Fluorescein-labeled neuronal targeting peptide formulation and related methods

Abstract

Disclosed herein is (a) Equation (I): A pharmaceutical composition comprising (b) a fluorescein-labeled neuronal targeting peptide of JPEG2026516124000034.jpg102128 or a pharmaceutically acceptable salt, solvate, hydrate, isomer, tautomer, racemate, or isotope thereof; (b) an additive comprising a pH adjuster, buffer, filler, tonicity adjuster, preservative, or any combination thereof; and (c) an aqueous medium, wherein the pH of the composition is in the range of about 7 to about 9, and the concentration of the peptide in the aqueous composition is in the range of about 25 mg / mL to about 200 mg / mL based on the total weight of the aqueous composition. Methods for manufacturing and using the pharmaceutical composition described herein are also disclosed.

Description

[Technical Field]

[0001] field Disclosed herein are fluorescein-labeled neuronal targeting peptide formulations and related methods, including their use in intraoperative neuronal visualization and other targeted surgical and nonsurgical applications. [Background technology]

[0002] background Human nerve cells and nerve preservation are important surgical goals; for example, accidental nerve transection can lead to significant pathological conditions such as chronic pain and loss of function. Nerves are typically identified by their elongated, whitish appearance and relationship to neighboring structures, or by electrophysiological testing if they are motor nerves (sensory and autonomic nerves cannot be monitored by electrophysiology). However, such nerve identification during surgery can be difficult, especially in more complex surgical environments. Nerve structures may be obscured or distorted in complex surgical environments, such as those involving trauma, tumors, inflammation, previous surgeries, previous radiation treatments, or infections. Nerve visibility can be further limited in minimally invasive procedures such as endoscopy, which rely on small incisions with limited access. Indeed, accidental nerve damage is the leading complication associated with many surgeries using open and minimally invasive techniques.

[0003] These observations highlight the need for methods and tools to reliably and efficiently identify nerve cells or nerves. Electromyography (EMG) offers one approach for identifying motor nerves before direct exposure during surgery, or for confirming the identity of nerve cells or nerves in uncertain cases. However, a drawback of this technique is the lack of visual feedback to the surgeon. Even if a nerve is identified in one place due to accidental or intentional stimulation, there is no visual cue for the surgeon about how far the nerve is from the stimulation site or in which direction it moves away from the stimulation site. Furthermore, EMG tracks only motor nerves, not sensory or autonomic nerves. EMG may be useless for detecting temporary blockages of nerve cells or nerve conduction or neuromuscular transmission somewhere distal to the recording site. Such blockages are easily caused by nerve cell or nerve compression, trauma, local anesthetics, or neuromuscular blockers.

[0004] Other approaches involve labeling neurons or nerves that rely on retrograde or anterograde tracking of individually identified axonal pathways, primarily through the use of fluorescent dyes. However, methods for labeling neurons or nerves with locally applied fluorescent tracers can be limited. First, depending on where the dye is injected, this technique can only label one neuron or nerve fiber pathway at a time. Second, this technique results in limited labeling of the fluorescent dye along the axonal pathway because retrograde axonal tracers typically accumulate in the neuronal cell body. Third, retrograde transport is relatively slow (in millimeters per day), and therefore, labeling human neurons or nerves that are often longer than one meter, as in the case of sciatic nerve cells or nerves and their dendritic regions, takes a long time. Fourth, the application of fluorescent dyes to nerve impulse transmission targets, such as direct intramuscular injection for labeling motor neurons or nerves, typically results in a variable amount of tracer dye remaining at the injection site, which is messy. Since the dissection of nerve cells or nerves depends on the precise visualization of adjacent structures before they are encountered, a surgical site contaminated with fluorescent dyes is undesirable. Finally, the direct injection of fluorescent dyes itself can damage the target organ or the nerve cells or nerves of interest due to mechanical damage or extremely high local concentrations of the dye and medium at the injection site.

[0005] Despite these challenges, a promising approach is disclosed in WO2019 / 028281, the entirety of which is incorporated herein by reference, which provides an optimized peptide for human neuronal targeting and its use in image-guided surgery, diagnostics, and therapeutic delivery. In particular, the fluorescein-labeled neuronal targeting peptide known as "Bevonescein(ALM-488)" (i.e., Sequence ID No. 104 of WO2019 / 028281) (hereinafter also referred to here as the compound or peptide of formula (I)) is progressing to clinical trials (Phase 1 / 2 and Phase 3) to evaluate the safety, tolerability, and efficacy of this compound for intravenous (IV) administration in patients undergoing head and neck surgery. Figure 1 shows the structure of Bevonescein(ALM-488). [Overview of the project] [Problems that the invention aims to solve]

[0006] However, formulating Bevonescein (ALM-488) in a form suitable for use in these clinical trials has proven difficult, and the need remains to find a suitable formulation for IV administration. For this purpose, the present invention overcomes these difficulties and provides a formulation of Bevonescein (ALM-488) suitable for manufacture, storage, reconstitution, and IV infusion to patients. These and other aspects of the present invention will become apparent with reference to the following description. [Means for solving the problem]

[0007] overview In one embodiment, the present invention relates to (a) formula (I): [ka] The present invention provides an aqueous pharmaceutical composition comprising (b) a fluorescein-labeled neuronal targeting peptide, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, tautomer, racemate, or isotope thereof; (b) an additive comprising a pH adjuster, buffer, bulking agent, tonicity adjuster, preservative, or any combination thereof; and (c) an aqueous medium, wherein the pH of the composition is in the range of about 7 to about 9, and the concentration of the peptide in the aqueous composition is in the range of about 25 mg / mL to about 200 mg / mL based on the total weight of the aqueous composition.

[0008] Other embodiments relate to lyophilized pharmaceutical compositions and methods for producing lyophilized pharmaceutical compositions from the aqueous pharmaceutical compositions of the present invention.

[0009] Other embodiments relate to a reconstituted aqueous pharmaceutical composition and a method for forming a reconstituted aqueous pharmaceutical composition from the lyophilized pharmaceutical composition of the present invention.

[0010] Another aspect relates to a method for identifying nerve cells or nerves by bringing them into contact with the composition of the present invention.

[0011] Another aspect relates to a method of delivering at least one drug to a nerve cell or nerve by contacting the nerve cell or nerve with the composition of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] This patent or application includes drawings submitted in at least one color. Copies of this patent or application with color drawings are provided by the Office upon payment of the fees and requests.

[0013] [Figure 1] Structure of Bevonescein (ALM-488);

[0014] [Figure 2] HPLC chromatogram of a 0.25 mg / mL solution of ALM-488 as an HCl salt aqueous solution;

[0015] [Figure 3] Expanded view of the major peak in the HPLC chromatogram of FIG. 2;

[0016] [Figure 4] Flow diagram of the synthetic route used in the production of ALM-488;

[0017] [Figure 5] Thermogravimetric weight loss curve of the chloride salt of ALM-488;

[0018] [Figure 6] Plot showing the linear relationship between the peak area and amount of ALM-488 in the HPLC method of Table 3;

[0019] [Figure 7] pH titration curve showing how pH and the solubility of the HCl salt of ALM-488 are affected as a function of added NaOH;

[0020] [Figure 8] This is a superposition of pH titration curves showing how pH as a function of added NaOH and how the solubility of ALM-488's HCl and acetate salts are affected by pH;

[0021] [Figure 9] This is a superposition of magnified HPLC chromatograms of ALM-488 solutions at various pH values ​​stored at 40°C for 24 hours.

[0022] [Figure 10] This is a superposition of magnified chromatograms of ALM-488 solution (50 mg / mL) aqueous solutions analyzed initially and after 7 days at various storage temperatures;

[0023] [Figure 11] This is a pH titration curve showing how pH as a function of added tromethamine (Tris) and how the solubility of the HCl salt of ALM-488 are affected by pH;

[0024] [Figure 12] This is a pH titration curve showing how pH as a function of added meglumine (N-methylglucamine) and the solubility of the HCl salt of ALM-488 are affected by pH;

[0025] [Figure 13] This is a pH titration curve showing how pH as a function of added L-arginine and how the solubility of the HCl salt of ALM-488 is affected by pH;

[0026] [Figure 14] This is a superposition of the magnified HPLC chromatograms of Sample 8-4 from Example 8, taken during initial preparation, after storage at 40°C for 7 days, and after storage at 40°C for 14 days;

[0027] [Figure 15]This is a superposition of magnified HPLC chromatographs of samples 8-1 to 8-7 from Example 8 after storage at 40°C for 14 days;

[0028] [Figure 16] This is a superposition of DSC heating curves for arginine-containing ALM-488 formulations of samples 8-1, 8-3, 8-4, and 8-7;

[0029] [Figure 17] This is a superposition of DSC heating curves for the meglumine-containing ALM-488 formulation of sample 8-2;

[0030] [Figure 18] The freeze-drying process diagram for sample 10-1 is shown;

[0031] [Figure 19] The freeze-drying process diagram for sample 10-2 is shown;

[0032] [Figure 20] The freeze-drying process diagram for sample 10-3 is shown;

[0033] [Figure 21] Side views of the freeze-dried cakes of samples 10-1 and 10-2 are shown;

[0034] [Figure 22] This shows an overhead view of the freeze-dried cake removed from sample 10-2;

[0035] [Figure 23] The PXRD diffraction pattern of freeze-dried sample 10-1 is shown;

[0036] [Figure 24] The PXRD diffraction pattern of freeze-dried sample 10-2 is shown;

[0037] [Figure 25] The PXRD diffraction pattern of freeze-dried sample 10-3 is shown;

[0038] [Figure 26] The freeze-drying process diagram for Sample 11-1 is shown;

[0039] [Figure 27] This is a superposition of HPLC chromatography results of ALM-488 / arginine-trehalose freeze-dried products after storage at 40°C and 75% relative humidity for periods of 1 month, 2 months, and 3 months;

[0040] [Figure 28] Overlay of magnified HPLC chromatography images of ALM-488 / arginine-trehalose lyophilized products after storage at 40°C and 75% relative humidity for periods of 1 month, 2 months, and 3 months;

[0041] [Figure 29] The PXRD diffraction pattern of freeze-dried sample 10-3, stored for 3 months at 75% relative humidity in °C, is shown. [Modes for carrying out the invention]

[0042] Detailed description The present invention relates to liquid and solid compositions comprising the neuronal targeting peptide Bevonescein (ALM-488) (see Figure 1) that are suitable for administration to a target (e.g., intravenous administration) even after long-term storage of the composition at high temperature and / or high humidity. While the formulation of ALM-488 has proven difficult, the compositions and related methods of the present invention enable the safe long-term storage of this neuronal targeting peptide under less-than-ideal conditions without significant degradation, followed by intravenous administration in predictable and reproducible doses without the need for further purification or optimization. Examples 8-13 of this specification illustrate these advantages.

[0043] Terms and Definitions Before describing the present invention in further detail, it may be helpful to define some terms used herein. Further definitions are provided throughout this specification.

[0044] The term “essentially consisting of” is limited to materials or processes that specify the claimed scope and do not substantially affect the fundamental and novel features of the claimed invention. The singular expression here is understood to mean “one or more” of the listed components.

[0045] The use of options (e.g., "or") should be understood to mean one of the options, both, or any combination thereof.

[0046] The terms “contains” and “have” as used herein are synonymous, and these terms and their variations are intended to be interpreted non-restrictively. The term “contains” means the presence of a feature, integer, process, or component cited or described in a claim, but does not exclude the presence or addition of one or more other features, integers, processes, components, or groups thereof.

[0047] In this description, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value within the range described, and, where appropriate, fractions thereof (e.g., 1 / 10 and 1 / 100 of an integer), unless otherwise specified. Similarly, any numerical range described herein relating to any physical properties such as polymer subunits, size, or thickness is understood to include any integer within the range described, unless otherwise specified.

[0048] The ranges and quantities used herein can be expressed as "approximately" a specific value or range. "Approximately" also includes that exact amount. Therefore, for example, a pH value of "approximately 7" means a pH of approximately 7, and also means a pH of 7. In one embodiment, "approximately" means within 5% of a value. Therefore, a pH of "approximately 7" means a pH in the range of 6.65 to 7.35. In one embodiment, "approximately" means within 4% of a value. In one embodiment, "approximately" means within 3% of a value. In one embodiment, "approximately" means within 2% of a value. In one embodiment, "approximately" means within 1% of a value. In general, the term "approximately" includes an amount that is expected to be within the range of experimental error.

[0049] The term "ambient temperature" used here refers to room temperature, that is, approximately 20°C to 25°C (approximately 68°F to 77°F).

[0050] The term "aqueous medium" used here refers to a water-containing liquid suitable for intravenous injection into a subject.

[0051] The term "pH adjuster" as used herein refers to basic, acidic, or amphoteric organic compounds suitable for adjusting the pH of aqueous solutions.

[0052] The term "buffering agent" as used here refers to a basic, acidic, or amphoteric organic compound that, when present in an aqueous solution, prevents changes in the pH of the solution.

[0053] As used herein, the term "bulking agent" refers to a substance that may be added to a pharmaceutical product to provide it with structure, volume, and / or mass, thereby facilitating its more precise measurement and handling.

[0054] The term "tonic modifier" used here refers to a compound that modifies the osmotic pressure of a formulation, for example, to make it isotonic.

[0055] The term "preservative" as used herein refers to a substance that prevents or inhibits microbial growth and extends the shelf life of aqueous formulations.

[0056] The term "freeze-dried cake with good appearance" as used herein refers to a freeze-dried composition (i.e., freeze-dried product) that has a homogeneous, foamy plug appearance with little shrinkage and minimal cracking.

[0057] As used herein, the term "homogeneous" refers to a liquid solution consisting of only one phase (i.e., without a solid phase and without liquid delimiters). In one embodiment, the term "homogeneous" refers to an aqueous composition in which the solid-state neurotargeting peptide conjugate (i.e., Bevonescein (ALM-488)) is present in less than 5% by weight, based on filtration of the aqueous composition through a 0.22 micron Millipak Durapore® capsule filter unit.

[0058] The terms "freeze-drying," "to freeze-dry," and "freeze-dried" used herein refer to the process of first freezing the substance to be dried, and then removing the ice or freezing solvent by sublimation in a vacuum environment.

[0059] The term "freeze-dried material" used here refers to a freeze-dried solid.

[0060] The term "reconstituted composition" used herein refers to an aqueous composition formed by dissolving a freeze-dried material in an aqueous medium.

[0061] The term "salt" as used herein refers to the acid-addition or base-addition salt of the compounds described herein. "Salt" specifically includes "pharmaceutically acceptable salts."

[0062] As used herein, "pharmaceutically acceptable" means a substance that is relatively non-toxic (i.e., the toxicity of the substance far outweighs the benefits of the substance) without diminishing the biological activity or properties of the drugs described herein. In some cases, a pharmaceutically acceptable substance may be administered to an individual without significant undesirable biological effects or significant adverse interactions with any of the components of a composition containing it.

[0063] As used herein, “pharmaceutically acceptable additives” or “pharmaceutically acceptable carriers” refer to pharmaceutically acceptable substances, compositions, or media such as liquid or solid fillers, diluents, additives, solvents, or encapsulants that are non-toxic and non-inflammatory in the patient. Each additive must be “acceptable” in that it is compatible with the other components of the formulation and is not harmful to the patient. Additives may include, for example: anti-adhesion agents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), softeners, emulsifiers, fillers (diluents), film-forming agents or coatings, flavoring agents, fragrances, flow enhancers (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, or hydration water. Examples of pharmaceutical additives may include sterile liquids such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably used as additives, especially for injectable solutions. Suitable pharmaceutical additives are listed in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0064] As used herein, "central nervous system" or "CNS" refers to the brain, spinal cord, and retina.

[0065] The term "peripheral nervous system" or "PNS" used here refers to nerves and ganglia outside the central nervous system (CNS). The PNS is divided into the somatic nervous system and the autonomic nervous system. The somatic nervous system controls the entire voluntary muscle system and the processes of voluntary reflex arcs in the body, and consists of afferent or sensory nerves that relay sensations from the body to the central nervous system, and efferent or motor nerves that send commands from the CNS to the body and stimulate muscle contraction. The autonomic nervous system supplies smooth muscles and glands and therefore affects visceral functions. The autonomic nervous system operates mostly unconsciously and controls bodily functions such as heart rate, digestion, respiratory rate, pupillary response, urination, and sexual stimulation.

[0066] The term "nerve cell" as used here refers to an electrically excitable cell that processes and transmits information through electrical and chemical signaling. A typical nerve cell has a cell body (often called the nerve cell body), dendrites, and an axon.

[0067] The term "nerve" used here refers to a cable-like bundle containing nerve axons. Each nerve is a cord-like structure containing many axons. Each axon within a nerve is an extension of an individual nerve cell. Within a nerve, each axon is surrounded by a layer of connective tissue called the endoneurium. Axons are bundled into groups called fiber bundles, and each fiber bundle is encased in a layer of connective tissue called the perineurium. Each nerve is externally covered by a high-density sheath of connective tissue, the epineurium.

[0068] The term "conjugate" as used herein refers to a peptide conjugated to at least one cargo molecule, optionally via a linker.

[0069] The terms “individual,” “patient,” or “subject” are used interchangeably. Herein, they refer to any mammal (i.e., any species of any order, family, and genus within the taxonomic kingdom Animalia: phylum Chordata: subphylum Vertebrata: class Mammalia). In some embodiments, mammals include cattle, horses, sheep, pigs, cats, dogs, goats, mice, rats, rabbits, guinea pigs, non-human primates, or humans.

[0070] The terms “administer,” “to administer,” and “by administering” as used herein refer to methods that may be used to enable the delivery of a drug or composition to a desired site of biological activity. These methods include, but are not limited to, topical, oral, rectal, vaginal, nasal, inhalation, and non-enteral injection (e.g., intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, intravitreous, intradrip, or topical). Administration techniques, optionally used in conjunction with the drugs and methods described herein, include, for example, those disclosed in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa. Drug administration may be topical or systemic. In one embodiment, administration is via systemic intravenous injection into a human patient.

[0071] As used herein, the term “surgery” refers to any method that may be used to manipulate, change, or induce an effect through physical intervention. These methods include, but are not limited to, any procedure that may affect any nerve cells or nerves, such as open surgery, endoscopic surgery, laparoscopic surgery, minimally invasive surgery, robotic surgery, placement of retractors during spinal surgery, microscopic procedures, cardiac nerve or nerve ablation, epidural injection, intrathecal injection, nerve or nerve block, implantation of devices such as nerve cells or nerve stimulators, and implantation of pumps. In some embodiments, the subject of surgery is a human subject or human patient.

[0072] Pharmaceutical composition and manufacturing method In one embodiment, disclosed herein is a pharmaceutical composition comprising the neuronal targeting peptide conjugate “Bevonescein (ALM-488)” (i.e., Sequence ID No. 104 of WO2019 / 028281), shown below as formula (I), and pharmaceutically acceptable salts, solvates, hydrates, isomers, tautomers, racemates, or isotopes thereof. [ka]

[0073] In one embodiment, the purity of ALM-488 used in the preparation of the following aqueous and solid (e.g., freeze-dried) compositions is at least 95% by mass, based on the total mass of ALM-488. In other embodiments, the purity of ALM-488 is at least 96.0% by mass, or at least 96.5% by mass, or at least 97.0% by mass, or at least 97.5% by mass, or at least 98.0% by mass, or at least 98.5% by mass, or at least 99.0% by mass, based on the total mass of ALM-488.

