed or have a family history of the disorder.
  • a subject can be considered at risk for a disorder if a "risk factor" associated with that disorder is found to be associated with that subject.
  • a risk factor can include any activity, trait, event or property associated with a given disorder, for example, through statistical or epidemiological studies on a population of subjects.
  • a subject can thus be classified as being at risk for a disorder even if studies identifying the underlying risk factors did not include the subject specifically.
  • a subject undergoing heart surgery is at risk of transient cerebral ischemic attack because the frequency of transient cerebral ischemic attack is increased in a population of subjects who have undergone heart surgery as compared to a population of subjects who have not.
  • Other common risk factors for stroke include age, family history, gender, prior incidence of stroke, transient ischemic attack or heart attack, high blood pressure, smoking, diabetes, carotid or other artery disease, atrial fibrillation, other heart diseases such as heart disease, heart failure, dilated cardiomyopathy, heart valve disease and/or congenital heart defects; high blood cholesterol, and diets high in saturated fat, trans fat or cholesterol.
  • a lyophilized formulation after reconstitution is administered to a patient at risk of a disease but not yet having the disease in an amount, frequency and route sufficient to prevent, delay or inhibit development of at least one sign or symptom of the disease.
  • a prophylactically effective amount means an amount of agent sufficient significantly to prevent, inhibit or delay at least one sign or symptom of the disease in a population of patients (or animal models) at risk of the disease relative treated with the agent compared to a control population of patients (or animal models) at risk of the disease not treated with a chimeric agent of the invention.
  • the amount is also considered prophylactically effective if an individual treated patient achieves an outcome more favorable than the mean outcome in a control population of comparable patients not treated by methods of the invention.
  • a prophylactically effective regime involves the administration of a prophylactically effective dose at a frequency and route of administration needed to achieve the intended purpose.
  • a prophylactically effective dose for prophylaxis of stroke in a patient at imminent risk of stroke (e.g., a patient undergoing heart surgery), a single dose of agent is usually sufficient.
  • administration can be parenteral, intravenous, nasal, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular.
  • Intravenous administration is preferred for peptide agents.
  • a preferred dose of active agent is 2- 3mg kg and more preferably 2.6 mg/kg.
  • active agent e.g., Tat-NR2B9c
  • Indicated dosages should be understood as including the margin of error inherent in the accuracy with which dosages can be measured in a typical hospital setting. Such amounts are for single dose administration, i.e., one dose per episode of disease.
  • Active agents such as Tat-NR2B9c are preferably delivered by infusion into a blood vessel, more preferably by intravenous infusion.
  • the time of the infusion can affect both side effects (due e.g., to mast cell degranulation and histamine release) and efficacy.
  • side effects due e.g., to mast cell degranulation and histamine release
  • efficacy In general, for a given dosage level, a shorter infusion time is more likely to lead to histamine release. However, a shorter infusion time also may result in improved efficacy.
  • a preferred infusion time providing a balance between these considerations is 5-15 minutes and more preferably 10 min. Indicated times should be understood as including a marking of error of +/- 10%. Infusion times do not include any extra time for a wash out diffusion to wash out any remaining droplets from an initial diffusion that has otherwise proceeded to completion.
  • the infusion times for Tat-N 2B9c can also serve as a guide for other active agents.
  • Examples 1 -7 show that an acetate salt of TAT-N 2B9c can be formulated in lyophilized form with histidine and trehalose.
  • Example 10 shows that the chloride salt of TAT-NR2B9c offers significantly greater stability than the acetate salt in an otherwise identical lyophilized formulation.
  • Example 9 shows that the chloride salt of TAT-NR2B9c formulated in histidine and trehalose also gives improved stability relative to a previously described lyophilized formulation of TAT-NR2B9c from normal saline.
  • Example 1 Demonstration that Standard buffers and excipients do not interfere with the efficacy of T AT-NR2B9c in vivo.
  • liquid toxicology formulations were compounded targeted at a 20 mg/ ' raL concentration of TAT-NR2B9e.
  • Table 1 includes the vehicle composition, Lot Number, and the potency, purity and pH at the time of compounding. Approximately 5 mL of each formulation was vialed for testing. Vials were frozen at -20 to simulate transport or liquid storage conditions.
  • Table 1 composition of TAT-NR2B9c formulations for efficacy testing in vivo
  • formulation #2 The purity of formulation #2 is notably lower than the other formulations.
