Fluorescein-labeled nerve-targeting peptide formulations and related methods

Abstract

Disclosed herein are pharmaceutical compositions comprising (a) a fluorescein labeled nerve targeting peptide of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, isomer, tautomer, racemate or isotope thereof: (I) (b) an additive comprising a pH regulator, a buffer, a filler, a tonicity regulator, a preservative, or any combination thereof; and (c) an aqueous vehicle wherein the pH of the composition is from about 7 to about 9 and the concentration of the peptide in the aqueous composition is from about 25 mg / mL to about 200 mg / mL based on the total volume of the aqueous composition. Also disclosed are methods for making and using the pharmaceutical compositions described herein.

Description

Technical Field

[0001] This article discloses fluorescently labeled neurotargeting peptide formulations and related methods, including their use in intraoperative neuroimaging and other targeted surgical and medical applications. Background Technology

[0002] Preserving human neurons and nerves is a critical goal of surgery; for example, accidental nerve transection can lead to serious pathologies such as chronic pain and loss of function. Nerves are typically identified by their elongated, whitish appearance and relationship to nearby structures, or, if they are motor nerves, by electrophysiological studies (sensory and autonomic nerves cannot be monitored electrophysiologically). However, such nerve identification during surgery can be challenging, especially in more complex surgical settings. In complex surgical environments, such as those involving trauma, tumors, inflammation, previous surgery, previous radiation exposure, or infection, nerve structures may be obscured or distorted. Nerve visibility can be further limited in minimally invasive surgeries that rely on small incisions for limited access (e.g., endoscopy). Indeed, unintentional nerve injury is a major complication associated with many surgeries using open and minimally invasive techniques.

[0003] These observations highlight the need for reliable and efficient methods and tools for identifying neurons or nerves. Electromyography (EMG) provides a method for identifying motor nerves before direct exposure during surgery or for confirming neuronal or nerve identity in uncertain situations. However, a drawback of this technique is the lack of visual feedback to the surgeon. Even if a nerve is identified at a location by accidental or intentional stimulation, there is no visual guidance for the surgeon regarding how far the nerve is from the stimulation site or the direction of its travel away from the stimulation site. Furthermore, EMG only tracks motor nerves, not sensory or autonomic nerves. EMG may fail if neuronal or nerve conduction or neuromuscular transmission is temporarily blocked anywhere distal to the recording location. Such blockages can easily occur due to neuronal or nerve compression, trauma, local anesthetics, or neuromuscular blocking agents.

[0004] Other methods include neuronal or nerve labeling, which primarily relies on retrograde or anterograde tracing of individually identified axonal bundles using fluorescent dyes. However, methods of labeling neurons or nerves by locally applying fluorescent tracers can be limited. First, depending on the location where the dye has been injected, this technique can only label one neuron or nerve fiber bundle at a time. Second, this technique produces only limited labeling of the fluorescent dye along the axonal bundle because retrograde axonal tracers typically accumulate in the nerve cell body. Third, retrograde transport is relatively slow (on the order of millimeters per day), thus requiring long periods to label human neurons or nerves that are typically longer than one meter, such as in the case of sciatic neurons or nerves and their branches. Fourth, applying fluorescent dyes to targets innervated by nerves, such as through direct intramuscular injection to label motor neurons or nerves, often becomes chaotic, leaving variable amounts of tracer dye residue at the injection site. Since the anatomy of neurons or nerves depends on precise visualization of adjacent structures before they are located, surgical sites contaminated with fluorescent dye are desirable. Finally, direct injection of the fluorescent dye itself may damage the target organ or neurons 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 has been disclosed in WO2019 / 028281, the entire contents of which are incorporated herein by reference, providing optimized peptides for targeting human nerves and their uses in image-guided surgery, diagnostics, and therapeutic delivery. In particular, a fluorescently labeled neurotargeting peptide known as “Bevonescein (ALM-488)” (i.e., SEQ ID NO: 104 of WO2019 / 028281) (also referred to herein as the compound or peptide of formula (I)) has entered clinical trials (Phase 1 / 2 and Phase 3) to evaluate the safety, tolerability, and efficacy of this compound for intravenous (IV) administration to patients undergoing head and neck surgery. Figure 1 The structure of Bevonescein (ALM-488) is shown.

[0006] However, it has proven challenging to formulate Bevonescein (ALM-488) in a manner suitable for use in these clinical trials, and a suitable formulation for IV administration remains to be discovered. To this end, this disclosure has overcome these challenges and provides a formulation of Bevonescein (ALM-488) suitable for manufacture, storage, reconstitution, and IV infusion into patients. These and other aspects of the invention will become apparent from the following disclosure. Summary of the Invention

[0007] In one aspect, this disclosure provides 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:

[0008] (I)

[0009] (b) an additive comprising a pH adjuster, a buffer, a filler, a tension modifier, a preservative, or any combination thereof; and (c) an aqueous medium, wherein the pH of the composition is from about 7 to about 9, and the peptide is present in the aqueous medium based on the total volume of the aqueous composition.