[0074] In one embodiment, the ALM-488 peptide used in the preparation of the following aqueous and solid (e.g., lyophilized) compositions is in the form of a pharmaceutically acceptable salt. For example, a pharmaceutically acceptable salt may be formed from an inorganic acid, an organic acid, or a combination thereof. In one embodiment, a pharmaceutically acceptable salt may be an acetate, aspartate, benzoate, besylate, bromide, bicarbonate, carbonate, chloride, bisulfate, sulfate, camphor sulfonate, chloride, chlorotheophylline, citrate, ethanedisulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, malate These include iodates, malonates, mandelates, mesylates, methylsulfates, naphthoates, napsylates, nicotinates, nitrates, octadecanoates, oleates, oxalates, palmitates, pamoates, phosphates, hydrogen phosphates, dihydrogen phosphates, polygalacturonates, propions, stearates, succinates, sulfates, sulfosalicylates, tartrates, tosylates, trifluoroacetates, etc., or any combination thereof.

[0075] The pharmaceutical compositions herein are formulated using one or more physiologically acceptable carriers, including additives and adjuvants, that facilitate the processing of the compound of formula (I) into a pharmaceutically useful formulation. The appropriate formulation depends on the chosen route of administration. Outlines of the pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins, 1999), the full contents of which are incorporated herein by reference.

[0076] The compositions of the present invention (i.e., the aqueous pharmaceutical compositions, lyophilized pharmaceutical compositions, and reconstituted aqueous pharmaceutical compositions described below) may contain at least one pharmaceutically acceptable additive (also referred to herein as “additive”). Such additives may include, for example, pH adjusters, buffers, bulking agents, tonicity adjusters, preservatives, solvents, and combinations thereof. See, for example, Pramanick et al., Excipient selection in parenteral formulation development, Pharma Times, 2013, 45(3), 65-77, the full content of which is incorporated herein by reference.

[0077] pH adjusting agents that may be included in the composition of the present invention include, for example, alanine, arginine, cellulose, cellobiose, cyclodextrin, dextrose, polydextrose, erythritol, fructose, galactose, glucose, glycerin, glycine, histidine, inositol, lactose, maltose, mannitol, mannose, N-methylglucamine (meglumine), raffinose, ribose, sorbitol, sucrose, trehalose, tromethamine, xylitol, xylose, or any combination thereof.

[0078] Buffers that may be included in the composition of the present invention include, for example, calcium acetate, sodium acetate, potassium distartrate, sodium borate, calcium chloride, potassium chloride, sodium chloride, citric acid, potassium citrate, sodium citrate, lactic acid, potassium lactate, sodium lactate, ammonium phosphate (monobasic and dibasic), potassium phosphate (monobasic and dibasic), sodium phosphate (monobasic and dibasic), succinic acid, magnesium sulfate, or any combination thereof.

[0079] Buffers that may be included in the composition of the present invention include, for example, Tris buffer, Tris-Cl buffer, histidine buffer, TAE buffer, HEPES buffer, TBE buffer, sodium phosphate buffer, MES buffer, ammonium sulfate buffer, potassium phosphate buffer, potassium thiocyanate buffer, succinate buffer, tartaric acid buffer, DIPSO buffer, HEPPSO buffer, POPSO buffer, PIPES buffer, PBS buffer, MOPS buffer, acetate buffer, phosphate buffer, cacodylic acid buffer, glycine buffer, sulfate buffer, and This includes midazole buffer, guanidine hydrochloride buffer, phosphate-citric acid buffer, borate buffer, malonic acid buffer, 3-picoline buffer, 2-picoline buffer, 4-picoline buffer, 3,5-lutidine buffer, 3,4-lutidine buffer, 2,4-lutidine buffer, Aces buffer, diethylmalonic acid buffer, N-methylimidazole buffer, 1,2-dimethylimidazole buffer, TAPS buffer, bis-Tris buffer, L-arginine buffer, lactate buffer, glycolic acid buffer, etc., or any combination thereof.

[0080] The bulking agents that may be included in the composition of the present invention are, for example, alanine, arginine, calcium chloride, calcium sulfate, cellulose, dextran 40, dextrin (e.g., maltodextrin), dextrose, polydextrose, dicalcium phosphate dihydrate, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol (PEG), β-glucan, glycerin, diglycerin, polyglycerin, glycine, histidine, hydrocolloids (e.g., gum arabic, pectin, guar gum, alginic acid, carrageenan, xanthan gum, cellulose gum, etc.), inulin, kaolin, magnesium chloride, aluminum magnesium silicate, meg This includes lumin (N-methylglucamine), monosaccharides (e.g., glucose, galactose, mannose, fructose, inositol, ribose, xylose, etc.), disaccharides (e.g., lactose, sucrose, cellobiose, trehalose, maltose, etc.), trisaccharides (e.g., raffinose, etc.), polysaccharides (e.g., cyclodextrin, etc.), sugar alcohols (e.g., erythritol, xylitol, sorbitol, mannitol, maltitol, etc.), potassium chloride, propylene glycol (PG), polypropylene glycol, polyvinylpyrrolidone (PVP), poloxamer, silicon dioxide, sodium chloride, starch, titanium dioxide, tromethamine, or any combination thereof.

[0081] The tonicity modifiers that may be included in the composition of the present invention include, for example, alanine, arginine, calcium chloride, calcium sulfate, cellulose, dextran 40, dextrin (e.g., maltodextrin), dextrose, polydextrose, dicalcium phosphate dihydrate, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol (PEG), β-glucan, glycerin, diglycerin, polyglycerin, glycine, histidine, hydrocolloids (e.g., gum arabic, pectin, guar gum, alginic acid, carrageenan, xanthan gum, cellulose gum, etc.), inulin, kaolin, magnesium chloride, aluminum magnesium silicate, and This includes gluten (N-methylglucamine), monosaccharides (e.g., glucose, galactose, mannose, fructose, inositol, ribose, xylose, etc.), disaccharides (e.g., lactose, sucrose, cellobiose, trehalose, maltose, etc.), trisaccharides (e.g., raffinose, etc.), polysaccharides (e.g., cyclodextrin, etc.), sugar alcohols (e.g., erythritol, xylitol, sorbitol, mannitol, maltitol, etc.), potassium chloride, propylene glycol (PG), polypropylene glycol, polyvinylpyrrolidone (PVP), poloxamer, silicon dioxide, sodium chloride, starch, titanium dioxide, tromethamine, or any combination thereof.

[0082] The preservatives that may be included in the composition of the present invention include, for example, benzoic acid, sodium benzoate, benzyl alcohol, benzalkonium chloride, bronidol, bronopol, butylparaben, chlorobutanol, methyl cresol, chlorocresol, methylparaben, phenol, phenoxyethanol, phenylethyl alcohol, propylene glycol, propylparaben, sorbic acid, thimerosal, or any combination thereof.

[0083] In some embodiments, the compositions of the pharmaceutical invention (i.e., the aqueous pharmaceutical compositions, lyophilized pharmaceutical compositions, and reconstituted aqueous pharmaceutical compositions described below) may contain other agents or pharmaceuticals, carriers, adjuvants, such as preservatives, stabilizers, wetting agents or emulsifiers, dissolution accelerators, osmotic salts, and / or buffers. Furthermore, the pharmaceutical compositions may also contain other therapeutically valuable substances.

[0084] Aqueous pharmaceutical composition One embodiment relates to an aqueous pharmaceutical composition comprising (a) a fluorescein-labeled neurotargeting peptide of formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, isomer, tautomer, racemate, or isotope thereof; (b) an additive comprising a pH adjuster, buffer, filler, tonicity adjuster, preservative, or any combination thereof; and (c) an aqueous medium. In one embodiment, the pH of the aqueous composition is in the range of about 5 to about 12, and the concentration of the neurotargeting peptide in the aqueous composition is in the range of about 10 mg / mL to about 500 mg / mL, based on the total weight of the aqueous composition.

[0085] In one embodiment, the peptide of formula (I) (i.e., Bevonescein(ALM-488)) is in the form of the pharmaceutically acceptable salt described above. For example, in one embodiment, the aqueous pharmaceutical composition comprises an HCl salt of the neurotargeting peptide. The chloride content of the HCl salt may range from about 0.1% (w / w) to about 5.0% (w / w), or from about 0.1% (w / w) to about 1.0% (w / w), or from about 1.0% (w / w) to about 2.0% (w / w), or from about 2.0% (w / w) to about 3.0% (w / w), or from about 3.0% (w / w) to about 4.0% (w / w), or from about 4.0% (w / w) to about 5.0% (w / w), based on the total mass of the peptide in the aqueous composition.

[0086] In one embodiment, the molar ratio of peptide to pharmaceutically acceptable salt in the aqueous composition is in the range of about 1:1 to about 1:3, or about 1:1 to about 1:2, or about 1:1 to about 1:1.9, or about 1:1 to about 1:1.8, or about 1:1 to about 1.7, or about 1:1 to about 1:1.6, or about 1:1 to about 1:1.5, or about 1:1 to about 1:1.4, or about 1:1 to about 1:1.3, or about 1:1 to about 1:1.2.

[0087] In one embodiment, the additives to the aqueous composition include a pH adjuster, a buffer, a buffer solution, a volume extender, a tonicity adjuster, a preservative, at least one solvent, and combinations thereof. Such additives may include, for example, the above-mentioned pH adjuster, buffer, buffer solution, volume extender, tonicity adjuster, and preservative.

[0088] In one embodiment, the additives to the aqueous composition include alanine, arginine, cellulose, cellobiose, cyclodextrin, dextrose, erythritol, fructose, galactose, glucose, glycerin, glycine, histidine, inositol, lactose, maltose, mannitol, mannose, meglumine, raffinose, ribose, sorbitol, sucrose, trehalose, tromethamine, xylitol, xylose, or any combination thereof. In another embodiment, the additives include arginine, glycine, mannitol, meglumine, trehalose, tromethamine, or any combination thereof.

[0089] In one embodiment, the additives to the aqueous composition include a first additive comprising arginine, meglumine, glycine, or any combination thereof; and a second additive optionally comprising mannitol, trehalose, or both. In one embodiment, the mass ratio of the first additive to the second additive is approximately 10:1 to approximately 1:10, or approximately 10:1 to approximately 9:1, or approximately 9:1 to approximately 8:1, or approximately 8:1 to approximately 7:1, or approximately 7:1 to approximately 6:1, or approximately 6:1 to approximately 5:1, or approximately 5:1 to approximately 4:1, or approximately 4:1 to approximately 3:1, or approximately 3:1 to approximately 2:1, or approximately 2:1 to approximately 1:0, or approximately 1.8:1 to approximately 1.7:1, or approximately 1.7:1 to approximately 1.6:1, or approximately 1.6:1 to approximately 1.5:1, or approximately 1.5:1 to approximately 1.4:1, or approximately 1.4:1 to approximately 1.3:1, or approximately 1.3:1 to approximately 1.2:1, or approximately 1.2: The range is approximately 1 to 1.1:1, or approximately 1:1.1 to 1:1.2, or approximately 1:1 to 1:2.5, or approximately 1:1.2 to 1:1.3, or approximately 1:1.3 to 1:1.4, or approximately 1:1.4 to 1:1.5, or approximately 1:1.5 to 1:1.6, or approximately 1:1.6 to 1:1.8, or approximately 1:1.8 to 1:1.9, or approximately 1:1.9 to 1:2, or approximately 1:2 to 1:3, or approximately 1:3 to 1:4, or approximately 1:4 to 1:5, or approximately 1:5 to 1:6, or approximately 1:6 to 1:7, or approximately 1:7 to 1:8, or approximately 1:8 to 1:9, or approximately 1:9 to 1:10.

[0090] In one embodiment, the additive to the aqueous composition includes arginine, arginine-mannitol, arginine-trehalose, arginine-meglumine, meglumine, meglumine-glycine, or meglumine-mannitol. In another embodiment, the additive includes arginine-trehalose. In one embodiment, the mass ratio of arginine to trehalose is approximately 10:1 to 1:10, or approximately 10:1 to 9:1, or approximately 9:1 to 8:1, or approximately 8:1 to 7:1, or approximately 7:1 to 6:1, or approximately 6:1 to 5:1, or approximately 5:1 to 4:1, or approximately 4:1 to 3:1, or approximately 3:1 to 2:1, or approximately 2:1 to 1:0, or approximately 1.8:1 to 1.7:1, or approximately 1.7:1 to 1.6:1, or approximately 1.6:1 to 1.5:1, or approximately 1.5:1 to 1.4:1, or approximately 1.4:1 to 1.3:1, or approximately 1.3:1 to 1.2:1, or approximately 1.2: The range is approximately 1 to 1.1:1, or approximately 1:1.1 to 1:1.2, or approximately 1:1 to 1:2.5, or approximately 1:1.2 to 1:1.3, or approximately 1:1.3 to 1:1.4, or approximately 1:1.4 to 1:1.5, or approximately 1:1.5 to 1:1.6, or approximately 1:1.6 to 1:1.8, or approximately 1:1.8 to 1:1.9, or approximately 1:1.9 to 1:2, or approximately 1:2 to 1:3, or approximately 1:3 to 1:4, or approximately 1:4 to 1:5, or approximately 1:5 to 1:6, or approximately 1:6 to 1:7, or approximately 1:7 to 1:8, or approximately 1:8 to 1:9, or approximately 1:9 to 1:10.

[0091] In one embodiment, the mass ratio of peptide to additive in the aqueous composition is in the range of about 5:1 to about 1:5. In another embodiment, the mass ratio of peptide to additive in the aqueous composition is about 5:1 to about 4.5:1, or about 4.5:1 to about 4.0:1, or about 4.0:1 to about 3.5:1, or about 3.5:1 to about 3.0:1, or about 3.0:1 to about 2.5:1, or about 2.5:1 to about 2.0:1, or about 2.0:1 to about 1.5:1, or about 1.5:1 to about 1.0:1, or about 1. The ratios range from 0:1 to approximately 0.5:1, or from approximately 1:0.5 to approximately 1:1.0, or from approximately 1:1.0 to approximately 1:1.5, or from approximately 1:1.5 to approximately 1:2.0, or from approximately 1:2.0 to approximately 1:2.5, or from approximately 1:2.5 to approximately 1:3.0, or from approximately 1:3.0 to approximately 1:3.5, or from approximately 1:3.5 to approximately 1:4.0, or from approximately 1:4.0 to approximately 1:4.5, or from approximately 1:4.5 to approximately 1:4.0. In other embodiments, the mass ratio of peptide to additive is in the range of approximately 1.0 to approximately 1.5.

[0092] In one embodiment, the aqueous medium in the aqueous composition includes sterile water, saline solution, aqueous buffer solution, or any combination thereof. In another embodiment, the aqueous medium includes sterile water, physiological saline, half physiological saline, or any combination thereof.

[0093] In one embodiment, the pH of the aqueous composition is approximately 5 to approximately 12, or approximately 5 to approximately 6, or approximately 6 to approximately 7, or approximately 7.0 to approximately 7.1, or approximately 7.1 to approximately 7.2, or approximately 7.2 to approximately 7.3, or approximately 7.3 to approximately 7.4, or approximately 7.4 to approximately 7.5, or approximately 7.5 to approximately 7.6, or approximately 7.6 to approximately 7.7, or approximately 7.7 to approximately 7.8, or approximately 7.8 to approximately 7.9, or approximately 7.9 to approximately The pH ranges from 8.0, or approximately 8.0 to 8.1, or approximately 8.1 to 8.2, or approximately 8.2 to 8.3, or approximately 8.3 to 8.4, or approximately 8.4 to 8.5, or approximately 8.5 to 8.6, or approximately 8.6 to 8.7, or approximately 8.7 to 8.8, or approximately 8.8 to 8.9, or approximately 8.9 to 9.0, or approximately 9 to 10, or approximately 10 to 11, or approximately 11 to 12. In one embodiment, the pH of the aqueous composition is in the range of approximately 7.2 to 7.8.

[0094] In one embodiment, the concentration of the peptide (i.e., Bevonescein (ALM-488)) in the aqueous composition is approximately 20 mg / mL to approximately 30 mg / mL, or approximately 30 mg / mL to approximately 40 mg / mL, or approximately 40 mg / mL to approximately 50 mg / mL, or approximately 50 mg / mL to approximately 60 mg / mL, or approximately 60 mg / mL to approximately 70 mg / mL, or approximately 70 mg / mL to approximately 80 mg / mL, or approximately 80 mg / mL to approximately 90 mg / mL, or approximately 90 mg / mL to approximately 100 mg / mL, or approximately 100 mg / mL to approximately 110 mg / mL, or approximately 110 mg / mL to approximately 120 mg / mL, or approximately 120 mg / mL. The range is approximately 130 mg / mL, or approximately 130 mg / mL to approximately 140 mg / mL, or approximately 140 mg / mL to approximately 150 mg / mL, or approximately 150 mg / mL to approximately 160 mg / mL, or approximately 160 mg / mL to approximately 170 mg / mL, or approximately 170 mg / mL to approximately 180 mg / mL, or approximately 180 mg / mL to approximately 190 mg / mL, or approximately 190 mg / mL to approximately 200 mg / mL, or approximately 200 mg / mL to approximately 250 mg / mL, or approximately 250 mg / mL to approximately 300 mg / mL, or approximately 300 mg / mL to approximately 350 mg / mL, or approximately 350 mg / mL to approximately 400 mg / mL, or approximately 400 mg / mL to approximately 450 mg / mL. In one embodiment, the concentration of the peptide in the aqueous composition is in the range of about 25 mg / mL to about 200 mg / mL, based on the total volume of the aqueous composition. In another embodiment, the concentration of the peptide in the aqueous composition is in the range of about 40 mg / mL to about 75 mg / mL, based on the total volume of the aqueous composition.

[0095] In one embodiment, the density of the aqueous composition is in the range of about 0.6 g / mL to about 1.3 g / mL. In another embodiment, the density of the aqueous composition is in the range of about 0.6 g / mL to about 0.7 g / mL, or about 0.7 g / mL to about 0.8 g / mL, or about 0.8 g / mL to about 0.85 g / mL, or about 0.85 g / mL to about 0.90 g / mL, or about 0.90 g / mL to about 0.95 g / mL, or about 0.95 g / mL to about 1.0 g / mL, or about 1.0 g / mL to about 1.05 g / mL, or about 1.05 g / mL to about 1.10 g / mL, or about 1.10 g / mL to about 1.15 g / mL, or about 1.15 g / mL to about 1.20 g / mL, or about 1.20 g / mL to about 1.25 g / mL. In other embodiments, the density of the aqueous composition is in the range of about 1.0 g / mL to about 1.08 g / mL.

[0096] In one embodiment, the osmotic pressure of the aqueous composition is in the range of approximately 150 mOsm / kg to approximately 400 mOsm / kg. In another embodiment, the osmotic pressure of the aqueous composition is in the range of approximately 150 mOsm / kg to approximately 175 mOsm / kg, or approximately 175 mOsm / kg to approximately 200 mOsm / kg, or approximately 200 mOsm / kg to approximately 225 mOsm / kg, or approximately 225 mOsm / kg to approximately 250 mOsm / kg, or approximately 250 mOsm / kg to approximately 275 mOsm / kg, or approximately 275 mOsm / kg to approximately 300 mOsm / kg, or approximately 300 mOsm / kg to approximately 325 mOsm / kg, or approximately 325 mOsm / kg to approximately 350 mOsm / kg. In other embodiments, the osmotic pressure of the aqueous composition is in the range of approximately 280 mOsm / kg to approximately 300 mOsm / kg.