  • Formulations 1 -5 were tested in the 3-PIAL Vessel Occlusions (3PVO) model of stroke in rats. Rats subjected to stroke were given one of the formulations by intravenous
  • TTC triphenyltetrazolium chloride
  • An anal temperature probe was inserted, and the animal was placed on a heating pad maintained at about 37°C.
  • the skull was exposed via a midline incision and scraped free of tissue.
  • a 6- to 8-mm cranial window was made over the right somatosensory cortex (2 mm caudal and 5 mm lateral to bregma) by drilling a rectangle through the skull and lifting off the piece of skull while keeping the dura intact.
  • the 3 pial arteriolar middle cerebral artery branches were cauterized around the barrel cortex were selected and electrically cauterized through the dura. After the cauterizations, the scalp was sutured.
  • Each rat was returned to its individual cage under a heating lamp to maintain body temperature until the rat fully recovered. Food and water was supplied.
  • One hour after 3PVO ischemia the rats were injected with NA1 formulations at 3 nmol/g in -0.45 mL saline based upon rat weight. Doses were administered over 5 minutes.
  • Example 2 Determination of TAT-NR2B9c stability in different buffers and at different pH values
  • Results indicate improved stability for TAT-NR2B9c in liquid media buffered between pH 6.0 and pH 6.5. Degradation appears to increase outside of this range in either direction. Data generated with the MSA method showed clear degradation patterns that were both pH and buffering species dependent, and provided valuable insight into future formulation development. Results for main peak purity by % HPLC Area using the MSA method are provided in Table 2, while results for main peak purity by %HPLC Area using the TFA method are provided in Table 3.
  • results indicate that TAT-NR2B9c solution stability is best maintained in pH 6.0 to 6.5 buffered media and the vehicle is still well tolerated for administration by IV.
  • histidine and citrate buffering systems were able to maintain TAT-NR2B9c in an intact form even when kept at accelerated stability conditions of 25°C or 40°C for 1 week.
  • Example 3 Determination of TAT- R2B9c stability in histidme and citrate buffers and at different pH values with varying amounts of sodium chloride.
  • Example 4 Selection of bulking agents for TAT-NR2B9c to form a stable lyophilized cake
  • Mannitol, Trehalose and Dextran-40 maintain the pH at 6.5 well (Table 9) and there is approximately a 1% decrease in punty (Table 10) over 1 week as a liquid formation when stored at high temperature.
  • mannitol and trehalose are preferred bulking agents as they confer better stability to TAT-NR2B9c than the dextran-40 solutions ( Figure 3).
  • Example 5 Thermal analysis of bulking agents to facilitate design of lyophilization cycles
  • TAT-N 2B9c formulations At a TAT-N 2B9c concentration of 20 mg/mL, tested TAT-N 2B9c formulations showed a thermal profile characterized by a broad melting event with onset at a low temperature. This extended melt masked the crystallization event typically seen in mannitol formulations, and may indicate that a robust freeze drying cycle must be performed where the product never exceeds the glass transition temperature.
  • TAT-NR2B9c fill volume lower than 3 mL, which using a 90 mg/mL TAT-NR2B9c formulation would give provide 270 mg in a target vial.
  • a wide range of quantities may be required in a vial, but 270 mg would provide a 2.6 mg/kg dose for a 100 kg patient.
  • the target reconstitution concentration for patient administration is still 20 mg/mL (but can be from 1 mg/ml to 100 mg/ml)
  • a 20-mL lyophilization vial containing 270 mg of TAT-N 2B9c can be used with a reconstitution volume of 13.5 mL. Therefore, optimal volumes of liquid for lyophilization of TAT-NR2B9c in the vial would be between 2.5mL and 10 mL.
  • Tg's are shown in Table 12.
  • the 100 mg/mL TAT-NR2B9c in histidine, pH 6.5 was also evaluated by DSC and the data is included in Table 12.
  • formulations 5 and 11 are the most promising for the active product with respect to the Tg. Any bulking agent may be suitable for use in a placebo product, but Formulation 4 (Mannitol) will have the shortest cycle length if annealed, and may be the most desirable should the appearance match the active.
  • Formulation 5 from Table 12 (Trehalose) demonstrated good solution stability and lyophile chemical stability at accelerated conditions (data shown subsequently), but requires a longer lyophilization cycle at a fill configuration of 13.5 mL. This longer cycle length may not be ideal for commercial manufacture in the future, where a shorter cycle is desirable.