[0010] The concentration in the compound is from about 25 mg / mL to about 200 mg / mL.

[0011] Other aspects relate to lyophilized pharmaceutical compositions and methods for forming lyophilized pharmaceutical compositions from aqueous pharmaceutical compositions disclosed herein.

[0012] Other aspects relate to reconstituted aqueous pharmaceutical compositions and methods for forming reconstituted aqueous pharmaceutical compositions from lyophilized pharmaceutical compositions disclosed herein.

[0013] Other aspects relate to methods for identifying said neurons or nerves by bringing them into contact with the compositions of this disclosure.

[0014] Other aspects relate to methods of delivering at least one drug to said neuron or nerve by contacting the neuron or nerve with the composition of this disclosure. Attached Figure Description

[0015] This patent or application contains at least one color drawing. A copy of the color drawing disclosed in this patent or patent application will be provided by the Patent Office upon request and payment of the necessary fees.

[0016] Figure 1 It is the structure of Bevonescein (ALM-488); Figure 2 This is the HPLC chromatogram of a 0.25 mg / mL ALM-488HCl saline solution; Figure 3 yes Figure 2 A magnified view of the main peak in the HPLC chromatogram; Figure 4 This is a flowchart of the synthetic route used to produce ALM-488; Figure 5 These are the thermogravimetric analysis (TGA) curves of the chloride salt of ALM-488; Figure 6This is a graph showing the linear relationship between the peak area and amount of ALM-488 in the HPLC method shown in Table 3; Figure 7 It is a pH titration curve showing how pH, as a function of the addition of NaOH, and the solubility of the HCl salt of ALM-488 are affected by pH; Figure 8 It is a superposition of pH titration curves showing how pH, as a function of NaOH addition, and the solubility of ALM-488's HCl and acetate salts are affected by pH; Figure 9 It is a superposition of amplified HPLC chromatograms of ALM-488 solutions at different pH values ​​after storage at 40℃ for 24 hours; Figure 10 It is a superposition of the initial analysis of ALM-488 aqueous solution (50 mg / mL) and the magnified chromatograms of the analysis after 7 days at different storage temperatures; Figure 11 It is a pH titration curve showing how pH, as a function of the addition of tromethamine (Tris), and the solubility of the HCl salt of ALM-488 are affected by pH. Figure 12 It is a pH titration curve showing how pH, as a function of the addition of meglumine (N-methylglucosamine), and the solubility of the HCl salt of ALM-488 are affected by pH; Figure 13 It is a pH titration curve showing how pH, as a function of L-arginine addition, and the solubility of the HCl salt of ALM-488 are affected by pH. Figure 14 It is a superposition of the enlarged HPLC chromatograms of sample 8-4 of Example 8 after initial preparation and storage at 40°C for 7 days and 40°C for 14 days; Figure 15 It is a superposition of amplified HPLC chromatograms of samples 8-1 to 8-7 of Example 8 after being stored at 40°C for 14 days; Figure 16 It is a superposition of the DSC heating curves of ALM-488 formulation samples 8-1, 8-3, 8-4 and 8-7 containing arginine; Figure 17 It is a superposition of the DSC heating curves of ALM-488 formulation sample 8-2 containing meglumine; Figure 18 The freeze-drying process diagram for sample 10-1 is shown; Figure 19 The freeze-drying process diagram for sample 10-2 is shown; Figure 20 The freeze-drying process diagram for sample 10-3 is shown; Figure 21Side views of the freeze-dried cakes of samples 10-1 and 10-2 are shown; Figure 22 Showing a top view of the freeze-dried cake after sample 10-2 was removed; Figure 23 The PXRD diffraction pattern of freeze-dried sample 10-1 is shown. Figure 24 The PXRD diffraction pattern of freeze-dried sample 10⁻² is shown. Figure 25 The PXRD diffraction pattern of the lyophilized sample 10⁻³ is shown. Figure 26 The freeze-drying process diagram for sample 11-1 is shown; Figure 27 The superposition of HPLC chromatograms of ALM-488 / arginine-trehalose lyophilized product after storage at 40℃ and 75% relative humidity for 1 month, 2 months and 3 months; Figure 28 It is a superposition of amplified HPLC chromatograms of ALM-488 / arginine-trehalose lyophilized product after storage at 40℃ and 75% relative humidity for 1 month, 2 months and 3 months; Figure 29 The PXRD diffraction pattern of freeze-dried sample 10⁻³ after storage at ℃ and 75% relative humidity for three months is shown. Detailed Implementation

[0017] This disclosure relates to the neurotargeting peptide Bevonescein (ALM-488) (see [link]). Figure 1 The liquid and solid compositions of this disclosure are suitable for administration to a subject (e.g., intravenous administration) even after prolonged storage at elevated temperatures and / or humidity. Although the formulation of ALM-488 has proven challenging, the compositions and related methods of this disclosure enable this neurotargeting peptide to be safely stored for extended periods under less-than-ideal conditions without significant degradation, and then administered intravenously at predictable and repeatable doses without the need for further purification or optimization. Examples 8 through 13 of this disclosure illustrate these advantages.