[0097] In one embodiment, the volume of the aqueous composition is in the range of about 0.5 mL to about 5.0 mL. In another embodiment, the volume of the aqueous composition is in the range of about 0.5 mL to about 1.0 mL, or about 1.0 mL to about 1.5 mL, or about 1.5 mL to about 2.0 mL, or about 2.0 mL to about 2.5 mL, or about 2.5 mL to about 3.0 mL, or about 3.0 mL to about 3.5 mL, or about 3.5 mL to about 4.0 mL, or about 4.0 mL to about 4.5 mL. In yet another embodiment, the volume of the aqueous composition is in the range of about 1.8 mL to about 2.2 mL.

[0098] In one embodiment, the aqueous composition is filtered. In another embodiment, however, the aqueous composition is not filtered. In one embodiment, the aqueous composition is homogeneous.

[0099] In one embodiment, the aqueous composition contains at least 95% by mass of peptide (i.e., Bevonescein (ALM-488)) relative to the total mass of the aqueous composition after being stored at 40°C for 14 days. In another embodiment, the aqueous composition contains at least 96.0% by mass, or at least 96.5% by mass, or at least 97.0% by mass, or at least 97.5% by mass, or at least 98.0% by mass, or at least 98.5% by mass, or at least 99.0% by mass, or at least 99.5% by mass of peptide, relative to the total mass of the aqueous composition after being stored at 40°C for 14 days.

[0100] Freeze-dried pharmaceutical composition and method of manufacture Other embodiments relate to a lyophilized pharmaceutical composition comprising (a) a fluorescein-labeled neuronal targeting peptide of formula (I) (i.e., Bevonescein (ALM-488)) or a pharmaceutically acceptable salt, solvate, hydrate, isomer, tautomer, racemate, or isotope thereof; and (b) an additive comprising a pH adjuster, buffer, bulking agent, tonicity adjuster, preservative, or any combination thereof, wherein the water content of the lyophilized composition is in the range of about 0.2% (w / w) to about 2.0% (w / w) relative to the total mass of the lyophilized composition.

[0101] In one embodiment, when the freeze-dried composition is mixed with an aqueous medium at ambient temperature, it forms a homogeneous aqueous composition having a peptide (i.e., Bevonescein (ALM-488)) concentration of about 50 mg / mL to about 500 mg / mL. In one embodiment, the peptide concentration of the homogeneous aqueous composition formed from the freeze-dried composition is about 50 mg / mL to about 75 mg / mL, or about 75 mg / mL to about 100 mg / mL, or about 100 mg / mL to about 125 mg / mL, or about 125 mg / mL to about 150 mg / mL, or about 150 mg / mL to about 175 mg / mL, or about 175 mg / mL to about 200 mg / mL, or about 200 mg / mL to about 225 mg / mL, or about 225 mg / mL. The range is g / mL to approximately 250 mg / mL, or approximately 250 mg / mL to approximately 275 mg / mL, or approximately 275 mg / mL to approximately 300 mg / mL, or approximately 300 mg / mL to approximately 325 mg / mL, or approximately 325 mg / mL to approximately 350 mg / mL, or approximately 350 mg / mL to approximately 375 mg / mL, or approximately 375 mg / mL to approximately 400 mg / mL, or approximately 400 mg / mL to approximately 425 mg / mL, or approximately 425 mg / mL to approximately 450 mg / mL. In another embodiment, the freeze-dried composition, when mixed with an aqueous medium at ambient temperature, forms a homogeneous aqueous composition having a peptide concentration of 200 mg / mL or less.

[0102] In one embodiment, the freeze-dried composition is in the form of a freeze-dried cake having a good appearance.

[0103] In one embodiment, the lyophilized composition contains at least 95% by mass of peptide (i.e., Bevonescein (ALM-488)) after being stored for three months at 40°C and 75% relative humidity. In another embodiment, the lyophilized composition contains at least 95% by mass, or at least 95.5% by mass, or at least 96.0% by mass, or at least 96.5% by mass, or at least 97.0% by mass, or about 97.5% by mass, or at least 98.0% by mass, or at least 98.5% by mass, or at least 99.0% by mass, or at least 99.5% by mass of peptide (i.e., Bevonescein (ALM-488)) after being stored for three months at 40°C and 75% relative humidity.

[0104] In one embodiment, the peptide of formula (I) (i.e., Bevonescein(ALM-488)) is in the form of the pharmaceutically acceptable salt described above. For example, in one embodiment, the lyophilized composition contains an HCl salt of the peptide. The chloride content of the HCl salt may range from 0.1% (w / w) to about 5.0% (w / w), or about 0.1% (w / w) to about 1.0% (w / w), or about 1.0% (w / w) to about 2.0% (w / w), or about 2.0% (w / w) to about 3.0% (w / w), or about 3.0% (w / w) to about 4.0% (w / w), or about 4.0% (w / w) to about 5.0% (w / w), based on the total mass of the peptide in the lyophilized composition.

[0105] In one embodiment, the molar ratio of peptide to pharmaceutically acceptable salt in the freeze-dried composition is in the range of about 1:1 to about 1:3, or about 1:1 to about 1:2, or about 1:1 to about 1:1.9, or about 1:1 to about 1:1.8, or about 1:1 to about 1.7, or about 1:1 to about 1:1.6, or about 1:1 to about 1:1.5, or about 1:1 to about 1:1.4, or about 1:1 to about 1:1.3, or about 1:1 to about 1:1.2.

[0106] In one embodiment, the additives contained in the freeze-dried composition include pH adjusters, buffers, buffer solutions, bulking agents, tonicity adjusters, preservatives, and combinations thereof. Such additives may include, for example, the above-mentioned pH adjusters, buffers, buffer solutions, bulking agents, tonicity adjusters, and preservatives.

[0107] In one embodiment, the additives included in the freeze-dried composition include alanine, arginine, cellulose, cellobiose, cyclodextrin, dextrose, erythritol, fructose, galactose, glucose, glycerin, glycine, histidine, inositol, lactose, maltose, mannitol, mannose, meglumine, raffinose, ribose, sorbitol, sucrose, trehalose, tromethamine, xylitol, xylose, or any combination thereof. In one embodiment, the additives include arginine, glycine, mannitol, meglumine, trehalose, tromethamine, or any combination thereof.

[0108] In one embodiment, the additives included in the freeze-dried composition include a first additive comprising arginine, meglumine, glycine, or any combination thereof; and a second additive optionally comprising mannitol, trehalose, or both. In one embodiment, the mass ratio of the first additive to the second additive is approximately 10:1 to approximately 1:10, or approximately 10:1 to approximately 9:1, or approximately 9:1 to approximately 8:1, or approximately 8:1 to approximately 7:1, or approximately 7:1 to approximately 6:1, or approximately 6:1 to approximately 5:1, or approximately 5:1 to approximately 4:1, or approximately 4:1 to approximately 3:1, or approximately 3:1 to approximately 2:1, or approximately 2:1 to approximately 1:0, or approximately 1.8:1 to approximately 1.7:1, or approximately 1.7:1 to approximately 1.6:1, or approximately 1.6:1 to approximately 1.5:1, or approximately 1.5:1 to approximately 1.4:1, or approximately 1.4:1 to approximately 1.3:1, or approximately 1.3:1 to approximately 1.2:1, or approximately 1.2: The range is approximately 1 to 1.1:1, or approximately 1:1.1 to 1:1.2, or approximately 1:1 to 1:2.5, or approximately 1:1.2 to 1:1.3, or approximately 1:1.3 to 1:1.4, or approximately 1:1.4 to 1:1.5, or approximately 1:1.5 to 1:1.6, or approximately 1:1.6 to 1:1.8, or approximately 1:1.8 to 1:1.9, or approximately 1:1.9 to 1:2, or approximately 1:2 to 1:3, or approximately 1:3 to 1:4, or approximately 1:4 to 1:5, or approximately 1:5 to 1:6, or approximately 1:6 to 1:7, or approximately 1:7 to 1:8, or approximately 1:8 to 1:9, or approximately 1:9 to 1:10.

[0109] In one embodiment, the additives included in the freeze-dried composition include arginine, arginine-mannitol, arginine-trehalose, arginine-meglumine, meglumine, meglumine-glycine, or meglumine-mannitol. In another embodiment, the additive includes arginine-trehalose. In one embodiment, the mass ratio of arginine to trehalose is approximately 10:1 to 1:10, or approximately 10:1 to 9:1, or approximately 9:1 to 8:1, or approximately 8:1 to 7:1, or approximately 7:1 to 6:1, or approximately 6:1 to 5:1, or approximately 5:1 to 4:1, or approximately 4:1 to 3:1, or approximately 3:1 to 2:1, or approximately 2:1 to 1:0, or approximately 1.8:1 to 1.7:1, or approximately 1.7:1 to 1.6:1, or approximately 1.6:1 to 1.5:1, or approximately 1.5:1 to 1.4:1, or approximately 1.4:1 to 1.3:1, or approximately 1.3:1 to 1.2:1, or approximately 1.2: The range is approximately 1 to 1.1:1, or approximately 1:1.1 to 1:1.2, or approximately 1:1 to 1:2.5, or approximately 1:1.2 to 1:1.3, or approximately 1:1.3 to 1:1.4, or approximately 1:1.4 to 1:1.5, or approximately 1:1.5 to 1:1.6, or approximately 1:1.6 to 1:1.8, or approximately 1:1.8 to 1:1.9, or approximately 1:1.9 to 1:2, or approximately 1:2 to 1:3, or approximately 1:3 to 1:4, or approximately 1:4 to 1:5, or approximately 1:5 to 1:6, or approximately 1:6 to 1:7, or approximately 1:7 to 1:8, or approximately 1:8 to 1:9, or approximately 1:9 to 1:10.

[0110] In one embodiment, the mass ratio of peptide to additive in the lyophilized composition is in the range of about 5:1 to about 1:5. In one embodiment, the mass ratio of peptide to additive in the lyophilized composition is about 5:1 to about 4.5:1, or about 4.5:1 to about 4.0:1, or about 4.0:1 to about 3.5:1, or about 3.5:1 to about 3.0:1, or about 3.0:1 to about 2.5:1, or about 2.5:1 to about 2.0:1, or about 2.0:1 to about 1.5:1, or about 1.5:1 to about 1.0:1, or about 1 The ratios range from 0:1 to approximately 0.5:1, or approximately 1:0.5 to approximately 1:1.0, or approximately 1:1.0 to approximately 1:1.5, or approximately 1:1.5 to approximately 1:2.0, or approximately 1:2.0 to approximately 1:2.5, or approximately 1:2.5 to approximately 1:3.0, or approximately 1:3.0 to approximately 1:3.5, or approximately 1:3.5 to approximately 1:4.0, or approximately 1:4.0 to approximately 1:4.5, or approximately 1:4.5 to approximately 1:4.0. In other embodiments, the peptide-to-additive mass ratio is in the range of approximately 1.0 to approximately 1.5.

[0111] Other embodiments relate to methods for forming lyophilized pharmaceutical compositions by freeze-drying the aqueous compositions disclosed herein. In one embodiment, freeze-drying includes freezing the aqueous composition at a temperature of about -100°C to about -45°C to obtain a frozen solid composition. In one embodiment, the frozen solid composition is formed by freezing the aqueous composition at a temperature in the range of about -100°C to about -90°C, or about -90°C to about -80°C, or about -80°C to about -70°C, or about -70°C to about -60°C, or about -60°C to about -50°C. In another embodiment, freeze-drying includes freezing the aqueous composition at a temperature in the range of about -80°C to about -50°C to obtain a frozen solid composition.

[0112] In one embodiment, freeze-drying further includes annealing of the freeze-solid composition at a temperature in the range of about -40°C to about -20°C. In one embodiment, annealing occurs at a temperature in the range of about -40°C to about -35°C, or about -35°C to about -30°C, or about -30°C to about -25°C, or about -25°C to about -20°C.

[0113] In one embodiment, freeze-drying further includes drying the freeze-solid composition at a temperature in the range of about -20°C to room temperature and at a pressure in the range of about 100 mTorr to about 300 mTorr to obtain a freeze-dried composition. In one embodiment, the drying temperature is in the range of about -20°C to about -15°C, or about -15°C to about -10°C, or about -10°C to about -5°C, or about -5°C to about 0°C, or about 0°C to about 5°C, or about 5°C to about 10°C, or about 10°C to about 15°C, or about 15°C to about 20°C, or about 20°C to about 25°C, at a pressure in the range of about 100 mTorr to about 300 mTorr.

[0114] In one embodiment, drying includes (i) primary drying of the frozen solid composition at a temperature in the range of about -20°C to about 0°C at a primary pressure in the range of about 100 mTorr to about 300 mTorr; and (ii) secondary drying of the frozen solid composition at a temperature in the range of about 0°C to room temperature at a secondary pressure in the range of about 100 mTorr to about 300 mTorr.

[0115] Other embodiments include lyophilized pharmaceutical compositions formed by the lyophilization method described herein.

[0116] Reconstituted aqueous pharmaceutical composition and method of production Other embodiments relate to reconstituted aqueous pharmaceutical compositions comprising ALM-488 and its pharmaceutically acceptable salts, solvates, hydrates, isomers, tautomers, racemates, or isotopes, formed by combining the lyophilized compositions described herein with an aqueous medium.

[0117] In one embodiment, the aqueous medium used to prepare the reconstituted aqueous composition includes sterile water, saline solution, aqueous buffer solution, or any combination thereof. In another embodiment, the aqueous medium includes sterile water, physiological saline, half physiological saline, or any combination thereof.

[0118] In one embodiment, the reconstituted aqueous composition is filtered. In another embodiment, however, the reconstituted aqueous composition is not filtered. In one embodiment, the reconstituted composition is homogeneous.

[0119] In one embodiment, the peptide of formula (I) (i.e., Bevonescein(ALM-488)) contained in the reconstituted aqueous composition is in the form of the pharmaceutically acceptable salt described above. For example, in one embodiment, the reconstituted aqueous pharmaceutical composition contains an HCl salt of the neurotargeting peptide. The chloride content of the HCl salt may range from about 0.1% (w / w) to about 5.0% (w / w), or from about 0.1% (w / w) to about 1.0% (w / w), or from about 1.0% (w / w) to about 2.0% (w / w), or from about 2.0% (w / w) to about 3.0% (w / w), or from about 3.0% (w / w) to about 4.0% (w / w), or from about 4.0% (w / w) to about 5.0% (w / w), based on the total mass of the peptide in the reconstituted aqueous composition.

[0120] In one embodiment, the molar ratio of peptide to pharmaceutically acceptable salt in the reconstituted aqueous composition is in the range of about 1:1 to about 1:3, or about 1:1 to about 1:2, or about 1:1 to about 1:1.9, or about 1:1 to about 1:1.8, or about 1:1 to about 1.7, or about 1:1 to about 1:1.6, or about 1:1 to about 1:1.5, or about 1:1 to about 1:1.4, or about 1:1 to about 1:1.3, or about 1:1 to about 1:1.2.

[0121] In one embodiment, the additives in the reconstituted aqueous composition include a pH adjuster, a buffer, a buffer solution, a volume extender, a tonicity adjuster, a preservative, at least one solvent, and combinations thereof. Such additives may include, for example, the above-mentioned pH adjuster, buffer, buffer solution, volume extender, tonicity adjuster, and preservative.

[0122] In one embodiment, the additives in the reconstituted aqueous composition include alanine, arginine, cellulose, cellobiose, cyclodextrin, dextrose, erythritol, fructose, galactose, glucose, glycerin, glycine, histidine, inositol, lactose, maltose, mannitol, mannose, meglumine, raffinose, ribose, sorbitol, sucrose, trehalose, tromethamine, xylitol, xylose, or any combination thereof. In one embodiment, the additives include arginine, glycine, mannitol, meglumine, trehalose, tromethamine, or any combination thereof.

[0123] In one embodiment, the additives in the reconstituted aqueous composition include a first additive comprising arginine, meglumine, glycine, or any combination thereof; and a second additive optionally comprising mannitol, trehalose, or both. In one embodiment, the mass ratio of the first additive to the second additive is approximately 10:1 to approximately 1:10, or approximately 10:1 to approximately 9:1, or approximately 9:1 to approximately 8:1, or approximately 8:1 to approximately 7:1, or approximately 7:1 to approximately 6:1, or approximately 6:1 to approximately 5:1, or approximately 5:1 to approximately 4:1, or approximately 4:1 to approximately 3:1, or approximately 3:1 to approximately 2:1, or approximately 2:1 to approximately 1:0, or approximately 1.8:1 to approximately 1.7:1, or approximately 1.7:1 to approximately 1.6:1, or approximately 1.6:1 to approximately 1.5:1, or approximately 1.5:1 to approximately 1.4:1, or approximately 1.4:1 to approximately 1.3:1, or approximately 1.3:1 to approximately 1.2:1, or approximately 1.2: The range is approximately 1 to 1.1:1, or approximately 1:1.1 to 1:1.2, or approximately 1:1 to 1:2.5, or approximately 1:1.2 to 1:1.3, or approximately 1:1.3 to 1:1.4, or approximately 1:1.4 to 1:1.5, or approximately 1:1.5 to 1:1.6, or approximately 1:1.6 to 1:1.8, or approximately 1:1.8 to 1:1.9, or approximately 1:1.9 to 1:2, or approximately 1:2 to 1:3, or approximately 1:3 to 1:4, or approximately 1:4 to 1:5, or approximately 1:5 to 1:6, or approximately 1:6 to 1:7, or approximately 1:7 to 1:8, or approximately 1:8 to 1:9, or approximately 1:9 to 1:10.

[0124] In one embodiment, the additive in the reconstituted aqueous composition includes arginine, arginine-mannitol, arginine-trehalose, arginine-meglumine, meglumine, meglumine-glycine, or meglumine-mannitol. In another embodiment, the additive includes arginine-trehalose. In one embodiment, the mass ratio of arginine to trehalose is approximately 10:1 to 1:10, or approximately 10:1 to 9:1, or approximately 9:1 to 8:1, or approximately 8:1 to 7:1, or approximately 7:1 to 6:1, or approximately 6:1 to 5:1, or approximately 5:1 to 4:1, or approximately 4:1 to 3:1, or approximately 3:1 to 2:1, or approximately 2:1 to 1:0, or approximately 1.8:1 to 1.7:1, or approximately 1.7:1 to 1.6:1, or approximately 1.6:1 to 1.5:1, or approximately 1.5:1 to 1.4:1, or approximately 1.4:1 to 1.3:1, or approximately 1.3:1 to 1.2:1, or approximately 1.2: The range is approximately 1 to 1.1:1, or approximately 1:1.1 to 1:1.2, or approximately 1:1 to 1:2.5, or approximately 1:1.2 to 1:1.3, or approximately 1:1.3 to 1:1.4, or approximately 1:1.4 to 1:1.5, or approximately 1:1.5 to 1:1.6, or approximately 1:1.6 to 1:1.8, or approximately 1:1.8 to 1:1.9, or approximately 1:1.9 to 1:2, or approximately 1:2 to 1:3, or approximately 1:3 to 1:4, or approximately 1:4 to 1:5, or approximately 1:5 to 1:6, or approximately 1:6 to 1:7, or approximately 1:7 to 1:8, or approximately 1:8 to 1:9, or approximately 1:9 to 1:10.