  • Formulation 1 from Table 12 (without a bulking agent, at 100 mg/mL TAT-NR2B9c) has a higher glass transition temperature than Formulation 5, allowing for a warmer, shorter cycle.
  • a decreased fill volume will significantly shorten the run time as there will be less ice to sublimate from each vial.
  • TAT-NR2B9c drug product was lyophilized to evaluate solid state stability after 1 week at 25°C, 40°C, and 60°C.
  • the drug product vehicles are described in Table 13 and are listed with the respective glass transition temperature and water content results.
  • the pH, TAT-NR2B9c amount and TAT- NR2B9c purity results are described in Tables 14-16.
  • dextran-40 in the bulking agent allows for a warmer primary drying temperature, but dextran-40 as a bulking agent demonstrated the poorest stability.
  • trehalose is a preferred bulking agent.
  • TAT-NR2B9c drug product was lyophilized to evaluate setting the shelf temperature at 5°C during primary drying.
  • TAT-NR2B9c was compounded at an active concentration of 27 mg/mL in 50 mM Histidine, pH 6.5 and 120 mM Trehalose.
  • the cycle parameters are outlined in Table 7.
  • Four 20-mL glass lyophilization vials were filled with 10 mL. Two vials were probed with temperature probes.
  • TAT-NR2B9c drug product was lyophilized to evaluate solid state stability after 1 week storage at 25°C and 60°C.
  • Reconstitution time was approximately 1.5 minutes compared to less than 10 seconds in the previous formulations.
  • the increased reconstitution time is most likely due to the increased concentrations of TAT-NR2B9c and histidine. Further tests showed good stability of TAT- N 2B9c with histidine buffer concentrations of 50 and 75 mM, with shortened resuspension times. Also, due to the 2-mL vial size used for this study, only 1 niL of water was added to the lyophile. The actual reconstitution volume is 4.5 mL in this small scale configuration. The reconstitution time will most likely improve when a larger volume of diluent is used.
  • Vials were placed on stability at 25°C and 60°C and tested after 1 week of storage.
  • the trehalose sample gave a more elegant cake.
  • the lyophilization cycle was run conservatively over 5 days, with a primary drying temperature of -32°. Based on the temperature probe data, the cycle can be shortened, demonstrating that with the higher concentration and lower fill volume the optimized cycle will be shorter.
  • trehalose Based on tins accelerated stability data, trehalose demonstrates a stabilizing effect on the TAT-NR2B9c formulation which improves the chemical stability of the lyophile. It is surprising that trehalose is able to confer this stabilizing effect while other standard bulking agents such as dextran and mannitol used for other peptides do not.
  • the reduced fill volume minimizes the competing evaporative cooling of the surrounding vials and minimizes the resistance to the sublimating water.
  • Vials were reconstituted with 13.5 niL of water. The time to dissolve is listed in Table 21. The active lyophile re-suspended immediately, but was cloudy for 17.6 sec before becoming a clear, colorless solution. All placebos were a clear, colorless solution.
  • a preferred commercial formulation for TAT-NR2B9c, prelyophilization would be 20-100 mM Histidine, 120 mM Trehalose pH 6.5. Trehalose concentrations can be increased without a loss of stability or cake elegance but resuspension times. Examination of increased Trehalose in cake formation and placebo matching by visual appearance and resuspension time.
  • Trehalose was tested with and without TAT-NR2B9c, and at either 3 or 5 mL fill volumes.
  • Figure 7B shows the appearance of the active formulations listed above.
  • Example 7 Stability of lyophilized 27 ⁇ mg TAT-NR2B9c in 20 mM Histidme buffer pH 6,5 and 120 mM trehalose
  • TAT-NR2B9c drug product was formulated at 90 mg/mL in 20 mM Histidine pH 6.5 and 120 mM trehalose and lyophilized to evaluate solid state stability after 4 weeks at -20°C, 40°C, and 60°C.
  • Table 25 shows the lyophilization conditions.
  • Impurities interpolated using the Arrnehius equation are less than 0.5% after 16 months at 25°C or 23 months at 5°C, and less than 2% for >60 months at room temperature and many years at 5°C.
  • this and related formulations are suitable for room temperature storage of lyophilized drug product.
  • the stability of TAT-NR2B9c could be increased by either adding a scavenger or other excipients to reduce acetyiation of TAT-NR2B9c in the lyophilized state or by reducing or removing the acetate so that there is a reduced chance of acetyiation.