[0018] Terms and Definitions

[0019] Before elaborating on this disclosure in more detail, providing definitions for certain terms used herein may aid in understanding. Other definitions are set forth throughout this disclosure.

[0020] The term "consistent essentially of..." limits the scope of the claim to the specified materials or steps, and those that do not materially affect the essential and novel features of the claimed invention. It should be understood that the term "an" or "a" as used herein refers to "one or more" of the listed components.

[0021] The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination of alternatives.

[0022] As used herein, the terms “comprising / including” and “having” are used synonymously, and these terms and variations thereof are intended to be interpreted as non-limiting. The term “comprising / including” means the presence of a feature, integer, step, or component mentioned in the claims, but it does not exclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.

[0023] In this specification, unless otherwise stated, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the range, and, where appropriate, fractions thereof (e.g., one-tenth and one-hundredth of an integer). Furthermore, unless otherwise stated, any numerical ranges relating to any physical characteristic, such as polymer subunits, size, or thickness, as described herein shall be understood to include any integer within the range.

[0024] As used herein, ranges and quantities can be expressed as “about” a specific value or range. “About” also includes an exact quantity. Thus, for example, a pH value of “about 7” means about 7 pH, and also refers to pH 7. In some embodiments, “about” means within 5% of that value. Therefore, a pH of “about 7” means a pH between 6.65 and 7.35. In some embodiments, “about” means within 4% of that value. In some embodiments, “about” means within 3% of that value. In some embodiments, “about” means within 2% of that value. In some embodiments, “about” means within 1% of that value. Generally, the term “about” includes the amount expected within experimental error.

[0025] As used in this article, the term “ambient temperature” refers to room temperature, which is approximately 20°C to approximately 25°C (approximately 68°F to approximately 77°F).

[0026] As used herein, the term "aqueous medium" refers to an aqueous liquid suitable for intravenous injection into a subject.

[0027] As used herein, the term "pH adjuster" refers to an alkaline, acidic, or amphoteric organic compound suitable for adjusting the pH of aqueous solutions.

[0028] As used herein, the term "buffer" refers to an alkaline, acidic, or amphoteric organic compound that, when contained in an aqueous solution, resists changes in the pH of that aqueous solution.

[0029] As used herein, the term "filler" refers to a reagent that can be added to a pharmaceutical product to provide that pharmaceutical product with structure, volume and / or mass, thereby facilitating its more accurate measurement and handling.

[0030] As used herein, the term "tension modifier" refers to a compound that alters the osmolality of a formulation, for example, to make it isotonic.

[0031] As used herein, the term "preservative" refers to a substance that prevents or inhibits the growth of microorganisms and prolongs the shelf life of aqueous preparations.

[0032] As used herein, the term "well-formed freeze-dried cake" refers to a freeze-dried composition (i.e., a freeze-dried product) that has a uniform foam plug appearance with minimal shrinkage and cracking.

[0033] As used herein, the term "homogeneous" refers to a liquid solution having only a single phase (i.e., no solid phase and no liquid separation). In some embodiments, the term "homogeneous" refers to an aqueous composition containing less than 5% by weight of a solid neurotargeting peptide conjugate (i.e., Bevonescein (ALM-488)), based on the aqueous composition passing through a 0.22-micron Millipak Durapore. ® Filtration by the capsule filter unit.

[0034] As used herein, the terms “freeze-drying,” “freeze-dried,” and “lyophilized” refer to a method by which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment.

[0035] As used in this article, the term "lyophilized product" refers to a freeze-dried solid.

[0036] As used herein, the term "reconstituted composition" refers to an aqueous composition formed by dissolving a lyophilized material in an aqueous medium.

[0037] As used herein, the term "salt" refers to the acid addition salt or base addition salt of the compounds described herein. "Salt" specifically includes "pharmaceutically acceptable salts".

[0038] As used herein, the term "pharmaceutically acceptable" means a material that does not eliminate the biological activity or properties of the reagents described herein and is relatively non-toxic (i.e., the toxicity of the material significantly outweighs its benefits). In some cases, pharmaceutically acceptable materials may be administered to individuals without causing significant undesirable biological effects or obvious interactions with any component of a composition containing the material in a harmful manner.