[0125] In one embodiment, the mass ratio of peptide to additive in the reconstituted aqueous composition is in the range of about 5:1 to about 1:5. In another embodiment, the mass ratio of peptide to additive in the aqueous composition is about 5:1 to about 4.5:1, or about 4.5:1 to about 4.0:1, or about 4.0:1 to about 3.5:1, or about 3.5:1 to about 3.0:1, or about 3.0:1 to about 2.5:1, or about 2.5:1 to about 2.0:1, or about 2.0:1 to about 1.5:1, or about 1.5:1 to about 1.0:1, or about 1. The ratios range from 0:1 to approximately 0.5:1, or from approximately 1:0.5 to approximately 1:1.0, or from approximately 1:1.0 to approximately 1:1.5, or from approximately 1:1.5 to approximately 1:2.0, or from approximately 1:2.0 to approximately 1:2.5, or from approximately 1:2.5 to approximately 1:3.0, or from approximately 1:3.0 to approximately 1:3.5, or from approximately 1:3.5 to approximately 1:4.0, or from approximately 1:4.0 to approximately 1:4.5, or from approximately 1:4.5 to approximately 1:4.0. In other embodiments, the mass ratio of peptide to additive is in the range of approximately 1.0 to approximately 1.5.

[0126] In one embodiment, the pH of the reconstituted aqueous composition is in the range of about 5 to about 12. In one embodiment, the pH of the reconstituted aqueous composition is about 5 to about 12, or about 5 to about 6, or about 6 to about 7, or about 7.0 to about 7.1, or about 7.1 to about 7.2, or about 7.2 to about 7.3, or about 7.3 to about 7.4, or about 7.4 to about 7.5, or about 7.5 to about 7.6, or about 7.6 to about 7.7, or about 7.7 to about 7.8, or about 7.8 to about 7.9, or about 7.9 The pH ranges from approximately 8.0, or approximately 8.0 to 8.1, or approximately 8.1 to 8.2, or approximately 8.2 to 8.3, or approximately 8.3 to 8.4, or approximately 8.4 to 8.5, or approximately 8.5 to 8.6, or approximately 8.6 to 8.7, or approximately 8.7 to 8.8, or approximately 8.8 to 8.9, or approximately 8.9 to 9.0, or approximately 9 to 10, or approximately 10 to 11, or approximately 11 to 12. In one embodiment, the pH of the reconstituted aqueous composition is in the range of approximately 7.2 to 7.8.

[0127] In one embodiment, the concentration of the peptide (i.e., Bevonescein (ALM-488)) in the reconstituted aqueous composition is in the range of about 10 mg / mL to about 500 mg / mL, based on the total weight of the aqueous composition. In another embodiment, the concentration of the peptide in the reconstituted aqueous composition is about 20 mg / mL to about 30 mg / mL, or about 30 mg / mL to about 40 mg / mL, or about 40 mg / mL to about 50 mg / mL, or about 50 mg / mL to about 60 mg / mL, or about 60 mg / mL to about 70 mg / mL, or about 70 mg / mL to about 80 mg / mL, or about 80 mg / mL to about 90 mg / mL, or about 90 mg / mL to about 100 mg / mL, or about 100 mg / mL to about 110 mg / mL, or about 110 mg / mL to about 120 mg / mL, or about 120 mg / mL to about 130 mg / mL, or about 130 mg / mL to about 1 Based on the total weight of the aqueous composition, the concentration is in the range of 40 mg / mL, or approximately 140 mg / mL to approximately 150 mg / mL, or approximately 150 mg / mL to approximately 160 mg / mL, or approximately 160 mg / mL to approximately 170 mg / mL, or approximately 170 mg / mL to approximately 180 mg / mL, or approximately 180 mg / mL to approximately 190 mg / mL, or approximately 190 mg / mL to approximately 200 mg / mL, or approximately 200 mg / mL to approximately 250 mg / mL, or approximately 250 mg / mL to approximately 300 mg / mL, or approximately 300 mg / mL to approximately 350 mg / mL, or approximately 350 mg / mL to approximately 400 mg / mL, or approximately 400 mg / mL to approximately 450 mg / mL. In one embodiment, the concentration of the peptide in the reconstituted aqueous composition is in the range of approximately 25 mg / mL to approximately 200 mg / mL, based on the total volume of the aqueous composition. In other embodiments, the concentration of the peptide in the reconstituted aqueous composition is in the range of about 40 mg / mL to about 75 mg / mL, based on the total volume of the aqueous composition.

[0128] In one embodiment, the density of the reconstituted aqueous composition is in the range of about 0.6 g / mL to about 1.3 g / mL. In another embodiment, the density of the reconstituted aqueous composition is in the range of about 0.6 g / mL to about 0.7 g / mL, or about 0.7 g / mL to about 0.8 g / mL, or about 0.8 g / mL to about 0.85 g / mL, or about 0.85 g / mL to about 0.90 g / mL, or about 0.90 g / mL to about 0.95 g / mL, or about 0.95 g / mL to about 1.0 g / mL, or about 1.0 g / mL to about 1.05 g / mL, or about 1.05 g / mL to about 1.10 g / mL, or about 1.10 g / mL to about 1.15 g / mL, or about 1.15 g / mL to about 1.20 g / mL, or about 1.20 g / mL to about 1.25 g / mL. In other embodiments, the density of the reconstituted aqueous composition is in the range of about 1.0 g / mL to about 1.08 g / mL.

[0129] In one embodiment, the osmotic pressure of the reconstituted aqueous composition is in the range of about 150 mOsm / kg to about 400 mOsm / kg. In another embodiment, the osmotic pressure of the reconstituted aqueous composition is in the range of about 150 mOsm / kg to about 175 mOsm / kg, or about 175 mOsm / kg to about 200 mOsm / kg, or about 200 mOsm / kg to about 225 mOsm / kg, or about 225 mOsm / kg to about 250 mOsm / kg, or about 250 mOsm / kg to about 275 mOsm / kg, or about 275 mOsm / kg to about 300 mOsm / kg, or about 300 mOsm / kg to about 325 mOsm / kg, or about 325 mOsm / kg to about 350 mOsm / kg. In other embodiments, the osmotic pressure of the reconstituted aqueous composition is in the range of approximately 280 mOsm / kg to approximately 300 mOsm / kg.

[0130] In one embodiment, the volume of the reconstituted aqueous composition is in the range of about 0.5 mL to about 5.0 mL. In another embodiment, the volume of the reconstituted aqueous composition is in the range of about 0.5 mL to about 1.0 mL, or about 1.0 mL to about 1.5 mL, or about 1.5 mL to about 2.0 mL, or about 2.0 mL to about 2.5 mL, or about 2.5 mL to about 3.0 mL, or about 3.0 mL to about 3.5 mL, or about 3.5 mL to about 4.0 mL, or about 4.0 mL to about 4.5 mL. In yet another embodiment, the volume of the reconstituted aqueous composition is in the range of about 1.8 mL to about 2.2 mL.

[0131] In one embodiment, the reconstituted aqueous composition contains at least 95% by mass of peptide (i.e., Bevonescein (ALM-488)) relative to the total mass of the reconstituted aqueous composition after being stored at 40°C for 14 days. In another embodiment, the reconstituted aqueous composition contains at least 96.0% by mass, or at least 96.5% by mass, or at least 97.0% by mass, or at least 97.5% by mass, or at least 98.0% by mass, or at least 98.5% by mass, or at least 99.0% by mass, or at least 99.5% by mass of peptide, relative to the total mass of the reconstituted aqueous composition after being stored at 40°C for 14 days.

[0132] Another embodiment relates to a method for forming a reconstituted aqueous composition by combining the lyophilized composition disclosed herein with an aqueous medium.

[0133] In one embodiment, the method includes stirring or agitating a mixture of a freeze-dried composition and an aqueous medium to obtain a reconstituted aqueous composition.

[0134] In one embodiment, the method includes heating a mixed mixture.

[0135] In one embodiment, the lyophilized composition used in the preparation of the reconstituted aqueous composition is in the form of a lyophilized cake having a good appearance.

[0136] How to use Other embodiments relate to methods for labeling or identifying nerve cells or neurons by contacting them with a Bevonescein (ALM-488)-containing composition disclosed herein. In one embodiment, nerve cells or neurons are contacted with a free fluorescent moiety in combination with the composition. In one embodiment, the fluorescent moiety includes a fluorescent protein, a fluorescent peptide, a fluorophore, or any combination thereof. In one embodiment, the contact occurs in vivo. In another embodiment, the contact occurs in vitro. In one embodiment, the nerve cells or neurons are human nerve cells or neurons.

[0137] In one embodiment, the neuronal targeting peptide ALM-488 is administered in combination with a fluorescent moiety (e.g., a neuronal targeting peptide conjugate and an unconjugated fluorescent moiety, or a "free" fluorescent moiety) (simultaneously, together, or sequentially). In one embodiment, the fluorescent moiety is fluorescein, for example, carboxyfluorescein.

[0138] Other embodiments relate to a method for delivering at least one drug to nerve cells or nerves by contacting the nerve cells or nerves with the ALM-488-containing composition disclosed herein. In one embodiment, the delivery of at least one drug to nerve cells or nerves is carried out by administering the composition to a subject that requires it. In one embodiment, the subject is a human subject. In one embodiment, the composition is administered to the subject before performing a surgical procedure on the subject. In one embodiment, the surgical procedure is, for example, a cancer surgery such as prostate cancer surgery.

[0139] In one embodiment of the method described herein, the neuronal targeting peptide ALM-488 is administered to subjects described herein, including humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In one embodiment, the subjects are mammals. In one embodiment, the subjects are primates. In one embodiment, the subjects are humans. In one embodiment, the human subjects are children (under 21 years of age), adults (22 to 65 years of age), or elderly (65 years of age and older).

[0140] Some embodiments of the methods of use described herein (e.g., labeling or identification of nerve cells or nerves, delivery of drugs to nerve cells or nerves) may be performed before or during surgery on the subject. In some embodiments, the surgery relates to traumatic injury of the subject. In some embodiments, the surgery relates to infection of the subject. In some embodiments, the surgery is reconstructive surgery on the subject, e.g., cosmetic surgery or reconstructive surgery.

[0141] In one embodiment, the surgery is of the head, neck, skull base, spinal cord, prostate, heart, kidneys, hands, arms, feet, legs, lower abdomen, or gynecological surgery. In one embodiment, the surgery is of the head and neck. In one embodiment, the surgery is of the gastrointestinal tract (GI) or genitourinary tract (GU).

[0142] In one embodiment, the surgery is cancer surgery. In one embodiment, the cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, pancreatic cancer, stomach cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial carcinoma, lung cancer, melanoma, testicular germ cell tumor, mesothelioma, and esophageal cancer. In one embodiment, the cancer is prostate cancer.

[0143] In one embodiment, the method includes administering the ALM-488-containing composition disclosed herein to a subject scheduled to undergo surgery. In another embodiment, the method includes administering it to a subject currently undergoing surgery. In another embodiment, the ALM-488-containing composition disclosed herein is administered systemically to a patient, for example, by intravenous injection. In another embodiment, the neurotargeting peptide conjugate disclosed herein is administered topically to a patient.

[0144] In one embodiment, the neuroactive targeting peptide ALM-488 is delivered to a target via a drug delivery medium or carrier. In one embodiment, the delivery medium consists of natural or synthetic substances or both. In one embodiment, the delivery medium may include nanoparticles, microparticles, polymer micelles, nanocapsules, dendrimers, large PEGs, nanogels, liposomes, fullerenes, nanostructured lipid carriers, nanoshells, quantum dots, protein-based nanocarriers (e.g., albumin, elastin, gliadin, regumin, zein, soy protein, milk protein, whey-based nanocarriers), organic nanocarriers (e.g., gelatin, dextran, guar gum, chitosan, collagen), polysaccharide-based carriers (e.g., dextran, chitosan, pectin), lipid emulsions, or combinations thereof.

[0145] In one embodiment, the ALM-488-containing composition disclosed herein is administered to a subject by any suitable route of administration, including but not limited to topical, oral, rectal, vaginal, nasal, inhalation, and non-enteral (intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, intravitreous, and intravenous infusion) administration. In one embodiment, the pharmaceutical composition disclosed herein is administered to a subject topically or systemically. In one embodiment, the pharmaceutical composition disclosed herein is administered intravenously, for example, by systemic intravenous injection.

[0146] Formulations suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions (e.g., lyophilized compositions described herein). Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or media include water, ethanol, polyols (such as propylene glycol, polyethylene glycol, glycerol, and cremophor), suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. Other commonly used surfactants, e.g., tween, span, and other emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solids, liquids, or other dosage forms may also be used for formulation purposes. Bioavailability enhancers may include osmotic or permeability enhancers. See, for example, Muheem et al., 2016, Saudi Pharm. J. 24, 413-428; Brayden et al. 2020, Adv Drug Deliv Rev. 157, 2-36; Ibrahim et al. 2020, J. Pham. Sci. 28, 403-416. In some formulations, appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of a surfactant. Formulations suitable for subcutaneous injection may also include optional additives such as preservatives, humectants, emulsifiers, and dispersants, which are known in the art. In some embodiments, formulations suitable for intravenous injection may be prepared in aqueous solutions such as saline buffer and other physiologically compatible buffers known in the art.

[0147] Non-enteral injections may optionally include bolus injections or continuous infusions. Injectable formulations may optionally be presented in unit dosage forms, e.g., ampoules or multi-dose containers with added preservatives. In one embodiment, the ALM-488-containing composition described herein is in a form suitable for non-enteral injection as a sterile suspension, solution, or emulsion in an oily or aqueous medium, and comprises formulation agents such as suspending agents, stabilizers, and / or dispersants. Pharmaceutical formulations for non-enteral administration comprise an aqueous solution of the activator in a water-soluble form. Furthermore, the suspension may optionally be prepared as a suitable oily injection suspension (e.g., from the lyophilized composition disclosed herein).

[0148] In one embodiment, the ALM-488-containing composition disclosed herein may be administered orally. Suitable dosage forms for oral administration may be solid or liquid and may include, for example, pills, capsules, lozenges, tablets, caplets, gel caplets (gel caps), syrups, aqueous suspensions or solutions, chewable forms, swallowable forms, soluble forms, effervescent forms, granular forms, and oral liquid solutions. In certain embodiments, the dosage form is a solid dosage form, more specifically including tablets or capsules, or administered orally by a medium or carrier disclosed herein.

[0149] In one embodiment, the ALM-488-containing pharmaceutical composition described herein is in a unit dosage form suitable for a single dose of a precise amount. In the unit dosage form, the formulation is divided into unit doses containing an appropriate amount of the activator disclosed herein. In one embodiment, the unit dose is in the form of a package containing individual amounts of the formulation. Non-limiting examples include packaged tablets or capsules, and powder in vials or ampoules. In one embodiment, the aqueous suspension composition is packaged in a single-dose non-re-sealable container. Alternatively, a multi-dose re-sealable container is used, in which case the composition typically contains a preservative. As just one example, formulations for non-enteral injection are presented in unit dosage forms including, but not limited to, ampoules or multi-dose containers with added preservatives.

[0150] As described above, one embodiment of the present invention relates to a method for targeted drug delivery. In one embodiment, the ALM-488-containing composition disclosed herein delivers a drug to a specific target. In one embodiment, the composition delivers a drug to nerve cells or nerves.

[0151] In one embodiment, the drug is an agent that reduces pain (the perception of pain or the action of pain-stimulating substances). In one embodiment, the drug is an anesthetic. In one embodiment, the drug is benzocaine; calticaine; cincocaine; cyclomethicaine; lidocaine; prilocaine; propoxycaine; proparacaine; tetracaine; tocainide; and trimecaine; or a combination thereof.