  • TAT-N 2B9c Based on the stability, resuspension times, and lyophilization times, a preferred commercial formulation for TAT-N 2B9c is 20-100 niM Histidme, 120 mM Trehalose pH 6.5. Trehalose concentrations can be increased without a loss of stability or cake elegance but resuspension times increase with increased trehalose concentration.
  • Example 8 Development and Stability of a Chloride salt of TAT-NR2B9c (TAT-NR2B9c- Cl)
  • TAT-NR2B9c is effective when administered to potential victims of stroke and other neurological disorders, and there is a high value to early treatment of these disorders, a formulation that is stable outside of hospital conditions would be valuable in helping the millions of people affected by these disorders every year.
  • a drug could be stored in ambulances, or small clinics, doctor's offices or even be available to individuals, and provided to subjects having neurological disorders such as stroke or other diseases amenable to neuroprotective agents much earlier than they might be administered if they had to travel to a hospital for treatment.
  • YMC ODS-A CI 8 Column, 5 ⁇ , 12 ⁇ ; Flow rate: 1.5 mL/ ' min.; Wavelength: 210 nm;
  • TAT-NR2B9c-Cl was compounded in 20 mM histidine and 120 tnM trehalose, pH 6.5, for direct comparison to a previous preferred lyophilized formulation under the same lyophilization conditions at 270 mg/vial (active weight).
  • the stability of this formulation was also compared to the previously disclosed formulation of TAT-NR2B9c-Ac resuspended in saline and lyophilized.
  • a small batch of TAT-NR2B9c-acetate drug product was formulated at 90 mg/mL in saline (no other excipients) and lyophilized at 270 mg/vial. Stability of the formulations in the lyophilized state was evaluated after 4 weeks at -20°C (control), 40°C and 60°C.
  • Table 28 Stability of TAT-NR2B9c-Cl (20 mM histidine, 120 mM trehalose) versus TAT- NR2B9c-Ac (saline) at 11 months
  • Table 27 shows the relative area of each peak observed in each HFLC assay at each temperature after 1 month of storage and at each relative retention time (peak identity) for direct comparison.
  • the starting purities are very similar (98% for TAT-NR2B9c-Cl vs 97.93% for TAT-NR2B9c-Ac by the TFA method, and 99.3% vs 99.13% for the methanesulfonic acid (MSA) method.
  • MSA methanesulfonic acid
  • the TAT- N 2B9c-Cl salt retains 98.6% of the main peak whereas the TAT-NR2B9c-Ac saline formulation only retains 96.8% of the starting material.
  • the difference is even more striking, where the TAT-N 2B9c-Cl salt retains 99.4% of its starting material whereas the TAT-NR2B9c-Ac saline formation only retains 97.2% of its starting material.
  • Table 28 shows the relative area of each peak observed in each HPLC assay at in the same samples after 1 months at the different storage temperatures. These data support the improved stability of the NA-1 CI salt formulation versus the NA-1 -Ac formulation in chloride. Comparing the area of the main NA-1 peaks in the TFA assay at 40°C, which is the accelerated condition by ICH guidance to support room temperature storage of a lyophilized drug, the NA-1- Cl retains 98.14% purity from a starting purity of 98.58, while the NA-1 -Ac in saline has dropped from 98.57 pure to 96.48, which is below the purity specification of 97% and would not be considered stable after about a year.
  • example purity limits for a drug for humans may be 97% purity with no uncharacterized impurity over 0.5% at the rated storage condition.
  • Arrenhms equation we can use the 40°C and 60°C stability data to predict the drop in purity of the mam peak or the growth of the observed impurities at different storage temperatures (e.g., 25°C or 37 U C versus the observed 40 U C or 60°C data). Assuming that the drug would be stored at ambient temperature, it would be useful to demonstrate stability at 37°C because there would be many environments without refrigeration where the ambient temperature in the summer could be in this range.
  • the saline formulation would be predicted to have a shelf life of 29.8 months (or a little over 2 years), whereas the TAT-NR2B9c-Cl formulation would have a shelf life of -500 months, or approximately 41 years. Tins is a significant improvement in stability.
  • the largest impurity growth after 1 month for the TAT-NR2B9c-Cl formulation is 0.17 to 0.43 (RRT 0.99 by the TFA method), whereas the largest impurity growth for the TAT-NR2B9c-Ac saline formulation is 0.05-0.72 (1.13 RRT by the TFA method).
  • the saline formulation is predicted to have a shelf life of - 4.8 months at 37 J C whereas the shelf life of the TAT-NR2B9c-Cl composition is predicted to be >10 years.