[0039] As used herein, "pharmaceuticalally acceptable excipient" or "pharmaceuticalally acceptable carrier" refers to a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, and which is non-toxic and non-inflammatory to patients. Each excipient must be "acceptable" in terms of compatibility with other components of the formulation and harmlessness to patients. Excipients may include, for example, anti-adhesives, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents), film-forming agents or coatings, flavorings, fragrances, flow enhancers (flow improvers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, or hydrates. Examples of pharmaceutical excipients may be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water or aqueous solutions of saline and aqueous solutions of glucose and glycerol are preferred as excipients, especially for injectable solutions. Suitable pharmaceutical excipients are described in EWMartin's "Remington's Pharmaceutical Sciences".

[0040] As used in this article, "central nervous system" or "CNS" refers to the brain and spinal cord, as well as the retina.

[0041] As used in this article, the "peripheral nervous system" or "PNS" refers to the 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 all voluntary muscle systems and voluntary reflex processes in the body, and is composed of afferent or sensory nerves and efferent or motor nerves. Afferent or sensory nerves transmit sensations from the body to the central nervous system, while efferent or motor nerves send instructions from the CNS to the body to stimulate muscle contraction. The autonomic nervous system innervates smooth muscles and glands, thereby affecting the function of internal organs. The autonomic nervous system operates largely unconsciously and regulates bodily functions such as heart rate, digestion, respiratory rate, pupillary response, urination, and sexual arousal.

[0042] As used in this article, a "neuron" is an electrically excitable cell that processes and transmits information through the conduction of electrical and chemical signals. A typical neuron has a cell body (usually called the cell body), dendrites, and axons.

[0043] As used in this article, "nerve" refers to a closed, cable-like bundle of nerve axons. Each nerve is a cord-like structure containing many axons. Each axon within a nerve is an extension of a single neuron. Within a nerve, each axon is surrounded by a layer of connective tissue called the endoneurium. Axons are bundled together to form groups called nerve bundles, and each nerve bundle is wrapped in a layer of connective tissue called the perineurium. Each nerve is covered laterally by a dense sheath of connective tissue (ependymal).

[0044] As used herein, the term "coupler" refers to a peptide attached (optionally, via a linker) to at least one cargo molecule.

[0045] The terms “individual,” “patient,” or “object” are used interchangeably. As used herein, they refer to any mammal (i.e., any species of any order, family, and genus within the taxonomic kingdom Animalia: phylum Chordata: Vertebrates: class Mammalia). In some embodiments, mammals are cattle, horses, sheep, pigs, cats, dogs, goats, mice, rats, rabbits, guinea pigs, non-human primates, or humans.

[0046] As used herein, the terms “administration,” “dosing,” “giving,” etc., refer to methods that can be used to deliver a pharmaceutical agent or composition to a desired biological site of action. These methods include, but are not limited to, local, oral, rectal, vaginal, nasal, inhalation, and parenteral administration (e.g., intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, intravitreal, infusion, or local). Administration techniques optionally used with the pharmaceutical agents and methods described herein include, for example, those discussed in Goodman and Gilman, *The Pharmacological Basis of Therapeutics*, current ed.; Pergamon; and Remington's, *Pharmaceutical Sciences* (current edition), Mack Publishing Co., Easton, Pa. The administration of a pharmaceutical agent can be local or systemic. In some embodiments, administration is via systemic intravenous injection into a human patient.

[0047] As used herein, the term "surgery" refers to any method that can be used to manipulate, alter, or induce effects through physical intervention. These methods include, but are not limited to, open surgery, endoscopic surgery, laparoscopic surgery, minimally invasive surgery, robotic surgery, any procedure that may affect any neuron or nerve, such as placement of a retractor during spinal surgery, microsurgery, cardiac neuron or nerve ablation, epidural injection, intrathecal injection, neuronal or nerve block, implantation of devices such as neuronal or neurostimulators, and implantation of pumps. In some embodiments, the subject of the surgery is a human subject or a human patient.

[0048] Pharmaceutical Compositions and Preparation Methods

[0049] In some embodiments, this document discloses pharmaceutical compositions comprising a neurotargeting peptide conjugate “Bevonescein (ALM-488)” (i.e., SEQ ID NO: 104 of WO2019 / 028281) as shown in formula (I), and pharmaceutically acceptable salts, solvates, hydrates, isomers, tautomers, racemates, or isotopes thereof:

[0050] (I)

[0051] In some embodiments, the purity of ALM-488 used to prepare the aqueous and solid (e.g., lyophilized) compositions described below 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, or at least 99.5% by mass, based on the total mass of ALM-488.