[0152] In one embodiment, the drug is an agent that modulates the death of nerve cells or nerves (e.g., through apoptosis or necrosis). In one embodiment, the drug is a cytotoxic agent. In one embodiment, the drug is methotrexate (Rheumatrex®, ametopterin); cyclophosphamide (Citoxane®); thalidomide (THALIDOMID®); paclitaxel; pemetrexed; pentostatin; pipobromane; picantrone; plicamycin; procarbazine; proteasome inhibitors (e.g., bortezomib); larcitrexed; rebeccamycin; rubitecan; SN-38; salinos Polaramide A; Satraplatin; Streptozocin; Swinesonin; Thaliquidal; Taxane; Tegafur-uracil; Temozolomide; Testolactone; tbioTEPA; Thioguanine; Topotecan; Trabectedin; Tretinoin; Triplatin tetranitrate; Tris(2-chloroethyl)amine; Troxacitabine; Uracil mustard; Barrubicin; Vinblastine; Vincristine; Vinorelbine; Vorinostat; Zoskidal; or combinations thereof. In one embodiment, the drug is an apoptosis promoter. In one embodiment, the drug is an anti-apoptotic agent. In one embodiment, the drug is minocycline; SB-203580 (4-(4-fluorophenyl)-2-(4-methylsulfumylphenyl)-5-(4-pyridyl)lH-imidazole); PD 169316 (4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-lH-imidazole); SB 202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)lH-imidazole); RWJ 67657 (4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-lH-imidazole-2-yl]-3-butin-1-ol); SB 220025(5-(2-amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidiniryl)imidazole);D-JNKI-1((D)-hJIP 175_i 57-DPrO-DPrO-(D)-HIV-TAT57-48);AM-111(Auris);SP600125 (Anthra[l,9-cd]pyrazole-6(2H)-one); JNK inhibitor I ((L)-HIV-T AT48-57-PP-JBD20); JNK Inhibitor III ((L)-HIV-TAT47-57-gaba-c-Junδ33-57); AS601245 (l,3-benzothiazole-2-yl(2-[[2-(3-pyridinyl)ethyl]amino]-4-pyrimidinyl)acetonitrile); JNK Inhibitor VI (H2N-RPKRPTTLNLF-NH2); JNK Inhibitor VIII (N-(4-amino-5-cyano-6-ethoxypyridine-2-yl)-2-(2,5-dimethoxyphenyl)acetamide); JNK Inhibitor IX (N-(3-cyano-4,5,6,7-tetrahydro-1-benzothien-2-yl)-1-naphthamide); dicumarol (3,3'-methylenebis(4-hydroxycoumarin)); SC-236 (4-[5-(4 -chlorophenyl)-3-(trifluoromethyl)-1H-pyrazole-1-yl]benzene-sulfonamide); CEP-1347 (Cephalon); CEP-11004 (Cephalon); artificial protein containing at least a portion of Bcl-2 polypeptide; recombinant FNK; V5 (also known as Bax inhibitor peptide V5); Bax channel blocker ((±)-1-(3,6-dibromocarbazole-9-yl)-3-piperazine-1-ylpropan-2-ol); Bax inhibitor peptide P5 (also known as Bax inhibitor peptide P5); Kp7-6; FAIM(S) (Fas apoptosis inhibitor molecule - short); FAIM(L) (Fas apoptosis inhibitor molecule - long); Fas:Fc; FAP-1; N0K2; F2051; Fl 926;F2928;ZB4;Fas M3 mAb;EGF;740 YP;SC 3036(KKHTDDGYMPMSPGVA);PI 3-kinase activator (Santa Cruz Biotechnology, Inc.);Pam3Cys((S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH, trihydrochloride);Actl(NF-κB activator 1);anti-DcB antibody;acetyl-11-keto-β-boswellic acid;androglaphoride;Phenethyl caffeate (CAPE); Gliotoxin; Isohelenin; NEMO-binding domain-binding peptide (DRQIKIWFQNRRMKWKKTALDWSWLQTE); NF-κB activator (6-amino-4-(4-phenoxyphenylethylamino)quinazoline); NF-κB activator II (4-methyl-Nl-(3-phenylpropyl)benzene-l,2-diamine); NF-κB activator III (3-chloro-4-nitro-N-(5-nitro-2-thiazolyl)-benzamide); NF-κB activator IV ((E)-2-fluoro-4'-methoxystilbene); NF-κB activator V (5-hydroxy-(2,6-diisopropylphenyl)-lH-isoindole-l,3-dione); NF-κB SN50 (AA VALLP A VLLALLAP) VQRKRQKLMP); Oridonin; Parthenolide; PPM-18 (2-Benzoylamino-l,4-Naphthoquinone); Rol06-9920; Sulfasalazine; TIRAP Inhibitor Peptide (RQIKIWFNRRMKWKKLQLRD AAPGG AIVS); Withaferin A; Ougonin; BAY 11-7082 ((E)3-[(4-Methylphenyl)sulfonyl]-2-Propennitrile); BAY 11-7085((E)3-[(4-t-butylphenyl)sulfonyl]-2-propennitrile);(E)-capsaicin; aurothiomalate (ATM or AuTM); evodiamine; hypoestoxide; IKK inhibitor III (BMS-345541); IKK inhibitor VII; IKK inhibitor X; IKK inhibitor II; IKK-2 inhibitor IV; IKK-2 inhibitor V; IKK-2 inhibitor VI; IKK-2 inhibitor (SC-514); IκB kinase inhibitor peptide; IKK-3 inhibitor LX; ARRY-797(Array BioPharma); SB-220025 (5-(2-amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidiniryl)imidazole); SB-239063 (trans-4-[4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazole-1-yl]cyclohexanol);SB-202190(4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole); JX-401([2-methoxy-4-(methylthio)benzoyl]-4-(phenylmethyl)piperidine); PD-169316(4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole); SKF-86002(6-(4-fluorophenyl)-2,3-dihydro-5-(4-pyridinyl)imidazo[2,lb]thiazole dihydrochloride); SB-200646(N-(l-methyl-1H-indole-5-yl)-N'-3-pyridinylurea); CMPD -I(2'-Fluoro-N-(4-hydroxyphenyl)-[1,1'-biphenyl]-4-butanamide);EO-1428((2-methylphenyl)-[4-[(2-amino-4-bromophenyl)amino]-2-chlorophenyl]methanone);SB-253080(4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazole-4-yl]pyridine);SD-169(1H-indole-5-carboxamide);SB-203580(4-(4-fluorophenyl)-2-(4-methylsulfomylphenyl)-5-(4-pyridyl)-1H-imidazole);TZP-101(Tranzyme Pharma); TZP-102 (Tranzyme Pharma); GHRP-6 (Growth Hormone-Releasing Peptide-6); GHRP-2 (Growth Hormone-Releasing Peptide-2); EX-1314 (Elixir Pharmaceuticals); MK-677 (Merck); L-692,429 (Butanamide, 3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1-((2'-(lH-tetrazole-5-yl)(l,l'-biphenyl)-4-yl)methyl)-lH-1-benzazepine-3-yl)-,(R)-); EP1572 (Aib-DTrp-DgTφ-CHO); Diltiazem; Diltiazem metabolites; BRE (Brain and Genital Proteins); Verapamil; Nimodipine; Diltiazem; Omega-Conotoxin; GVIA; Amlodipine; Felodipine; Lacidipine; Mibefuradil; NPPB (5-Nitro-2-(3-phenylpropylamino)benzoic acid); Flunarizine;Erythropoietin; Piperine; Hemin; Brazilin; zV AD-FMK (benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone); z-LEHD-FMK (benzyloxycarbonyl-Leu-Glu(OMe)-His-Asp(OMe)-fluoromethyl ketone); BD-FMK (boc-aspartyl(Ome)-fluoromethyl ketone); Ac-LEHD-CHO (N-acetyl-Leu-Glu-His-Asp-CHO); Ac-IETD-CHO (N-acetyl-Ile-Glu-Thr-Asp-CHO); z-IETD-FMK (benzyloxycarbonyl-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethyl ketone); FAM-LE HD-FMK (Benzyloxycarbonyl Leu-Glu-His-Asp-fluoromethyl ketone); FAM-LETD-FMK (Benzyloxycarbonyl Leu-Glu-Thr-Asp-fluoromethyl ketone); Q-VD-OPH (Quinoline-VaI-ASp-CH2-O-Ph); XIAP; cIAP-1; cIAP-2; ML-IAP; ILP-2; NAIP; Survivin; Brace; IAPL-3; Fortilin; Leupeptin; PD-150606 (3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid); MDL-28170 (Z-Val-Phe-CHO); Carpeptin; Acetyl-carpastatin; MG 132(N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(IS)-1-formyl-3-methylbutyl]-L-leucinamide);MYODUR;BN 82270(Ipsen);BN 2204(Ipsen);AHLi-11(Quark Pharmaceuticals), mdm2 protein, pifislin-α(l-(4-methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazolyl)etanone); trans-stilbene; cis-stilbene; resveratrol; piciatannol; lapontin; deoxylapontin; butein; chalcone; isoliquitigen; butein; 4,2',4'-trihydroxychalcone; 3,4,2',4',6'-pentahydroxychalcone; flavone; morin; fisetin; luteolin; quercetin; kaempferol; apigenin;Gossipetin; Myricetin; 6-Hydroxyapigenin; 5-Hydroxyflavone; 5,7,3',4',5'-Pentahydroxyflavone; 3,7,3',4',5'-Pentahydroxy-flavone; 3,6,3',4'-Tetrahydroxyflavone; 7,3',4',5'-Tetrahydroxyflavone; 3,6,2',4'-Tetrahydroxy-flavone; 7,4'-Dihydroxyflavone; 7,8,3',4'-Tetrahydroxyflavone; 3,6,2',3'-Tetrahydroxyflavone; 4'-Hydroxyflavone; 5-Hydroxyflavone; 5,4'-Dihydroxyflavone; 5,7-Dihydroxyflavone; Daidzein; Genistein; Naringenin; Flavanone; 3,5, 7,3',4'-Pentahydroxyflavanone; Pelargonidine chloride; Cyanidin chloride; Delphinidin chloride; (-)-Epicatechin (Hydroxy moiety: 3,5,7,3',4'; (-)-Catechin (Hydroxy moiety: 3,5,7,3',4); (-)-Gallocatechin (Hydroxy moiety: 3,5,7,3',4',5); (+)-Catechin (Hydroxy moiety: 3,5,7,3',4'; (+)-Epicatechin (Hydroxy moiety: 3,5,7,3',4'; Hinokitiol (β-Thujaplicin; 2-Hydroxy-4-Isopropyl-2,4,6-Cycloheptatrien-1-one); L-(+)-Ergothioneine ((S)-α-Carboxy-2,3-Dihydro-N,N,N-Trimethyl-2; -Thioxo-1H-imidazole 4-ethanaminonium intramolecular salt); caffeate phenyl ester; MCI-186(3-methyl-1-phenyl-2-pyrazolin-5-one); HBED(N,N'-di-(2-hydroxybenzyl)ethylene-diamine-N,N'-diacetic acid*H2O); ambroxol(trans-4-(2-amino-3,5-dibromobenzylamino)-cyclohexane-HCl; and U-83836E((-)-2-((4-(2,6-di-1-pyrrolidinyl-4 -Pyrimidinyl)-1-piperzainyl)-methyl)-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol>>2HCl);β-1-5-methylnicotinamide-2'-deoxyribose;3-D-1'-5-methylnicotinamide-2'-deoxyribofuranoside;3-1'-4,5-dimethylnicotinamide-2'-deoxyribose;3-D-1'-4,5-dimethylnicotinamide-2'-deoxyribofuranoside;1-naphthyl P Pl(1-(1,1-dimethylethyl)-3-(l-naphthalenyl)-lH-pyrazolo[3,4-d]pyrimidine-4-amine); Lavenderstine A(5-[[(2,5-dihydroxyphenyl)methyl][(2-hydroxyphenyl)methyl]amino]-2-hydroxybenzoic acid); MNS(3,4-methylenedioxy-b-nitrostyrene); Ppl(l-(l,l-dimethylethyl)-1-(4-methylphenyl)-lH-pyrazolo[3,4-d]pyrimidine-4-amine); PP 2(3-(4-chlorophenyl)-1-(l,l-dimethylethyl)-lH-pyrazolo[3,4-d]pyrimidine-4-amine); KX1-004 (Kinex); KX1-005 (Kinex); KX1-136 (Kinex); KX1-174 (Kinex); KX1-141 (Kinex); KX2-328 (Kinex); KX1-306 (Kinex); KX1-329 (Kinex); KX2-391 (Kinex); KX2-377 (Kinex); ZD4190 (Astra Zeneca; N-(4-bromo-2-fluorophenyl)-6-methoxy-7-(2-(lH-l,2,3-triazole-1-yl)ethoxy)quinazoline-4-amine); AP22408 (Ariad Pharmaceuticals);AP23236 (Ariad Pharmaceuticals); AP23451 (Ariad Pharmaceuticals); AP23464 (Ariad Pharmaceuticals); AZD0530 (Astra Zeneca); AZM475271 (M475271; Astra Zeneca); Dasatinib (N-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)-piperazin-1-yl)-2-methylpyrimidine-4-ylamino)thiazole-5-carboxamide); GN963 (trans-4-(6,7-dimethoxy-quinoxaline-2-ylamino)cyclohexanol sulfate); Bosutinib (4-((2,4-dichloro-5-methoxyphenyl)-amino)-6-methoxy-7-(3-(4-methyl-1-piperazinyl)propoxy)-3-quinoline carbononitrile); or combinations thereof.

[0153] In one embodiment, the drug is an agent that reduces unwanted nerve cells or nerve impulses. In another embodiment, the drug reduces one or more symptoms of dyskinesia or synkinesis. In another embodiment, the drug is carbamazepine, oxycarboazepine, phenytein, valproic acid, sodium valoproate, cinnarizine, flunarizine, or nimodipine, or a combination thereof.

[0154] In one embodiment, the drug is an agent that promotes the regeneration of nerve cells or nerve tissue. In another embodiment, the drug is a growth factor. In yet another embodiment, the drug is selected from brain-derived neurotrophic factor (BDNF); ciliary neurotrophic factor (CNTF); glial cell line-derived neurotrophic factor (GDNF); neurotrophin-3; neurotrophin-4; fibroblast growth factor (FGF) receptor; insulin-like growth factor (IGF); or a combination thereof.

[0155] In one embodiment, disclosed herein is a method for delivering a photosensitive agent to human nerve cells or neurons, comprising contacting the human nerve cells or neurons with the ALM-488-containing composition disclosed herein. In one embodiment, the method further comprises exposing the human nerve cells or neurons to a light source that activates the photosensitive agent contained in ALM-488, thereby inducing ablation or death of the human nerve cells or neurons. Upon exposure to light of a specific wavelength, the photosensitive agent reacts with molecular oxygen to produce cytotoxic singlet oxygen.

[0156] In one embodiment, the ALM-488-containing composition disclosed herein can be used for the treatment of prostate cancer in a subject. Autonomic nerve impulse transmission can contribute to prostate cancer growth and metastasis through photo-induced ablation of local autonomic nerves. Therefore, local autonomic nerves may be a viable target for prostate cancer treatment. In one embodiment, renovascular hypertension can be treated in a subject by photo-induced ablation of sympathetic nerves in the renal blood vessels. In one embodiment, the ALM-488-containing composition disclosed herein can be used for the treatment of hyperhidrosis. In one embodiment, the ALM-488-containing composition disclosed herein can be used for the treatment of cardiac arrhythmias. In one embodiment, the ALM-488-containing composition disclosed herein can be used for the treatment of pathological muscle spasms (e.g., Meige syndrome, hemifacial spasm, torticollis).

[0157] In one embodiment, the ALM-488-containing composition disclosed herein may be administered in combination with a second neuronal targeting peptide conjugate (simultaneously, together, or sequentially).

[0158] Other embodiments relate to the use of the ALM-488-containing composition disclosed herein in the manufacture of a pharmaceutical for labeling or identifying nerve cells or nerves in a subject. Other embodiments relate to the ALM-488-containing composition disclosed herein for use for labeling or identifying nerve cells or nerves in a subject. Other embodiments relate to the use of the ALM-488-containing composition disclosed herein in the manufacture of a pharmaceutical for delivering at least one drug to nerve cells or nerves in a subject. Other embodiments relate to the ALM-488-containing composition disclosed herein for use to deliver at least one drug to nerve cells or nerves in a subject.

[0159] While aspects of the present invention are shown and described throughout this disclosure, it will be apparent to those skilled in the art that such aspects are provided merely as examples. Those skilled in the art will readily conceive of numerous modifications, changes, and substitutions without departing from this disclosure. It should be understood that various alternatives to the aspects of the present invention described throughout this disclosure may be used in carrying out the invention. The following claims define the scope of the present invention, and the methods and structures within those claims, as well as their equivalents, are intended to be covered thereby.

[0160] A combination of the various embodiments described above may provide a particular embodiment. U.S. patents, U.S. patent application publications, U.S. patent specifications, foreign patents, foreign patent applications, and non-patent literature referenced herein and / or listed in the application datasheet are incorporated herein by reference as a whole. The aspects of the embodiments may be modified as necessary to provide further embodiments using concepts from various patents, applications, and publications. [Examples]

[0161] Abbreviation Ac2O: Acetic anhydride; ACN: Acetonitrile; DIC: N,N'-diisopropylcarbodiimide; DIEA: N,N-diisopropylethylamine; DMF: N,N-dimethylformamide; DSC: Differential Scanning Calorimetry; EDT: Ethane-1,2-dithiol; 5-FAM: 5-carboxyfluorescein; Fmoc:9-Fluorenylmethyloxycarbonyl; FDM: Freeze-drying microscope; HBTU: N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate; HNS: Half physiological saline; HOBt: Hydroxybenzotriazole; HPLC: High-performance (high-speed) liquid chromatography; MBHA: 4-methylbenzhydrylamine; MeOH: methanol; NS: Physiological saline solution; Pip: piperidine; PVDF: Polyvinyl fluoride; RP: Out of phase; SPPS: Solid-phase peptide synthesis; TFA: Trifluoroacetic acid; TIS: Triisopropylsilane; UPLC: Ultra-high-performance liquid chromatography;

[0162] Device The equipment used here is shown in Table 1. [Table 1]

[0163] material The materials used here are shown in Table 2 below. [Table 2]

[0164] HPLC conditions The HPLC conditions for the purity analysis of ALM-488 are summarized in Table 3 below. [Table 3]

[0165] Figure 2 shows the HPLC chromatogram of a 0.25 mg / mL solution of ALM-488 (as an aqueous solution of the HCl salt) analyzed using the HPLC conditions in Table 3. Figure 3 is a magnified view of the major peaks in the HPLC chromatogram of Figure 2. Under the HPLC conditions in Table 3 (as shown in Figures 2 and 3), ALM-488 had a retention time of approximately 10 minutes, and the major impurities eluted with retention times of approximately 0.96 (0.15 area%) and approximately 1.06 (0.11 area%).

[0166] Example 1 : Manufacturing of crude ALM-488 A. Overview of the peptide synthesis process In the embodiment of Example 1, the ALM-488 peptide is synthesized using a solid support MBHA resin, starting from the C-terminus of the peptide sequence and ending at the N-terminus. Figure 4 shows a flow diagram of the solid support synthesis used in the production of ALM-488.

[0167] Rink amide linker, followed by Fmoc-Gly-OH, is coupled to the MBHA resin. Then, 5-FAM is coupled to the resulting resin. To couple the next Fmoc amino acid in the sequence, the Fmoc protecting group of the preceding amino acid (from the peptide bound to the resin) is removed by deprotection using 20% ​​piperidine / DMF. Then, the resulting amino acid is coupled to the N-terminal group of the preceding amino acid. The manufacturing process uses two coupling methods. The primary coupling method is the DIC method for the initial coupling of all amino acids, and the secondary coupling method is the HBTU method for amino acid recoupling if necessary.

[0168] B. Resin expansion and cleaning Before coupling to the Rink amide linker, the starting resin is expanded and washed. Then, the resin is distributed into the reaction vessel, and DMF is added to expand it (DMF to starting resin ratio is 7-12 mL / g). The reaction mixture is then stirred for at least 2 hours and drained.

[0169] Next, add more DMF to the vessel, then stir, and add 5% DIEA. After stirring for 10-20 minutes, drain the reaction vessel and add more DMF. Then, stir the reaction vessel for at least 2 minutes, and then drain the solvent. Wash the resin two more times by repeating the washing steps described above. Perform a ninhydrin test to confirm that the resulting resin is free of residual amines and / or amino acids.

[0170] C. Fmoc-Rink amide linker packing The molar ratio of Fmoc-Rink amide linker / HOBt (or HOBt-A) / DIC / synthesis scale was 1.5:1.5:1.5:1. The desired amounts of Fmoc-Rink amide linker and HOBt were dissolved in DMF in an amino acid (AA) mixed container. After the Fmoc-Rink amide linker / HOBt solution was completely dissolved, it was then transferred to the reaction vessel. The reaction vessel was then stirred, and DIC was slowly added (the acceptable range of DIC was ±2.0 mL). The reaction mixture was then stirred for 2 to 72 hours, and the first ninhydrin test was performed at least 2 hours from the start of stirring. If the first ninhydrin test was negative, the process continued with the further DMF washing step described below. If the first ninhydrin test was positive, the reaction time was extended, and the second ninhydrin test was performed at least 1 hour after the first ninhydrin test. The ninhydrin test was continued until the test was negative.

[0171] After the ninhydrin test yields a negative result, the resin is washed three more times with DMF, stirring for at least 2 minutes after each wash. After each DMF wash, the solvent is drained from the reaction vessel. Then, additional DMF is added to the resin and the resulting mixture is stirred. During stirring, 5% DIEA and 5% Ac2O are added, and the reaction mixture is stirred for 15–25 minutes. Then, the resin is washed four more times with DMF, stirring for at least 2 minutes after each wash, and the solvent is drained from the reaction vessel after each wash.

[0172] D. Intermediate deprotection and cleaning Remove the Fmoc protecting group using a 20% Pip / DMF deprotection solution. Add the deprotection solution to the reaction vessel containing the peptide resin and stir the resulting suspension for 5-10 minutes. After this time, drain the solvent from the reaction vessel and add further deprotection solution. Stir the resulting suspension for a further 20-30 minutes, then drain the solvent from the vessel. Wash the resin a total of eight times using DMF, stirring for at least 2 minutes after each wash. After each of the eight washes, drain the solvent from the reaction vessel. Perform a ninhydrin test for each AA cycle, and stop the process after the ninhydrin test is negative.