  • the TAT-NR2B9c-Cl composition is a significant improvement over the saline formulation.
  • TAT-NR2B9c-Cl and TAT-NR2B9c-Ac were both formulated at 90 mg/mL in 20 mM Histidiiie, 120 mM trehalose pH 6.5. Three milliliter aliquots (270 mg) of each were lyophilized under the same program as presented supra, and on completion were placed into constant temperature and humidity incubators at -20°C (control), 40°C and 60°C. After 4 weeks, the stability of each formulation was tested using two orthogonal HPLC methods - one with TFA as the carrier and one with MSA as the carrier.
  • Table 29 shows the relative area of each peak observed in each of the two HPLC assays at each temperature after 1 month of storage and at each relative retention time (RRT; peak identity) for direct comparison.
  • the left panel represents the TAT-NR2B9c ⁇ Ac formulation and the right panel shows the results of the TAT ⁇ NR2B9c-Cl salt formulation.
  • the peak at an RRT of 1 corresponds to the intact TAT-NR2B9c peptide in each assay and column.
  • the starting purities are very similar (98% for TAT-NR2B9c ⁇ Cl vs 98.3% for TAT-NR2B9c-Ac by the TFA method, and 99.3% vs 99,2% respectively for the MSA method).
  • the TAT-NR2B9c-Cl composition is substantially more stable than the TAT- R2B9c-Ac formulation by both methods.
  • the TAT-NR2B9c-Cl salt retains 98.6% of the main peak (96.64% divided by 98% starting) whereas the TAT-NR2B9c-Ac saline formulation only retains 96% of the starting material (94.44% divided by 98.3% starting purity).
  • the difference is even more striking, where the TAT-NR2B9c-Cl salt retains 99.4% of its starting material (98.68%/99.3%) whereas the TAT-NR2B9c-Ac saline formation only retains 95.6% of its starting material (94.81%/99.2%).
  • the TAT-NR2B9c-acetate salt has larger impurities and is thus less stable.
  • the chloride salt of TAT- NR2B9c is surprisingly more stable than the acetate salt under identical conditions.
  • Table 30 shows the relative area of each peak observed in each HPLC assay at in the same samples after 1 months at the different storage temperatures. These data support the improved stability of the NA-1 CI salt formulation versus the NA-1 -Ac salt when formulated in the exact same buffer (20 mM Histidine/120 mM Trehalose pH 6.5).
  • the NA-1 -CI Comparing the area of the mam NA-1 peaks in the TFA assay at 40°C, which is the accelerated condition by ICH guidance to support room temperature storage of a lyophilized drug, the NA-1 -CI retains 98.14% purity from a starting purity of 98.58, while the NA-1 -Ac in saline has dropped from 98.57 pure to 95.91, which is below the purity specification of 97% and would not be considered stable. The same trend is observed for the MSA assay from 99.3% pure to 98.71 pure for NA-1 -CI and 96.85% for NA-1 -Ac in saline.
  • NA-1 chloride salt is predicted to be surprisingly and significantly more stable than the acetate salt in the same buffer.
  • Table 29 Stability of TAT-NR2B9c-Ac versus TAT-NR2B9c-Cl lyophilized in 20 mM Histidine 120 mM Trehalose pH 6.5 at 4 weeks
  • Table 30 Stability of T AT-NR2B9c- Ac versus TAT-NR2B9c-CI lyophilized in 20 mM

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CA2950395A CA2950395C (en) 2014-05-28 2015-05-28 Chloride salt of tat-nr2b9c
CN201580028269.3A CN106661126B (zh) 2014-05-28 2015-05-28 TAT-NR2B9c的氯盐
KR1020217031792A KR102407856B1 (ko) 2014-05-28 2015-05-28 Tat-nr2b9c의 클로라이드 염
HRP20200462TT HRP20200462T1 (hr) 2014-05-28 2015-05-28 Kloridna sol tat-nr2b9c
DK15799583.8T DK3149048T3 (en) 2014-05-28 2015-05-28 Chloridsalt af tat-nr2b9c
EP20152821.3A EP3757134B1 (en) 2014-05-28 2015-05-28 Chloride salt of tat-nr2b9c
BR112016027814A BR112016027814A2 (pt) 2014-05-28 2015-05-28 sal de cloreto de tat-nr2b9c
US15/313,940 US10206973B2 (en) 2014-05-28 2015-05-28 Chloride salt of TAT-NR2B9c
MEP-2020-120A ME03790B (me) 2014-05-28 2015-05-28 Hloridna so tat-nr2b9c
MX2016015457A MX374958B (es) 2014-05-28 2015-05-28 Sal de cloruro de tat-nr2b9c.