[0052] In some embodiments, the ALM-488 peptide used to prepare the aqueous and solid (e.g., lyophilized) compositions described below 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 some embodiments, the pharmaceutically acceptable salts are acetates, aspartates, benzoates, benzenesulfonates, bromides, bicarbonates, carbonates, chlorides, bisulfates, sulfates, camphorsulfonates, chlorides, theophylline salts, citrates, ethanedisulfonates, fumarates, gluconate, gluconate, glucuronide, hippurate, iodides, hydroxyethylsulfonates, lactates, lacturonates, dodecyl sulfates, malates, maleates, malonates, mandelates, methanesulfonates, methyl sulfates, naphthates, naphthalenesulfonates, nicotinates, nitrates, octadecanoates, oleates, oxalates, palmitates, dihydroxynaphthalates, phosphates, hydrogen phosphates, dihydrogen phosphates, polygalacturonates, propionates, stearates, succinates, sulfates, sulfosalicylates, tartrates, toluenesulfonates, trifluoroacetates, etc., or any combination thereof.

[0053] The pharmaceutical compositions described herein are formulated using one or more physiologically acceptable carriers (including excipients and adjuvants) that facilitate the processing of the compound of formula (I) into a pharmaceutically usable preparation. A suitable formulation depends on the chosen route of administration. Overviews 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 entire contents of each of which are incorporated herein by reference.

[0054] The compositions disclosed herein (i.e., the aqueous pharmaceutical compositions, lyophilized pharmaceutical compositions, and reconstituted aqueous pharmaceutical compositions described below) may contain at least one pharmaceutically acceptable excipient (also referred to herein as an “additive”). Such additives may include, for example, pH adjusters, buffers, fillers, tension modifiers, 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 entire contents of which are incorporated herein by reference.

[0055] pH adjusters that may be included in the compositions disclosed herein include, for example, alanine, arginine, cellulose, cellobiose, cyclodextrin, dextrose, polydextrose, erythritol, fructose, galactose, glucose, glycerol, glycine, histidine, inositol, lactose, maltose, mannitol, mannose, N-methylglucosamine (glucosamine), raffinose, ribose, sorbitol, sucrose, trehalose, tromethamine, xylitol, xylose, etc., or any combination thereof.

[0056] Buffers that may be included in the compositions disclosed herein include, for example, calcium acetate, sodium acetate, potassium hydrogen tartrate, 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.

[0057] Buffers that may be included in the compositions disclosed herein 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, tartrate buffer, DIPSO buffer, HEPPSO buffer, POPSO buffer, PIPES buffer, PBS buffer, MOPS buffer, acetate buffer, phosphate buffer, dimethylarsine buffer, glycine buffer, and sulfate buffer. Salt buffer, imidazole buffer, guanidine hydrochloride buffer, phosphate-citrate buffer, borate buffer, malonate buffer, 3-methylpyridine buffer, 2-methylpyridine buffer, 4-methylpyridine buffer, 3,5-dimethylpyridine buffer, 3,4-dimethylpyridine buffer, 2,4-dimethylpyridine buffer, Aces buffer, diethylmalonate buffer, N-methylimidazolium buffer, 1,2-dimethylimidazolium buffer, TAPS buffer, bis-Tris buffer, L-arginine buffer, lactate buffer, glycolate buffer, etc., or any combination thereof.

[0058] Fillers that may be included in the compositions disclosed herein include, for example, alanine, arginine, calcium chloride, calcium sulfate, cellulose, dextran 40, dextrin (e.g., maltodextrin), dextran, polydextrin, dicalcium phosphate dihydrate, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol (PEG), β-glucan, glycerol, diglycerol, polyglycerol, glycine, histidine, aqueous colloids (e.g., gum arabic, pectin, guar gum, alginate, carrageenan, xanthan gum, cellulose gum, etc.), inulin, kaolin, magnesium chloride, magnesium aluminum silicate, and glucosamine. Amines (N-methylglucosamine), 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, povidone (PVP), poloxamer, silicon dioxide, sodium chloride, starch, titanium dioxide, tromethamine, or any combination thereof.

[0059] Tension modifiers that may be included in the compositions disclosed herein include, for example, alanine, arginine, calcium chloride, calcium sulfate, cellulose, dextran 40, dextrin (e.g., maltodextrin), dextran, polydextrin, dicalcium phosphate dihydrate, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol (PEG), β-glucan, glycerol, diglycerol, polyglycerol, glycine, histidine, hydrocolloids (e.g., gum arabic, pectin, guar gum, alginate, carrageenan, xanthan gum, cellulose gum, etc.), inulin, kaolin, magnesium chloride, magnesium aluminum silicate, and glucosamine. Methylamine (N-methylglucosamine), 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, povidone (PVP), poloxamer, silicon dioxide, sodium chloride, starch, titanium dioxide, tromethamine, or any combination thereof.

[0060] Preservatives that may be included in the compositions disclosed herein include, for example, benzoic acid, sodium benzoate, benzyl alcohol, benzalkonium chloride, Bronidol, Bronopol, butylparaben, chlorobutanol, methyl cresol, chlorocresol, methylparaben, phenol, phenoxyethanol, phenethyl alcohol, propylene glycol, propylparaben, sorbic acid, thimerosal, etc., or any combination thereof.