[0173] E. Amino acid coupling and washing (DIC method) The molar ratio of Fmoc-amino acid / HOBt (or HOBt-A) / DIC / synthesis scale is 3:3:3:1, except for 5-Fam in AA cycle 1, which uses a molar ratio of 1.5:1.5:1.5:1. Dissolve the desired amounts of Fmoc-AA-OH and HOBt in DMF in an AA mixing container. Once the Fmoc-AA / HOBt solution is completely dissolved, transfer it to the reaction vessel and stir for 2-72 hours, slowly adding DIC during this time. Perform the ninhydrin test at least 2 hours after the start of the reaction. If the ninhydrin result is negative, the process may proceed to the next cycle or final wash (for cycle 1 only) after the DMF wash. If the ninhydrin test is positive, extend the reaction time and perform the second ninhydrin test at least 1 hour after the first test. For the DMF wash, wash the resin a total of three times with DMF, stirring for at least 2 minutes, then drain the solvent from the reaction vessel. If the ninhydrin test yields a negative result, the process proceeds to the next AA cycle. If the ninhydrin test yields a positive result, the amino acid recoupling process is performed.

[0174] F. Amino acid recoupling and washing During amino acid coupling, as described above, if a positive result is obtained in the ninhydrin test, the HBTU / DIEA recoupling method is followed. Dissolve the desired amount of amino acids and reagents in DMF in an AA mixing container (the acceptable range for AA, 5-FAM, and HBTU is ±2.0 g). After the amino acid and reagent solutions are completely dissolved, transfer the resulting mixture to a reaction vessel and stir for 1 to 3 hours, during which time DIEA is slowly added. Perform the ninhydrin test at least 1 hour after the start time. If a positive result is obtained in the ninhydrin test, extend the reaction time and perform a further ninhydrin test at least 1 hour after the first test. If a negative result is obtained in the ninhydrin test, the process may proceed to the next cycle or final wash after DMF washing. For DMF washing, wash the resin three times with DMF, stirring for at least 2 minutes during each wash. After each wash, drain the solvent from the reaction vessel. If a negative result is obtained in the ninhydrin test, continue the next AA cycle using the same process. If the ninhydrin test yields consecutive positive results, perform the following capping process to inactivate the non-coupling amino groups (except for AA cycle 1).

[0175] G. Capping The continuous positive results of the ninhydrin test during the recoupling process described above are addressed by performing a capping process to inactivate the non-coupling amino groups. For this capping, DMF is added to the resin and the resulting mixture is stirred. During stirring, 5% DIEA and 5% Ac2O are added and the mixture is stirred for a total of 15-25 minutes. After the stirring time is complete, the resin is washed four times with DMF, stirring for at least 2 minutes between each wash.

[0176] H. Peptide manufacturing, synthesis, and cleavage processes After adding the final unit (i.e., the 5-FAM portion), the resulting resin is washed five times with MeOH, stirring for at least two minutes between each wash. For the final wash, the solvent is drained from the reaction vessel after each of the five washes. The peptide resin is washed with DMF, followed by MeOH, and the resulting resin is dried under vacuum. Weight loss due to drying is used as the criterion for completion of the drying process.

[0177] When the desired peptide resin is synthesized, the peptide is separated from the solid support, and all amino acid side chain protecting groups are removed to obtain the desired peptide. In Fmoc SPPS, this step is carried out by treating the peptidyl resin with TFA containing a scavenger containing EDT / TIS / water. The cleaved mixture is then precipitated, washed with ether, and dried to obtain the crude ALM-488 peptide.

[0178] Example 2 Purification of crude ALM-488 The identity of the crude peptide (ALM-488) from Example 1 was verified using mass spectrometry (MS), and its purity was analyzed using analytical C18 reversed-phase HPLC (RP-HPLC) under the HPLC method conditions summarized in Table 3 above.

[0179] A. Separation by HPLC Crude ALM-488 is dissolved in ACN and distilled water. To completely dissolve the crude ALM-488 in ACN / water, a small amount of ammonium acetate / hydroxyl hydroxide may be added if necessary. The resulting homogeneous mixture is then purified using RP-HPLC with various buffer systems. The buffer systems consist of an aqueous solution (Buffer A) combined with an organic phase modifier consisting of 100% acetonitrile (Buffer B).

[0180] Perform HPLC separation using the following steps: 1) Dilute the peptide solution by adding purified water or aqueous buffer solution; 2) Equilibrate the RP-HPLC column with at least two column volumes of aqueous buffer A and organic buffer B (acetonitrile); 3) Pack the diluted peptide solution into the RP-HPLC column; 4) Equilibrate the RP-HPLC column with at least two column volumes of aqueous buffer A and organic buffer B (acetonitrile); 5) Perform an HPLC gradient and collect the eluent when there is an increase in absorption or when the corresponding %B is reached for peptide elution; stop collection when the absorption drops to near its starting point; and 6) Wash the HPLC column with a high percentage of organic buffer B and aqueous buffer A, using at least two column volumes.

[0181] During each purification step, eluted fractions are collected and their purity is analyzed by UPLC / HPLC. Fractions that meet the first and second cut purity criteria are stored, while fractions below the second cut purity criteria are discarded. The stored first cut fraction is advanced to the next purification step, while the second cut fraction is combined for further HPLC purification.

[0182] B. Ion exchange of HPLC-purified ALM-488 Next, the combined first cut fraction of HPLC-purified ALM-488 is ion-exchanged using the following procedure to obtain ALM-488 chloride salt.

[0183] Dilute the HPLC-purified ALM-488 by adding purified water or aqueous buffer. Then, equilibrate the RP-HPLC column with at least 3 column volumes of aqueous buffer A (1% triethylamine phosphate aqueous solution) and organic buffer B (acetonitrile). Next, add the diluted peptide solution to the RP-HPLC column.

[0184] Next, the RP-HPLC column is equilibrated with (i) at least 3 column volumes of aqueous buffer A (1% triethylamine phosphate aqueous solution) and organic buffer B (acetonitrile), (ii) followed by at least 3 column volumes of aqueous buffer A (50 mM ammonium acetate aqueous solution) and organic buffer B (acetonitrile), (iii) followed by at least 3 column volumes of aqueous buffer A (1% acetic acid aqueous solution) and organic buffer B (acetonitrile), and (iv) followed by at least 3 column volumes of aqueous buffer A (0.01 N hydrochloric acid aqueous solution) and organic buffer B (acetonitrile).

[0185] Next, perform an HPLC gradient and collect the eluent when there is an increase in absorption or when the corresponding %B is reached for peptide elution. Stop collection when the absorption drops to near its starting point. Finally, wash the HPLC column with at least two column volumes of high-percentage organic buffer B and aqueous buffer A.

[0186] C. Freeze-dried ALM-488 chloride salt After subjecting the combined first cut fraction to the ion exchange procedure described above, the combined first cut is freeze-dried using a jar freeze-drying device. If freeze-drying of multiple batches is required to freeze-dry the combined first cut, these individual freeze-drying processes are then carried out for at least 12 hours, followed by final (combined) freeze-drying for at least 72 hours. The individual freeze-dried products may be stored at ≤-15°C before the final (combined) freeze-drying process.

[0187] Example 3 Analysis of purified ALM-488 By the above procedure, the chloride salt of ALM-488 is obtained as a yellow-orange solid. In other embodiments, the ion exchange process may be modified so that the ALM-488 product is in the form of a different salt, such as an acetate.

[0188] A. HPLC purity of purified ALM-488 As shown in Figures 2 and 3, the chloride salt of ALM-488 eluted with a retention time of approximately 10 minutes under the HPLC method shown in Table 3. Major impurities were detected at area levels of approximately 0.14% and 0.11% at relative retention times of approximately 0.96% and 1.06% (see Figure 3). Based on HPLC analysis, the purity of the chloride salt of ALM-488 was 98.95%.

[0189] B. Chloride content of purified ALM-488 The chloride content of ALM488 chloride salt was measured to be 3.57% w / w (3.67% w / w as HCl).

[0190] C. Thermogravimetric analysis of purified ALM-488 Thermogravimetric analysis (TGA) of the chloride salt ALM-488 showed a weight loss of 3.96% when the sample was heated to 100°C using a Perkin Elmer TGA 7 thermogravimetric analyzer. Figure 5 shows the thermogravimetric weight loss curve of the chloride salt ALM-488. As shown in Figure 5, there was a further gradual weight loss before the steep weight loss, likely due to decomposition that begins at around 200-250°C.

[0191] D. Water content of purified ALM-488 Karl Fischer titration was performed with the chloride salt of ALM-488 using a Mettler Toledo Stromboli automated oven sample changer. The instrument was standardized with Hydranal® water standard (Fluka). Solid ALM-488 samples were weighed into sample vials, heated to 100°C in the oven, and the water evaporated from the sample was transferred to the titrator cell by purging gas. The water content measured in the samples was 4.08%, which was consistent with the TGA results.

[0192] E. Peptide content of purified ALM-488 To correct for the measured chloride and water content in the chloride salt of ALM-488, the peptide content was calculated to be 91.28%. The total concentrations and amounts of ALM-488 described below are expressed in terms of pure peptide, without both counterions and water. To determine the amount of free peptide for weighing ALM-488 for experimental and product manufacturing purposes, a correction multiplier of 1.096 is applied to the chloride salt of ALM-488.

[0193] Linear relationship between the amount of F. ALM-488 and HPLC peak area Standard solutions of ALM-488 at various concentrations and various injection volumes were analyzed using the above HPLC method. The linear relationship between the amount of ALM-488 injected and the area of the main chromatographic peak is shown in Figure 6. At loading masses outside the range plotted in Figure 6 (>5 μg of ALM-488), the peak area did not increase proportionally to the loading. All samples were appropriately diluted to remain within the demonstrated linear range. The acceptable precision was obtained by repeated injection of the standard solution. For example, the relative standard deviation of the average peak area from six 4 μL injections of an approximately 1 mg / mL aqueous solution of ALM-488 was 0.35%.

[0194] Example 4 :Test of pH-dependent solubility of ALM-488 salt To understand the correlation between pH and solubility, tests were conducted using both the chloride and acetate salts of purified ALM-488.

[0195] A. Solubility of the HCl salt of ALM-488 based on pH The initial visual evaluation confirmed that the solubility of ALM-488, HCl salt, was >180 mg / mL at a solution pH of <pH2.

[0196] To determine whether a nominal solubility of 40-50 mg / mL or higher could be maintained as the pH increased by gradually adding NaOH, titration experiments were conducted using ALM-488, HCl salt. The starting concentration of ALM-488, HCl salt in water (approximately 55 mg / mL, approximately 24 mM) (in terms of pure peptide) had a solution pH of 1.7. Figure 7 shows a plot of the measured pH as a function of increasing NaOH concentration added to the mixture. At solution pH < 4, the peptide remained in solution at a concentration > 50 mg / mL. Above pH 4, the peptide precipitated. The degree of precipitation increased, resulting in an opaque dispersion in the pH range of approximately 4.5-5.5. Further addition of NaOH led to the purification of the mixture as the pH increased to approximately 6.5, and complete dissolution was observed at pH > 7. Dilution of the mixture by base addition reduced the peptide concentration to approximately 47 mg / mL at pH > 7. The shaded region in Figure 7 represents the pH range in which the ALM-488 peptide exhibits appropriate solubility. As shown in the titration curve, resistance to pH changes due to base addition was observed at pH values ​​of approximately 4.5 and 6.5, which may correspond to the pKa values ​​of glutamic acid and 5-FAM.

[0197] B. Solubility of ALM-488 chloride and acetate based on pH. Figure 8 shows a superposition of titration curves for ALM-488 hydrochloride and ALM-488 acetate. As shown in Figure 8, the pH of the initial ALM-488 acetate mixture (approximately 50 mg / mL) was significantly higher (approximately pH 4) compared to the initial pH of the hydrochloride (approximately pH 1.8). At an initial pH of approximately 4, the ALM-488 peptide in the acetate did not completely dissolve, forming an opaque orange dispersion. Raising the pH of the acetate mixture by adding NaOH dissolved the precipitate, resulting in a clear solution at pH > 7. The shaded region in Figure 8 (pH > 7) represents the pH range in which ALM-488 chloride and acetate are expected to exhibit appropriate solubility. As shown in Figure 8, the amount of NaOH required to adjust the pH of the ALM-488 acetate solution to approximately 7 (to obtain appropriate solubility and an acceptable pH for intravenous formulation) was lower than that required for ALM-488 hydrochloride.

[0198] Example 5 : Effect of pH on the stability of ALM-488 To understand the pH-stability correlation, solutions of ALM-488, HCl salt, were prepared at approximately 1 mg / mL in 50 mM sodium citrate (pH 4-6) or sodium phosphate buffer (pH 7-8) solution. As shown in Table 4 below, the dissolution of ALM-488 was rapid and complete in water (pH 3.5) and phosphate buffer (pH 7-8). However, in citrate buffer (pH 4-6), the solubility of ALM-488 was <1 mg / mL, resulting in incomplete dissolution and excess insoluble solid. The entire mixture was filtered through a 0.45 μm PVDF syringe filter before storage to remove the insoluble solid. The pH value of the filtered solution was measured, and the samples were analyzed by HPLC using the HPLC method conditions in Table 3. The table lists the measured concentrations of ALM-488 (after filtration) and the measured solution pH.

[0199] [Table 4]

[0200] As shown in Table 4, the solubility of ALM-488 decreased significantly as the solution pH approached the isoelectric point, reaching approximately 0.2 mg / mL at pH 5 (50 nM citrate buffer, pH = 6.01). The solution was then transferred to an HPLC sample vial and stored at 5°C, room temperature (22-23°C), and 40°C.

[0201] Table 5 below shows the concentration measurements of intact ALM-488 peptide quantified relative to standard solutions and the area % of the major peptide peak measured using the HPLC analysis method in Table 3. Figure 9 is a superposition of representative chromatograms of ALM-488 solutions at various solution pH values ​​after storage at 40°C for 24 hours. As shown in this superposition, impurities at RRT approximately 1.4 proliferated in the solution at low pH values ​​- most notably at approximately 0.5 area % in water (pH 3.46). At high pH values, impurities at RRT approximately 0.96 appeared to proliferate during storage.

[0202] [Table 5]

[0203] The results of Example 5 demonstrate that ALM-488 has appropriate solution stability at a neutral pH value for further development of lyophilized products.

[0204] Example 6 The effect of storage temperature on the stability of ALM-488 To evaluate whether ALM-488, the HCl salt, could be supplied in the form of a concentrated frozen solution, a 50 mg / mL solution of the HCl salt was evaluated in water, and its storage stability at various temperatures was assessed. Solutions with a pH of 1.87 were prepared in sterile water for injection, and these solutions were stored at various storage temperatures (-20°C, 5°C, and 40°C) and analyzed at various periods using the HPLC method shown in Table 3. Table 6 lists the chromatographic data for these sample solutions, and Figure 10 shows superimposed magnified chromatograms of the ALM-488 solution (50 mg / mL) aqueous solution analyzed initially and after 7 days at various storage temperatures.

[0205] [Table 6]

[0206] As shown in Table 6 and Figure 10, solutions stored at 40°C showed significant degradation, mainly resulting in the proliferation of impurities with RRTs of approximately 1.06 and 1.38. The data also suggest that the level of impurities with an RRT of approximately 0.97 (formed under high pH conditions) decreased in a temperature-dependent manner in acidic solutions. Less degradation was detected in samples stored at lower temperatures (-20°C and 5°C).

[0207] Example 7 Testing of the solubility of organic amine compounds in ALM-488 salts To evaluate formulations suitable for preparing approximately 50 mg / vial samples of ALM-488, and to evaluate samples with an ALM-488 concentration of approximately 50 mg / mL, formulations containing various organic amine compounds were tested. Each of these samples contained (i) ALM-488 hydrochloride, (ii) a suitable base for lyophilized formulations (to adjust the solution pH to an optimal 7-8 as described in Examples 4 and 5), and (iii) at least one organic amine compound functioning as a pH adjuster, volume expander, and / or tonicity adjuster. Three organic amine compounds were tested: tromethamine (Tris, pKa 8.06), N-methylglucamine (meglumine, pKa 9.6), and L-arginine (pKa 12.5). Solutions of ALM-488 at 55-60 mg / mL (pH 1.8-1.9) were titrated by gradually adding 750 mM arginine, 1 M Tris, or 1 M meglumine. Visual observation was similar to that shown in Figure 7. As shown in Figures 11, 12, and 13, increasing the pH first precipitated the ALM-488 peptide, followed by dissolution by sequential base addition at pH > 7. In all cases, the ALM-488 concentration in the final solution at pH 7–8 was >50 mg / mL.

[0208] Figure 11 is a pH titration curve showing how pH as a function of added tromethamine (Tris) and the solubility of the HCl salt of ALM-488 are affected by pH. The calculated total ALM-488 peptide concentration of the sample ranged from 55 mg / mL (23.8 mM) without Tris (pH 1.89) to 107 mg / mL with Tris (pH 7.3) and 49.2 mg / mL (21.3 mM). The shaded area in Figure 11 represents the pH range in which a clear solution was obtained. Precipitation occurred near the isoelectric point pH, and the solubility was <50 mg / mL in the pH range of approximately 4 to 7.

[0209] Figure 12 is a pH titration curve showing how pH as a function of added meglumine (N-methylglucamine) and the solubility of the HCl salt of ALM-488 are affected by pH. The calculated total ALM-488 peptide concentration of the sample ranged from 55 mg / mL (23.8 mM) without meglumine (pH 1.86) to 49.2 mg / mL (21.3 mM) at 107 mm tris (pH 7.88). The shaded area in Figure 12 represents the pH range in which a clear solution was obtained. Precipitation occurred near the isoelectric point pH, and the solubility was <50 mg / mL in the pH range of approximately 4 to 7.

[0210] Figure 13 is a pH titration curve showing how pH as a function of added L-arginine (N-methylglucamine) and the solubility of the HCl salt of ALM-488 are affected by pH. The calculated total ALM-488 peptide concentration of the sample ranged from 55 mg / mL (23.8 mM) without L-arginine (pH 1.86) to 51.7 mg / mL (22.4 mM) with 116 mM arginine (pH 7.61). The shaded area in Figure 13 represents the pH range in which a clear solution was obtained. Precipitation occurred near the isoelectric point pH, and the solubility was <50 mg / mL in the pH range of approximately 4 to 6.6.

[0211] As can be seen in the titration curves in Figures 11-13, an organic base concentration of approximately 110 mM resulted in a nearly neutral solution pH and appropriate drug solubility.

[0212] The bases investigated are used in commercially available pharmaceuticals. Arginine is present in approved IV products at significantly higher doses than the ALM-488 formulation assumed based on the test in Example 7. For example, the arginine dose from the maximum dose of Azactam® injection is 6.2 g / day, and from the maximum dose of Caldolor® injection it is 3.2 g / day. Tromethamine, when used as a base with ALM-488, requires amounts exceeding the typical amounts encountered when tromethamine is used as a buffer. However, the maximum daily dose of ketrolactromethamine via IV injection (120 mg) is equivalent to approximately 38 mg of tromethamine. Tham® (tromethamine injection) is a 300 mM (3.6%) solution of tromethamine used for metabolic acidosis and is administered by slow infusion at much higher doses. The total daily dose of Baxdela® (delafloxacin meglumine injection, 300 mg twice daily) is equivalent to a meglumine dose of 384 mg.