EP15799583.8A EP3149048B1 (en) 2014-05-28 2015-05-28 Chloride salt of tat-nr2b9c
KR1020227019189A KR102605540B1 (ko) 2014-05-28 2015-05-28 Tat-nr2b9c의 클로라이드 염
JP2016569655A JP6622222B2 (ja) 2014-05-28 2015-05-28 Ｔａｔ−ＮＲ２Ｂ９ｃの塩化物塩
SI201531132T SI3149048T1 (sl) 2014-05-28 2015-05-28 Kloridna sol TAT-NR2B9c
PL15799583T PL3149048T3 (pl) 2014-05-28 2015-05-28 SÓL CHLORKOWA TATNR2B9c
RU2016150678A RU2707885C2 (ru) 2014-05-28 2015-05-28 ХЛОРИДНАЯ СОЛЬ ПЕПТИДА TAT-NR2B9с, КОМПОЗИЦИЯ НА ЕГО ОСНОВЕ И СПОСОБЫ ИХ ПОЛУЧЕНИЯ
AU2015265487A AU2015265487B2 (en) 2014-05-28 2015-05-28 Chloride salt of TAT-NR2B9c
LTEP15799583.8T LT3149048T (lt) 2014-05-28 2015-05-28 Tat-nr2b9c chlorido druska
ES15799583T ES2784500T3 (es) 2014-05-28 2015-05-28 Sal de cloruro de Tat-NR2B9c
UAA201613470A UA123820C2 (uk) 2014-05-28 2015-05-28 Хлоридна сіль пептиду tat-nr2b9c
SM20200307T SMT202000307T1 (it) 2014-05-28 2015-05-28 Sale cloruro di tat-nr2b9c
KR1020167036752A KR102311038B1 (ko) 2014-05-28 2015-05-28 Tat-nr2b9c의 클로라이드 염
RS20200367A RS60625B1 (sr) 2014-05-28 2015-05-28 Hloridna so tat-nr2b9c
SG11201609911YA SG11201609911YA (en) 2014-05-28 2015-05-28 Chloride salt of tat-nr2b9c
IL249252A IL249252B (en) 2014-05-28 2016-11-28 chloride salt of tat–nr2b9c
ZA2016/08843A ZA201608843B (en) 2014-05-28 2016-12-21 Chloride salt of tat-nr2b9c
US16/128,423 US10758589B2 (en) 2014-05-28 2018-09-11 Chloride salt of TAT-NR2B9c
CY20201100251T CY1122793T1 (el) 2014-05-28 2020-03-18 Χλωριουχο αλας του tat-nr2b9c
US16/904,230 US11266714B2 (en) 2014-05-28 2020-06-17 Chloride salt of TAT-NR2B9C
AU2020267304A AU2020267304C1 (en) 2014-05-28 2020-11-13 Chloride salt of TAT-NR2B9c
US17/545,995 US11911438B2 (en) 2014-05-28 2021-12-08 Chloride salt of TAT-NR2B9C

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US10206973B2 (en) 2014-05-28 2019-02-19 Nono Inc. Chloride salt of TAT-NR2B9c
US10758589B2 (en) 2014-05-28 2020-09-01 Nono Inc. Chloride salt of TAT-NR2B9c
US11266714B2 (en) 2014-05-28 2022-03-08 Nono Inc. Chloride salt of TAT-NR2B9C
US12071458B2 (en) 2015-07-01 2024-08-27 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against ovarian cancer and other cancers
US20170002055A1 (en) * 2015-07-01 2017-01-05 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against ovarian cancer and other cancers
US11912749B2 (en) 2015-07-01 2024-02-27 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against ovarian cancer and other cancers
US11912748B2 (en) 2015-07-01 2024-02-27 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against ovarian cancer and other cancers
JP2020504713A (ja) * 2016-12-05 2020-02-13 ランカスター ユニバーシティ ビジネス エンタプライジズ リミテッドＬａｎｃａｓｔｅｒ Ｕｎｉｖｅｒｓｉｔｙ Ｂｕｓｉｎｅｓｓ Ｅｎｔｅｒｐｒｉｓｅｓ Ｌｔｄ 神経疾患の治療
US11851468B2 (en) 2016-12-05 2023-12-26 University Of Lancaster Treatment of neurological diseases
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