[0061] In some embodiments, the pharmaceutical compositions of this disclosure (i.e., the aqueous pharmaceutical compositions, lyophilized pharmaceutical compositions, and reconstituted aqueous pharmaceutical compositions described below) may contain other pharmaceuticals or agents, carriers, adjuvants such as preservatives, stabilizers, wetting agents or emulsifiers, solubilizers, salts and / or buffers for adjusting osmotic pressure. Furthermore, the pharmaceutical compositions may also contain other substances of therapeutic value.

[0062] Aqueous pharmaceutical compositions

[0063] 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, as described above, a pH adjuster, buffer, filler, tonicant, preservative, or any combination thereof; and (c) an aqueous mediator. In some embodiments, the aqueous composition has a pH of about 5 to about 12, and the concentration of the neurotargeting peptide in the aqueous composition is about 10 mg / mL to about 500 mg / mL based on the total volume of the aqueous composition.

[0064] In some embodiments, the peptide of formula (I) (i.e., Bevonescein (ALM-488)) is in the form of a pharmaceutically acceptable salt as described above. For example, in some embodiments, the aqueous pharmaceutical composition comprises an HCl salt of a neurotargeting peptide. Based on the total mass of the peptide in the aqueous composition, the chloride content of the HCl salt may be 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).

[0065] In some embodiments, the molar ratio of the peptide to a pharmaceutically acceptable salt in the aqueous composition is 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.

[0066] In some embodiments, the additives in the aqueous composition include pH adjusters, buffers, buffer solutions, fillers, tension modifiers, preservatives, at least one solvent, and combinations thereof. Such additives may include, for example, pH adjusters, buffers, buffer solutions, fillers, tension modifiers, and preservatives as described above.

[0067] In some embodiments, the additives in the aqueous composition include alanine, arginine, cellulose, cellobiose, cyclodextrin, dextrose, erythritol, fructose, galactose, glucose, glycerol, glycine, histidine, inositol, lactose, maltose, mannitol, mannose, meglumine, raffinose, ribose, sorbitol, sucrose, trehalose, tromethamine, xylitol, xylose, or any combination thereof. In some embodiments, the additives include arginine, glycine, mannitol, meglumine, trehalose, tromethamine, or any combination thereof.

[0068] In some embodiments, the additives in the aqueous composition include a first additive comprising arginine, meglumine, glycine, or any combination thereof; and an optional second additive comprising mannitol, trehalose, or both. In some embodiments, the mass ratio of the first additive to the second additive is about 10:1 to about 1:10, or about 10:1 to about 9:1, or about 9:1 to about 8:1, or about 8:1 to about 7:1, or about 7:1 to about 6:1, or about 6:1 to about 5:1, or about 5:1 to about 4:1, or about 4:1 to about 3:1, or about 3:1 to about 2:1, or about 2:1 to about 1:0, or about 1.8:1 to about 1.7:1, or about 1.7:1 to about 1.6:1, or about 1.6:1 to about 1.5:1, or about 1.5:1 to about 1.4:1, or about 1.4:1 to about 1.3:1, or about 1.3:1 to about 1.2:1, or about 1. 2:1 to about 1.1:1, or about 1:1.1 to about 1:1.2, or about 1:1 to about 1:2.5, or about 1:1.2 to about 1:1.3, or about 1:1.3 to about 1:1.4, or about 1:1.4 to about 1:1.5, or about 1:1.5 to about 1:1.6, or about 1:1.6 to about 1:1.8, or about 1:1.8 to about 1:1.9, or about 1:1.9 to about 1:2, or about 1:2 to about 1:3, or about 1:3 to about 1:4, or about 1:4 to about 1:5, or about 1:5 to about 1:6, or about 1:6 to about 1:7, or about 1:7 to about 1:8, or about 1:8 to about 1:9, or about 1:9 to about 1:10.

[0069] In some embodiments, the additives in the aqueous composition include arginine, arginine-mannitol, arginine-trehalose, arginine-glucamine, meglumine, meglumine-glycine, or meglumine-mannitol. In some embodiments, the additive includes arginine-trehalose. In some embodiments, the mass ratio of arginine to trehalose is about 10:1 to about 1:10, or about 10:1 to about 9:1, or about 9:1 to about 8:1, or about 8:1 to about 7:1, or about 7:1 to about 6:1, or about 6:1 to about 5:1, or about 5:1 to about 4:1, or about 4:1 to about 3:1, or about 3:1 to about 2:1, or about 2:1 to about 1:0, or about 1.8:1 to about 1.7:1, or about 1.7:1 to about 1.6:1, or about 1.6:1 to about 1.5:1, or about 1.5:1 to about 1.4:1, or about 1.4:1 to about 1.3:1, or about 1.3:1 to about 1.2:1, or about 1.2: 1 to about 1.1:1, or about 1:1.1 to about 1:1.2, or about 1:1 to about 1:2.5, or about 1:1.2 to about 1:1.3, or about 1:1.3 to about 1:1.4, or about 1:1.4 to about 1:1.5, or about 1:1.5 to about 1:1.6, or about 1:1.6 to about 1:1.8, or about 1:1.8 to about 1:1.9, or about 1:1.9 to about 1:2, or about 1:2 to about 1:3, or about 1:3 to about 1:4, or about 1:4 to about 1:5, or about 1:5 to about 1:6, or about 1:6 to about 1:7, or about 1:7 to about 1:8, or about 1:8 to about 1:9, or about 1:9 to about 1:10.