[0213] The expected doses of tromethamine (Tris), meglumine (N-methylglucamine), and L-arginine required for the preparation of a 50 mg ALM-488 formulation are predicted to be significantly lower than the amounts of these bases contained in the approved drug.

[0214] Example 8 ALM-488 composition for stability testing A series of ALM-488 sample formulations were prepared in the pH range of 7–8 using various organic amine additives and their combinations. Formulations using L-arginine, meglumine, glycine, mannitol, and trehalose dihydrate, and their combinations, were prepared, as summarized in Table 7. The formulations were designed to obtain a 25 mg / mL solution of ALM-488 with sufficient amounts of each base to adjust the solution pH to 7–8. A concentration of 50 mg / vial of ALM-488 peptide was achieved by filling each vial with 2 mL of solution. The lyophilized ALM-488 formulation is expected to be reconstituted with a 1 mL volume solution, thus yielding a 50 mg / mL solution of ALM-488 peptide. The sample formulations in Table 7 were designed to target an osmotic pressure of approximately 300 mOsm / kg in the final reconstituted 50 mg / mL solution.

[0215] [Table 7]

[0216] As shown in Table 7, the pH of all sample formulations was approximately 7.5. The solution osmotic pressure of samples 8-1 and 8-2, which contained only a single pH adjuster (L-arginine and meglumine, respectively), was approximately 70 mOsm / kg. However, the use of combinations of pH adjusters in samples 8-3 to 8-7 resulted in significantly higher solution osmotic pressures (148 to 151 mOsm / kg). The concentrations of the pH adjusters were calculated so that, when used in combination, they would produce an osmotic pressure value of 140 to 150 mOsm / kg for the pre-lyophilized solution (25 mg / mL ALM-488). When the resulting lyophilized material is reconstituted with water in a 50 mg / mL ALM-488 solution, the osmotic pressure is expected to be in the desirable range of approximately 280 to 300 mOsm / kg.

[0217] The sample solution was filtered through a 0.22-μm PVDF syringe filter, and a certain amount of these filtered solutions was placed in USP Type 1 amber glass vials, stoppered with a freeze-dried rubber stopper, and crimp-sealed. The obtained sample vials were stored at 5 °C and 40 °C. Then, after 7 days and 14 days of storage, the sample solution was diluted 100× with water and analyzed under the HPLC method conditions in Table 3.

[0218] Tables 8 to 11 summarize the chromatography data of Samples 8-1 to 8-7. Figure 14 is an overlay of the enlarged HPLC chromatograms of Sample 8-4 at the time of the first preparation, after storage at 40 °C for 7 days, and after storage at 40 °C for 14 days. Figure 15 is an overlay of the enlarged HPLC chromatograms of Samples 8-1 to 8-7 of Example 8 after storage at 40 °C for 14 days.

[0219]

Table 8

[0220]

Table 9

[0221]

Table 10

[0222]

Table 11

[0223] As shown in Tables 8-11, the main common impurities occurred at RRT approximately 0.97. Impurities at RRT approximately 1.20 appeared to be more prevalent in sample 8-3, which contained arginine and glycine (see Figure 15). With the exception of the arginine-glycine solution (sample 8-3), which appeared to have lost approximately 2% intact ALM-488, all sample formulations lost only approximately 1% intact ALM-488 after 14 days at 40°C, expressed as area % of the main peak.

[0224] Example 9 ALM-488 composition for stability testing Sample formulations 8-1 to 8-7 were subjected to DSC experiments at temperatures below ambient temperature. Solution samples (approximately 25 μL) were transferred to a DSC pan, sealed, and subjected to a cooling-heating cycle. In a typical run, the sample was cooled to -50°C and maintained isothermally to ensure maximum freezing and formation of the freeze-concentrated phase. After this, the sample was heated at 2°C / min to a temperature exceeding the ice melting event. A nitrogen gas purge of 40 mL / min was maintained in the DSC chamber. Meanwhile, a sharp exothermic reaction of ice crystallization was observed during the cooling of the sample formulations, but no solute crystallization event was observed.

[0225] The heating curves of the frozen solutions of the arginine-containing preparations (samples 8-1, 8-3, 8-4, and 8-7) are superimposed on Figure 16, and the heating curves of the frozen solutions of the meglumine-containing preparations (samples 8-2, 8-5, and 8-6) are superimposed on Figure 17.

[0226] As shown in Figure 16, the ALM488-arginine frozen preparation (8-1) exhibited a weak glass transition (Tg'), starting at approximately -16°C. When mannitol (8-4) or glycine (8-3) were included as further solutes, their crystallization appeared to be inhibited. Retention of mannitol (8-4) or glycine (8-3) in the frozen concentrated amorphous phase resulted in a decrease in Tg', starting at approximately -33°C and -34°C, respectively. The arginine-trehalose preparation (8-7) also retained its amorphous state during solution freezing and resulted in a decrease in Tg' (starting at approximately -28°C).

[0227] As shown in Figure 17, the ALM488-meglumine freezing formulation (8-2) did not clearly exhibit a strong glass transition temperature. Crystallization of the meglumine-mannitol formulation (8-6) and meglumine-glycine formulation (8-5) during freezing also appeared to be inhibited. This resulted in a single broad glass transition, with initiation temperatures of approximately -36°C and -37°C, respectively.

[0228] Heat treatment of the frozen solution at -15°C in DSC raises the Tg' onset by approximately 2°C, suggesting that crystallization is substantially inhibited by the co-solute, ALM488, and arginine / meglumine.

[0229] Table 12 below summarizes the DSC and FDM (freeze-dried microscopy) data for samples 8-1 to 8-7.

[0230] Lyostat 2 is a fully integrated freeze-drying microscope that enables the determination of critical parameters for freeze-drying formulations. Using this method, a liquid film of 2 μL of sample solution was placed between a quartz slide and a glass coverslip, and then sealed within the cryostage of the freeze-drying microscope. In a typical experiment, the sample was frozen to approximately -50°C with the help of liquid nitrogen and then held for 30 minutes to ensure maximum freezing of the solution. Once a frozen film was obtained, ice sublimation was initiated by reducing the chamber pressure to approximately 150 mTorr. This resulted in drying from the periphery to the center of the film. When the freeze-dried periphery region could be visualized in an image, the sample stage was heated at an appropriate rate, and images were captured at regular intervals to visualize the thermal events.

[0231] The initial visual evidence of the decay event (due to increased mobility from freeze-concentration) for samples 8-1 to 8-7, along with the glass transition onset temperature, is shown in Table 12. In each case, the visual observation of the decay event was observed at a much higher temperature than the onset of the broad glass transition event.

[0232] [Table 12]

[0233] Arginine sample preparation (8-1) was selected for use in the lyophilization test below because its use is approved in several non-enteral products and because it exhibited slightly higher glass transition and decay temperatures compared to other sample preparations. Arginine-glycine preparation (8-3) was not selected for further testing because it exhibited slightly lower solution stability compared to the other solutions.

[0234] Example 10 Initial freeze-drying test Sample ALM-488 formulations containing arginine (10-1), arginine-mannitol (10-2), and arginine-trehalose (10-3), summarized in Table 13 below, were prepared in amber glass vials for initial lyophilization testing. For all three samples, the molar ratio of ALM-488 peptide to HCl was approximately 1:2.5 (peptide / HCl), and the molar ratio of ALM-488 peptide to arginine was approximately 1:5 (peptide / arginine). Each sample was prepared in a 5 mL amber glass vial (20 mm opening, USPI type glass tube) with a 20 mm lyophilization stopper (FluoroTech V10-F597 4432 / 50, West Pharmaceutical Services) and a 20 mm flip-off aluminum crimp seal.

[0235] [Table 13] 1 Chloride content (3.57% w / w; 3.67% w / w as HCl) and water content (KFT) -4.08%, peptide purity = 98.95%; 50 mg of ALM-488 = (50 × 100) / (0.9895) × (100 - 4.08 - 3.67) = 54.78 mg of the drug substance. 2The densities of the compositions before lyophilization were determined to be 1.00 g / cc (Sample 10-1), 1.00 g / cc (Sample 10-2), and 1.01 g / cc (Sample 10-3). Using these densities, the total solution fill volume per vial was calculated as described in the table. Water was removed during the process.

[0236] A. Solution Preparation and Vial Filling For each sample, the ALM-488 peptide was first placed into a suitable container for solution preparation. Then, approximately 80% of the estimated batch volume of water was slowly added to the peptide, and the mixture was agitated to dissolve the drug. Next, the batch amount of arginine was added to the peptide solution while mixing to create a vortex. The addition of arginine first precipitates the drug, but mixing results in a clear orange solution. For compositions containing mannitol or trehalose, additional agents were then added to the solution and stirred to obtain a clear solution. The total solution weight was then adjusted with sterile water for injection. The resulting solution was then filtered through a 0.2 micron PVDF filter unit, and the filtered solution was sealed into vials within the lyophilization fill weight tolerance of ±2%.

[0237] B. Lyophilization The processing parameters for the three compositions are summarized in Table 14, and the corresponding lyophilization process diagrams are shown in Figures 18 - 20.

[0238] [Table 14]

[0239] All of Samples 10-1 to 10-3 resulted in lyophilized cakes with no evidence of collapse and a good appearance. Figures 21 and 22 are images of the lyophilized products from ALM-488 Sample Formulations 10-1 and 10-2. Figure 21 shows a side view of the lyophilized cake, and Figure 22 shows an overhead view of the removed lyophilized cake of ALM-488 Sample Formulation 10-2.

[0240] C. Water Content of Lyophilized Samples The water content of the lyophilized samples 10-1 to 10-3 was measured using a Mettler Toledo DL39 coulometric Karl Fischer titrator equipped with a Mettler Toledo Stromboli automatic oven sample changer. The instrument was standardized with Hydranal® water standard (Fluka). Solid lyophilized samples were weighed into sample vials, heated to 100°C in an oven, and the water evaporated from the sample was transferred to the titrator cell using a purging gas. The measured water content in each lyophilized batch is shown in Table 15 below. The water content was low, confirming the appropriateness of the lyophilization process. The mannitol-containing lyophilized sample (10-2) had a higher residual water content than the other two batches.

[0241] [Table 15]

[0242] D. Powder X-ray diffraction The X-ray diffraction patterns of the lyophilized solid products of ALM-488 formulation samples 10-1, 10-2, and 10-3 were measured using a wide-angle X-ray diffractometer (Bruker D8 Advance). Diffraction patterns were recorded over an angular range of 5–45° 2θ. Scanning was performed in step scanning mode with a step size of 0.005° 2θ and a measurement time of 0.25 seconds / step.

[0243] As shown in Figures 23 and 25, the PXRD patterns of the lyophilized samples 10-1 (arginine) and 10-3 (arginine-trehalose) show amorphous halos. In contrast, as shown in Figure 24, the PXRD pattern of the lyophilized sample 10-2 (arginine-mannitol) shows characteristic peaks of crystalline mannitol. There was clear evidence of mannitol hemihydrate, along with peaks characteristic of the δ- and β-anhydrous forms of mannitol. The presence of mannitol hemihydrate can be explained in part by the high residual water content in the lyophilized sample 10-2, as shown in Table 15.

[0244] The PXRD pattern of sample 10-2 in Figure 24 suggests partial crystallization of mannitol during cooling and / or subsequent heat treatment. However, the mannitol hemihydrate remains completely dehydrated during secondary drying and is retained in the final freeze-dried product. It has been reported that a higher temperature drying step is required to dehydrate the mannitol hemihydrate formed during freeze-drying.

[0245] E. Pharmaceutical Assays The content and purity of ALM-488 peptide in the lyophilized product vials of samples 10-1, 10-2, and 10-3 were evaluated using the HPLC method conditions in Table 3. 1 mL of purified water was added to each lyophilized product vial to completely dissolve the contents. The resulting solution was then transferred to a 200 mL volumetric flask. Each vial was thoroughly rinsed with water, and the rinse solution was transferred to the flask before adjusting the volume to 200 mL with water. The resulting solutions (approximately 0.25 mg / mL of ALM488) were individually injected into the HPLC system as described above and analyzed. The lyophilization process appeared to slightly increase the levels of some impurities. As shown in Table 16, the impurities previously observed in the solution (see RRT approximately 0.73, RRT approximately 1.16, and RRT approximately 1.20 and 8-11 in Table 6) were also found in the lyophilized products of samples 10-1 to 10-3.

[0246] [Table 16]

[0247] F. Reconstituted solution composition Next, the reconstituted solution compositions were prepared using samples 10-1, 10-2, and 10-3, and the target reconstituted solution concentration was a 50 mg / mL solution obtained by reconstituting the contents of the lyophilized samples to a final volume of 1 mL. The compositions of the reconstituted solutions are summarized in Table 17 below. Solutions of these compositions were prepared by volume, and their weight was used to estimate the density of the reconstituted solutions (listed in Table 17). This allowed for the determination of the theoretical amount of water to be added to each vial to obtain a reconstituted solution volume of 1 mL / vial, and therefore an ALM-488 concentration of 50 mg / mL.

[0248] [Table 17] 1 The lyophilization and amorphization of trehalose dihydrate by freeze-drying yields a theoretical amount of 50.4 mg of anhydrous trehalose in the final freeze-dried vial. The original solution is prepared with 56.4 mg of trehalose dihydrate per vial. 2 The amount of water is based on the theoretical amount needed to obtain a final reconstituted solution volume of 1 mL.

[0249] G. Reconstitution of freeze-dried samples Lyophilized solids from samples 10-1, 10-2, and 10-3 were reconstituted by immobilizing and adding reconstitution fluids (summarized in Table 18). The lyophilized compositions of samples 10-2 and 10-3 contained amounts of mannitol or trehalose calculated to produce osmotic pressure values ​​similar to human plasma upon reconstitution with water. The lyophilized composition of sample 10-1 was isotonic and reconstituted with physiological saline (NS; 0.9% NaCl) or half physiological saline (HNS; 0.45% NaCl). Based on the osmotic pressures of the pre-lyophilized compositions (see Table 7, samples 8-1, 8-4, and 8-7), an acceptable osmotic pressure was achieved, as expected, when reconstituted with 0.45% NaCl.

[0250] [Table 18]

[0251] In all samples 10-1, 10-2, and 10-3, the lyophilized solids dissolved immediately, becoming clear solutions in less than one minute after the addition of the reconstitution fluid.

[0252] H. HPLC analysis and stability of reconstituted samples The reconstituted solutions of samples 10-1, 10-2, and 10-3, prepared as described above, were analyzed by HPLC using the method conditions in Table 3 to determine the ALM-488 content and evaluate the level of impurities. Certain amounts of these samples were stored at 5°C and 25°C, and then analyzed after 24 hours to confirm the stability of the reconstituted solutions during short-term storage. Tables 19A, 19B, and 19C contain HPLC data for the reconstituted samples obtained from the reconstitution of lyophilized samples 10-1, 10-2, and 10-3, both immediately after reconstitution and after storage. As shown in Tables 19A, 19B, and 19C, no significant detectable growth of any impurities was observed during short-term storage of the reconstituted samples.

[0253] [Table 19]

[0254] [Table 20]

[0255] [Table 21]

[0256] Example 11 :ALM-488 / Arginine-Trehalose-containing batch preparation A. Preparation of ALM-488 / arginine-trehalose freeze-dried product (Sample 11-1) A series of batch formulations containing arginine-trehalose, specifically ALM-488,HCl salts, were prepared under the conditions of Examples 1 and 2, as summarized in Table 20. The amounts in Table 20 represent the amount of ALM-488 product per vial (50 mg / vial), and the ALM-488 concentration of the pre-lyophilized solution was 25 mg / mL. The batch formulations are collectively referred to as Sample 11-1. The chloride content of ALM-488,HCl salt was 3.67% (expressed as HCl). After correction for water, the peptide:HCl molar ratio was 1:2.5.

[0257] The batch solution was prepared as described above, filtered through a 0.22 micron PVDF filter, then packed into a series of 5 mL amber glass vials (2 mL / vial), and subsequently lyophilized. Lyophilized sample 11-1 was prepared using the same lyophilization process as for sample 10-3, which was carried out at a primary drying shelf temperature of -10°C for 17 hours, followed by a 30-minute gradient to a secondary drying temperature of 25°C, and then a 3.5-hour secondary drying at a +25°C shelf. The chamber pressure was maintained at approximately 150 mTorr during both the primary and secondary drying stages. A process diagram of the lyophilization process for sample 11-1 is shown in Figure 26.

[0258] [Table 22] 1 Chloride content (3.57% w / w; 3.67% w / w as HCl) and water content (KFT) -4.08%, peptide purity -98.95%. 50 mg of ALM488 = (50 × 100) / (0.9895) × (100 - 4.08 - 3.67) = 54.78 mg of the drug substance. 2 The density of the composition before freeze-drying was determined to be 1.02 g / cc. Using this density, the total solution volume per vial was calculated as shown in the table. Water was removed during processing.

[0259] B. Reconstitution of ALM-488 / arginine-trehalose preparation (Sample 11-2) A 1 mL volume / vial (50 mg / mL) reconstituted formulation of ALM-488 / arginine-trehalose named Sample 11-2 was prepared from Sample 11-1. Addition of 920 μL of sterile water to the vial of Sample 11-1 (50 mg / vial) resulted in a reconstituted formulation having the composition and properties shown in Table 21, where the concentration of the ALM-488 peptide was 50 mg / mL.

[0260]

Table 23

[0261] Example 12 : Filtration Suitability Test Using ALM-488 / Arginine-Trehalose To evaluate whether filtration of the ALM-488 / arginine-trehalose formulation before lyophilization is necessary or advantageous, both filtered and unfiltered ALM-488 / arginine-trehalose samples before lyophilization were prepared in the same manner as described in Example 11. The unfiltered sample before lyophilization was named Sample 12-1 and the filtered sample before lyophilization was named Sample 12-2. Sample 12-2 was filtered through a 0.22 micron Millipak Durapore® capsule filter unit after complete dissolution of the sample formulation was visually observed.

[0262] Samples 12-1 and 12-2 were analyzed by HPLC for drug content and purity using the HPLC method conditions in Table 3. Table 22 lists the levels (area %) of the main peaks, significant drug-related impurities, and calculated drug concentrations in the solution. As seen in Table 22, there were no significant differences in impurity levels or drug content in the solution (bottom row) between the unfiltered (12-1) and filtered (12-2) solution samples. This data indicates that complete dissolution of the drug occurred during the formulation process and there was no apparent significant incompatibility with the filter material of the 0.22 micron Millipak Durapore® capsule filter unit.

[0263] [Table 24]

[0264] Example 13 Long-term stable freeze-dried product of ALM-488 Lyophilized ALM-488 samples 13-1 (arginine), 13-2 (arginine-mannitol), and 13-3 (arginine-trehalose), prepared in the same manner as the corresponding samples 10-1 (arginine), 10-2 (arginine-mannitol), and 10-3 (arginine-trehalose) in Example 10, were stored at various temperatures and relative humidities for up to 3 months. The resulting lyophilized samples 13-1, 13-2, and 13-3 were then reconstituted (hydrated) and tested for purity using the HPLC method conditions in Table 3. Lyophilized ALM-488 / arginine-trehalose sample 13-3 was also tested for water content using Karl Fischer titration and subjected to PXRD analysis.