[0070] In some embodiments, the mass ratio of peptide to additive in the aqueous composition is from about 5:1 to about 1:5. In some embodiments, the mass ratio of peptide to additive in the aqueous composition is from about 5:1 to about 4.5:1, or from about 4.5:1 to about 4.0:1, or from about 4.0:1 to about 3.5:1, or from about 3.5:1 to about 3.0:1, or from about 3.0:1 to about 2.5:1, or from about 2.5:1 to about 2.0:1, or from about 2.0:1 to about 1.5:1, or from about 1.5:1 to about 1.0:1, or from about 1... The ratio of peptide to additive is from about 0.0:1 to about 0.5:1, or about 1:0.5 to about 1:1.0, or about 1:1.0 to about 1:1.5, or about 1:1.5 to about 1:2.0, or about 1:2.0 to about 1:2.5, or about 1:2.5 to about 1:3.0, or about 1:3.0 to about 1:3.5, or about 1:3.5 to about 1:4.0, or about 1:4.0 to about 1:4.5, or about 1:4.5 to about 1:4.0. In other embodiments, the mass ratio of peptide to additive is from about 1.0 to about 1.5.

[0071] In some embodiments, the aqueous medium in the aqueous composition includes sterile water, saline solution, aqueous buffer solution, or any combination thereof. In some embodiments, the aqueous medium includes sterile water, physiological saline, half-concentration physiological saline, or any combination thereof.

[0072] In some embodiments, the pH of the 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 is approximately 8.0, or approximately 8.0 to approximately 8.1, or approximately 8.1 to approximately 8.2, or approximately 8.2 to approximately 8.3, or approximately 8.3 to approximately 8.4, or approximately 8.4 to approximately 8.5, or approximately 8.5 to approximately 8.6, or approximately 8.6 to approximately 8.7, or approximately 8.7 to approximately 8.8, or approximately 8.8 to approximately 8.9, or approximately 8.9 to approximately 9.0, or approximately 9 to approximately 10, or approximately 10 to approximately 11, or approximately 11 to approximately 12. In some embodiments, the pH of the aqueous composition is approximately 7.2 to approximately 7.8.

[0073] In some embodiments, based on the total volume of the aqueous composition, the concentration of the peptide (i.e., Bevonescein (ALM-488)) in the 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 140 mg / mL, or about 140 mg / mL to about 150 mg / mL, or about 150 mg / mL... The concentration of peptides in the aqueous composition is from about 160 mg / mL to about 170 mg / mL, or about 170 mg / mL to about 180 mg / mL, or about 180 mg / mL to about 190 mg / mL, or about 190 mg / mL to about 200 mg / mL, or about 200 mg / mL to about 250 mg / mL, or about 250 mg / mL to about 300 mg / mL, or about 300 mg / mL to about 350 mg / mL, or about 350 mg / mL to about 400 mg / mL, or about 400 mg / mL to about 450 mg / mL. In some embodiments, the peptide concentration in the aqueous composition is from about 25 mg / mL to about 200 mg / mL based on the total volume of the aqueous composition. In other embodiments, the peptide concentration in the aqueous composition is from about 40 mg / mL to about 75 mg / mL based on the total volume of the aqueous composition.

[0074] In some embodiments, the density of the aqueous composition is from about 0.6 g / mL to about 1.3 g / mL. In some embodiments, the density of the aqueous composition is from about 0.6 g / mL to about 0.7 g / mL, or from about 0.7 g / mL to about 0.8 g / mL, or from about 0.8 g / mL to about 0.85 g / mL, or from about 0.85 g / mL to about 0.90 g / mL, or from about 0.90 g / mL to about 0.95 g / mL, or from about 0.95 g / mL to about 1.0 g / mL, or from about 1.0 g / mL to about 1.05 g / mL, or from about 1.05 g / mL to about 1.10 g / mL, or from about 1.10 g / mL to about 1.15 g / mL, or from about 1.15 g / mL to about 1.20 g / mL, or from about 1.20 g / mL to about 1.25 g / mL. In other embodiments, the density of the aqueous composition is from about 1.0 g / mL to about 1.08 g / mL.