[0265] A. Effect of long-term storage on the HPLC purity of freeze-dried ALM-488 / arginine (Sample 13-1) The lyophilized ALM-488 / arginine sample 13-1 (corresponding to sample 10-1 in Example 10) was divided into three HPLC vials and stored for three months at (a) 40°C and 75% relative humidity (sample 13-1-a), (b) 25°C and 60% relative humidity (sample 13-1-b), and (c) 5°C for three months (sample 13-1-c). Each sample was then tested for purity using the HPLC method conditions in Table 3. Table 23 below summarizes the HPLC purity data for samples 13-1-a, 13-1-b, and 13-1-c compared to the initial (pre-storage) sample 13-1. As shown in Table 23, the overall degree of degradation was low in all stored samples (<0.6% loss of intact drug after three months at 40°C / 75%RH).

[0266] [Table 25]

[0267] B. Effect of long-term storage on the HPLC purity of freeze-dried ALM-488 / arginine-mannitol (Sample 13-2) Lyophilized ALM-488 / arginine-mannitol sample 13-2 (corresponding to sample 10-2 in Example 10) was divided into three HPLC vials and stored for three months at (a) 40°C and 75% relative humidity (sample 13-2-a), (b) 25°C and 60% relative humidity (sample 13-2-b), and (c) 5°C (sample 13-2-c). Each sample was then tested for purity using the HPLC method conditions in Table 3. Table 24 below summarizes the HPLC purity data for samples 13-2-a, 13-2-b, and 13-2-c compared to the initial (pre-storage) sample 13-2. As shown in Table 24, the proportion of ALM-488 measured after storage appears to increase in all samples. However, the presence of mannitol hemihydrate in sample 10-2 raises concerns about the risk of dehydration during long-term storage and its potential impact on subsequent stability.

[0268] [Table 26]

[0269] C. Effect of long-term storage on the HPLC purity of freeze-dried ALM-488 / arginine-trehalose (Sample 13-3) The freeze-dried ALM-488 / arginine-trehalose sample 13-3 (corresponding to sample 10-3 in Example 10) was divided into three HPLC vials and stored for the following periods: (a) 1 month at 25°C and 60% relative humidity (sample 13-3-a), (b) 3 months at 25°C and 60% relative humidity (sample 13-3-b), (c) 3 months at 25°C and 60% relative humidity (sample 13-3-c), (d) 2 months at 5°C (sample 13-2-d), (e) 3 months at 5°C (sample 13-2-e), (f) 1 month at 40°C and 75% relative humidity (sample 13-3-f), (g) 2 months at 40°C and 75% relative humidity (sample 13-3-g), and (h) 3 months at 40°C and 75% relative humidity (sample 13-3-h). Next, each sample was tested for purity using the HPLC method conditions in Table 3. Table 25 below summarizes the HPLC purity data for samples 13-3-a, 13-3-b, 13-3-c, 13-3-d, and 13-3-3e compared to the initial (pre-storage) sample 13-3.

[0270] [Table 27]

[0271] Table 26 below summarizes the HPLC purity data for samples 13-3-f, 13-3-g, and 13-3-h compared to the initial (pre-storage) sample 13-3.

[0272] [Table 28]

[0273] As shown in Tables 25 and 26, the overall degree of degradation of the total ALM-488 / arginine-trehalose lyophilized product was low (<0.6% loss of intact drug after 3 months at 40°C / 75%RH). The main degradation product appeared as a shoulder of the main peak (RRT approximately 0.97) and was particularly amplified in samples under 40°C / 75%RH and 25°C / 60%RH conditions.

[0274] Figures 27 and 28 show superimposed HPLC chromatograms of the lyophilized sample 13-3 (ALM-488 / arginine-trehalose) initially and after storage at 40°C / 75%RH for 1 month, 2 months, and 3 months. The chromatograms show the growth of primary impurities (RRT approximately 0.97) as shoulders of the main peak during storage.

[0275] A comparison of the long-term storage results of samples 13-1 (arginine), 13-2 (arginine-mannitol), and 13-3 (arginine-trehalose) leads to the conclusion that arginine-trehalose sample 13-3 is the best candidate for further development. Sample 13-3 is advantageous over sample 13-1 because the use of a tonicity regulator (trehalose) allows for fine-tuning of the pH and solubility characteristics of the pre-freeze-drying and reconstituted formulations. Despite sample 13-2 showing good long-term stability over three months, sample 13-3 is advantageous over sample 13-2 because the presence of mannitol hemihydrate in sample 13-2 is thought to raise the risk of dehydration during long-term storage and its potential impact on subsequent stability.

[0276] D. Effects of water content in sample 13-3 on long-term storage The ALM-488 / arginine-trehalose freeze-dried sample 13-3 was stored at 40°C and 75% relative humidity for 3 months and then tested using the Karl Fischer titration method described in Example 10. After this long-term storage, the water content of sample 10-3 increased from an initial value of approximately 0.7% w / w to approximately 1.8% w / w. This increase is due to a combination of water transfer from the rubber stopper to the amorphous dry pharmaceutical and water vapor transfer from the stopper to the humid external environment.

[0277] E. Effects of long-term storage on powder X-ray diffraction of sample 10-3 The ALM-488 / arginine-trehalose lyophilized sample 13-3 was stored at 40°C and 75% relative humidity for 3 months, and then tested using the PXRD method described above in Example 10. As shown in Figure 29, the lyophilized sample 10-3 retained its amorphous characteristics after being stored at high temperature and high humidity. Furthermore, the obtained lyophilized sample showed rapid dissolution into a clear solution within 1 minute of adding water to the sample vial.

[0278] These and other variations may be made into embodiments in light of the foregoing. In general, the terms used in the following claims should not be construed as limiting the scope of claims to any particular embodiment disclosed in the specification and claims, but rather as including all possible embodiments in line with the equivalent complete scope to which such claims are entitled. Accordingly, the claims are not limited by this disclosure. This application claims priority under U.S. Provisional Application 63 / 501,593, filed with the U.S. Patent and Trademark Office on 11 May 2023, which is incorporated herein by reference in its entirety and disclosures.

Claims

1. (a) Equation (I): 【Chemistry 1】 Fluorescein-labeled neuronal targeting peptides or their pharmaceutically acceptable salts, solvates, hydrates, isomers, tautomers, racemates, or isotopes; (b) Additives including pH adjusters, buffers, bulking agents, tonicity adjusters, preservatives, or any combination thereof; and (c) Aqueous medium An aqueous pharmaceutical composition comprising: The pH of the composition is approximately 7 to 9; and The concentration of peptides in the aqueous composition is in the range of approximately 25 mg / mL to approximately 200 mg / mL, based on the total weight of the aqueous composition. Aqueous pharmaceutical composition.

2. The aqueous composition according to claim 1, comprising a pharmaceutically acceptable salt of a peptide.

3. The aqueous composition according to claim 1 or 2, comprising the HCl salt of a peptide.

4. The aqueous composition according to claim 3, wherein the chloride content of the HCl salt is in the range of about 3.0% (w / w) to about 4.0% (w / w) based on the total mass of the peptide in the aqueous composition.

5. The aqueous composition according to any one of claims 2 to 4, wherein the molar ratio of peptide to pharmaceutically acceptable salt is in the range of about 1:1 to about 1:

3.

6. The aqueous composition according to any one of claims 1 to 5, wherein the additive comprises a pH adjuster, a volume extender, a tonicity adjuster, or any combination thereof.

7. The aqueous composition according to any one of claims 1 to 6, wherein the additive comprises alanine, arginine, cellulose, cellobiose, cyclodextrin, dextrose, erythritol, fructose, galactose, glucose, glycerin, glycine, histidine, inositol, lactose, maltose, mannitol, mannose, meglumine, raffinose, ribose, sorbitol, sucrose, trehalose, tromethamine, xylitol, xylose, or any combination thereof.

8. The aqueous composition according to any one of claims 1 to 6, wherein the additive comprises arginine, glycine, mannitol, meglumine, trehalose, tromethamine, or any combination thereof.

9. Additives: A first additive comprising arginine, meglumine, glycine, or any combination thereof; and A second additive containing mannitol, trehalose, or both may be added as desired. An aqueous composition according to any one of claims 1 to 8, comprising

10. The aqueous composition according to claim 9, wherein the mass ratio of the first additive to the second additive is in the range of about 1:1 to about 1:

5.

11. The aqueous composition according to any one of claims 1 to 9, wherein the additive comprises arginine, arginine-mannitol, arginine-trehalose, arginine-meglumine, meglumine, meglumine-glycine, or meglumine-mannitol.

12. The aqueous composition according to any one of claims 1 to 11, wherein the additive comprises arginine-trehalose.

13. The aqueous composition according to claim 12, wherein the mass ratio of arginine to trehalose is in the range of about 1:2 to about 1:

3.

14. The aqueous composition according to any one of claims 1 to 13, wherein the mass ratio of peptide to additive is in the range of about 1.0 to about 1.

5.

15. The aqueous composition according to any one of claims 1 to 14, wherein the aqueous medium comprises sterile water, saline solution, aqueous buffer solution, or any combination thereof.

16. The aqueous composition according to any one of claims 1 to 15, wherein the aqueous medium comprises sterile water, physiological saline, half physiological saline, or any combination thereof.

17. The aqueous composition according to any one of claims 1 to 16, wherein the pH of the aqueous composition is in the range of about 7.2 to about 7.

8.

18. The aqueous composition according to any one of claims 1 to 17, wherein the concentration of the peptide in the aqueous composition is in the range of about 40 mg / mL to about 75 mg / mL.

19. The aqueous composition according to any one of claims 1 to 18, wherein the density of the aqueous composition is in the range of about 0.85 g / mL to about 1.15 g / mL.

20. The aqueous composition according to any one of claims 1 to 19, wherein the osmotic pressure of the aqueous composition is in the range of about 200 mOsm / kg to about 350 mOsm / kg.

21. The aqueous composition according to any one of claims 1 to 20, wherein the volume of the aqueous composition is in the range of about 1.8 mL to about 2.2 mL.

22. An aqueous composition according to any one of claims 1 to 21, wherein the aqueous composition is filtered.

23. The aqueous composition according to any one of claims 1 to 21, wherein the aqueous composition is not filtered.

24. The aqueous composition according to any one of claims 1 to 23, wherein, after being stored at 40°C for 14 days, it contains at least 98.5% by mass of the total mass of the aqueous composition.

25. A freeze-dried pharmaceutical composition formed by freeze-drying an aqueous composition according to any one of claims 1 to 24.

26. (a) Equation (I): 【Chemistry 2】 Fluorescein-labeled neuronal targeting peptides, or their pharmaceutically acceptable salts, solvates, hydrates, isomers, tautomers, racemates, or isotopes; and (b) Additives including pH adjusters, buffers, fillers, tonicity adjusters, preservatives, or any combination thereof A freeze-dried pharmaceutical composition comprising, Here, the water content of the freeze-dried composition is in the range of approximately 0.2% (w / w) to approximately 2.0% (w / w) relative to the total mass of the freeze-dried composition. Freeze-dried pharmaceutical composition.

27. The freeze-dried composition according to claim 26, wherein when the freeze-dried composition is mixed with an aqueous medium at ambient temperature, it forms a homogeneous aqueous composition having a peptide concentration of 200 mg / mL or less.

28. The freeze-dried composition according to claim 26 or 27, wherein the freeze-dried composition is in the form of a freeze-dried cake having a good appearance.

29. The freeze-dried composition according to any one of claims 26 to 28, wherein the freeze-dried composition contains at least 98.5% by mass of peptide after being stored at 40°C and 75% relative humidity for three months.

30. A freeze-dried composition according to any one of claims 26 to 29, comprising a pharmaceutically acceptable salt of a peptide.

31. A freeze-dried composition according to any one of claims 26 to 30, comprising the HCl salt of a peptide.

32. The freeze-dried composition according to claim 31, wherein the chloride content of the HCl salt is in the range of about 3.0% (w / w) to about 4.0% (w / w) based on the total mass of the HCl salt of the peptide.

33. The freeze-dried composition according to claim 31 or 32, wherein the molar ratio of peptide to pharmaceutically acceptable salt is in the range of about 1:1 to about 1:

3.

34. The freeze-dried composition according to any one of claims 26 to 33, wherein the additive comprises a pH adjuster, a volume extender, a tonicity adjuster, or any combination thereof.

35. A freeze-dried composition according to any one of claims 26 to 34, wherein the additive comprises alanine, arginine, cellulose, cellobiose, cyclodextrin, dextrose, erythritol, fructose, galactose, glucose, glycerin, glycine, histidine, inositol, lactose, maltose, mannitol, mannose, meglumine, raffinose, ribose, sorbitol, sucrose, trehalose, tromethamine, xylitol, xylose, or any combination thereof.

36. A freeze-dried composition according to any one of claims 26 to 35, wherein the additive comprises arginine, glycine, mannitol, meglumine, trehalose, tromethamine, or any combination thereof.

37. A freeze-dried composition according to any one of claims 26 to 36, comprising a first additive containing arginine, meglumine, or glycine, and optionally a second additive containing mannitol or trehalose.

38. The freeze-dried composition according to claim 37, wherein the mass ratio of the first additive to the second additive is in the range of about 1:1 to about 1:

5.

39. A freeze-dried composition according to any one of claims 26 to 38, wherein the additive comprises arginine, arginine-mannitol, arginine-trehalose, arginine-meglumine, meglumine, meglumine-glycine, or meglumine-mannitol.

40. A freeze-dried composition according to any one of claims 26 to 39, wherein the additive comprises arginine-trehalose.

41. The freeze-dried composition according to claim 40, wherein the mass ratio of arginine to trehalose is in the range of about 1:2 to about 1:

3.

42. A freeze-dried composition according to any one of claims 26 to 41, wherein the mass ratio of peptide to additive is in the range of about 1.0 to about 1.

5.

43. A reconstituted aqueous pharmaceutical composition formed by combining any freeze-dried composition of claims 26 to 42 with an aqueous medium.

44. The reconstituted aqueous composition according to claim 43, wherein the aqueous medium comprises sterile water, saline solution, aqueous buffer solution, or any combination thereof.

45. The reconstituted aqueous composition according to claim 43, wherein the aqueous medium comprises sterile water, physiological saline, half physiological saline, or any combination thereof.

46. The reconstituted aqueous composition according to any one of claims 43 to 45, wherein the reconstituted composition is homogeneous.

47. The reconstituted aqueous composition according to any one of claims 43 to 46, wherein the pH of the reconstituted composition is in the range of about 7 to about 9.

48. The reconstituted aqueous composition according to any one of claims 43 to 47, wherein the concentration of the peptide in the reconstituted composition is in the range of about 25 mg / mL to about 200 mg / mL based on the total volume of the reconstituted composition.

49. The reconstituted aqueous composition according to any one of claims 43 to 48, wherein the density of the reconstituted composition is in the range of about 0.85 g / mL to about 1.15 g / mL.

50. The reconstituted aqueous composition according to any one of claims 43 to 49, wherein the osmotic pressure of the reconstituted composition is in the range of about 200 mOsm / kg to about 350 mOsm / kg.

51. A reconstituted aqueous composition according to any one of claims 43 to 50, wherein the volume of the reconstituted composition is in the range of about 1.8 mL to about 2.2 mL.

52. A reconstituted aqueous composition according to any one of claims 43 to 51, wherein the reconstituted composition is filtered.

53. The reconstituted aqueous composition according to any one of claims 43 to 52, wherein the reconstituted composition is unfiltered.

54. A reconstituted aqueous composition according to any one of claims 43 to 53, wherein, after being stored at 40°C for 14 days, it contains at least 98.5% by mass of peptides relative to the total mass of the reconstituted composition.

55. A method for forming a freeze-dried pharmaceutical composition, comprising freeze-drying at least one aqueous composition according to claims 1 to 24 to obtain a freeze-dried product.

56. The method according to claim 55, wherein freeze-drying includes freezing an aqueous composition at a temperature of about -80°C to about -50°C to obtain a frozen solid composition.

57. The method according to claim 56, wherein the freeze-drying further comprises annealing of the frozen solid composition at a temperature in the range of about -40°C to about -20°C.

58. The method according to claim 56 or 57, wherein the freeze-drying further comprises drying the freeze-solid composition at a temperature in the range of about -20°C to room temperature and at a pressure in the range of about 100 mTorr to about 300 mTorr to obtain a freeze-dried composition.

59. Dryness: Primary drying of frozen solid compositions at a primary pressure of approximately 100 mTorr to approximately 300 mTorr at a temperature in the range of approximately -20°C to approximately 0°C; and Secondary drying of frozen solid compositions at a secondary pressure of approximately 100 mTorr to approximately 300 mTorr at a temperature range of approximately 0°C to room temperature. The method according to claim 58, including the method described in claim 58.

60. A freeze-dried pharmaceutical composition formed by any of the methods of claims 55 to 59.

61. A method for forming a reconstituted aqueous pharmaceutical composition, comprising combining a freeze-dried composition according to any of claims 26 to 42 with an aqueous medium.

62. The method according to claim 61, further comprising filtration of the reconstituted aqueous composition.

63. The method according to claim 61, wherein the reconstituted aqueous composition is not filtered.

64. The method according to any one of claims 61 to 63, wherein the reconstituted aqueous composition is homogeneous.

65. The method according to any one of claims 61 to 64, wherein the aqueous medium comprises sterile water, saline solution, aqueous buffer solution, or any combination thereof.

66. The method according to any one of claims 61 to 64, wherein the aqueous medium comprises sterile water, physiological saline, half physiological saline, or any combination thereof.

67. The method according to any one of claims 61 to 66, wherein the pH of the reconstituted aqueous composition is in the range of about 7 to about 9.

68. The method according to any one of claims 61 to 67, wherein the concentration of the peptide in the reconstituted aqueous composition is in the range of about 25 mg / mL to about 200 mg / mL based on the total volume of the reconstituted composition.

69. The method according to any one of claims 61 to 68, wherein the density of the reconstituted aqueous composition is in the range of about 0.85 g / mL to about 1.15 g / mL.

70. A method composition according to any one of claims 61 to 69, wherein the osmotic pressure of the reconstituted aqueous composition is in the range of about 200 mOsm / kg to about 350 mOsm / kg.

71. The method according to any one of claims 61 to 70, wherein the volume of the reconstituted aqueous composition is in the range of about 1.8 mL to about 2.2 mL.

72. The method according to any one of claims 61 to 71, wherein the reconstituted aqueous composition contains at least 98.5% by mass of peptides, measured relative to the total mass of the reconstituted aqueous composition after being stored at 40°C for 14 days.

73. A method for identifying human nerve cells or nerves, comprising contacting human nerve cells or nerves with any composition of claims 1 to 54.

74. The method according to claim 73, wherein human nerve cells or nerves are brought into contact with a free fluorescent portion in combination with the composition.

75. A method for delivering a drug to human nerve cells or nerves, comprising contacting human nerve cells or nerves with any composition of claims 1 to 54.

76. The method according to any one of claims 73 to 75, comprising administering the composition to a human subject in need thereof.

77. The method according to any one of claims 73 to 76, comprising administering the composition by systemic intravenous injection.

78. The method according to any one of claims 73 to 77, comprising administering the composition before a surgical procedure.

79. The method according to claim 78, wherein the surgical procedure is a cancer surgery.

80. The method according to claim 79, wherein the surgical procedure is a prostate cancer surgery.

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