[0075] In some embodiments, the weight permeation concentration of the aqueous composition is from about 150 mOsm / kg to about 400 mOsm / kg. In some embodiments, the weight permeation concentration of the aqueous composition is from about 150 mOsm / kg to about 175 mOsm / kg, or from about 175 mOsm / kg to about 200 mOsm / kg, or from about 200 mOsm / kg to about 225 mOsm / kg, or from about 225 mOsm / kg to about 250 mOsm / kg, or from about 250 mOsm / kg to about 275 mOsm / kg, or from about 275 mOsm / kg to about 300 mOsm / kg, or from about 300 mOsm / kg to about 325 mOsm / kg, or from about 325 mOsm / kg to about 350 mOsm / kg. In other embodiments, the weight permeation concentration of the aqueous composition is from about 280 mOsm / kg to about 300 mOsm / kg.

[0076] In some embodiments, the volume of the aqueous composition is from about 0.5 mL to about 5.0 mL. In some embodiments, the volume of the aqueous composition is from about 0.5 mL to about 1.0 mL, or from about 1.0 mL to about 1.5 mL, or from about 1.5 mL to about 2.0 mL, or from about 2.0 mL to about 2.5 mL, or from about 2.5 mL to about 3.0 mL, or from about 3.0 mL to about 3.5 mL, or from about 3.5 mL to about 4.0 mL, or from about 4.0 mL to about 4.5 mL. In other embodiments, the volume of the aqueous composition is from about 1.8 mL to about 2.2 mL.

[0077] In some embodiments, the aqueous composition is filtered. In other embodiments, the aqueous composition is unfiltered. In some embodiments, the aqueous composition is homogeneous.

[0078] In some embodiments, the aqueous composition comprises at least 95% by mass of a peptide (i.e., Bevonescein (ALM-488)), as measured relative to the total mass of the aqueous composition after storage at 40°C for 14 days. In some embodiments, the aqueous composition comprises 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 a peptide, as measured relative to the total mass of the aqueous composition after storage at 40°C for 14 days.

[0079] Lyophilized pharmaceutical compositions and preparation methods

[0080] Another embodiment relates to a lyophilized pharmaceutical composition comprising (a) a fluorescein-labeled neurotargeting 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, filler, tension modifier, preservative, or any combination thereof, wherein the water content of the lyophilized composition is from about 0.2% (w / w) to about 2.0% (w / w) relative to the total mass of the lyophilized composition.

[0081] In some embodiments, when combined with an aqueous medium at ambient temperature, the lyophilized composition forms a homogeneous aqueous composition having a peptide concentration of about 50 mg / mL to about 500 mg / mL (i.e., Bevonescein (ALM-488)). In some embodiments, the peptide concentration of the homogeneous aqueous composition formed from the lyophilized 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 to about 250 mg / mL, or about 250 mg / mL to about 275 mg / mL, or about 275 mg / mL to about 300 mg / mL, or about 300 mg / mL to about 325 mg / mL, or about 325 mg / mL to about 350 mg / mL, or about 350 mg / mL to about 375 mg / mL, or about 375 mg / mL to about 400 mg / mL, or about 400 mg / mL... The concentration is approximately 425 mg / mL to about 425 mg / mL, or approximately 450 mg / mL to about 425 mg / mL. In other embodiments, when combined with an aqueous medium at ambient temperature, the lyophilized composition forms a homogeneous aqueous composition having a peptide concentration of 200 mg / mL or lower.

[0082] In some embodiments, the freeze-dried composition is in the form of a well-formed freeze-dried cake.

[0083] In some embodiments, after being stored at 40°C and 75% relative humidity for 3 months, the lyophilized composition contains at least 95% by mass of a peptide (i.e., Bevonescein (ALM-488)). In some embodiments, after being stored at 40°C and 75% relative humidity for 3 months, the lyophilized composition contains at least 95% by mass, or about 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 about at least 99.0% by mass, or at least 99.5% by mass of a peptide (i.e., Bevonescein (ALM-488)).

[0084] In some embodiments, the peptide of formula (I) (i.e., Bevonescein (ALM-488)) is in the form of a pharmaceutically acceptable salt as described above. For example, in some embodiments, the lyophilized composition comprises an HCl salt of the peptide. Based on the total mass of the peptide in the lyophilized composition, the chloride content of the HCl salt may be 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).

[0085] In some embodiments, the molar ratio of the peptide to a pharmaceutically acceptable salt in the lyophilized composition is 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.

[0086] In some embodiments, the additives included in the lyophilized composition include pH adjusters, buffers, buffer solutions, fillers, tension modifiers, preservatives, and combinations thereof. Such additives may include, for example, pH adjusters, buffers, buffer solutions, fillers, tension modifiers, and preservatives as described above.

[0087] In some embodiments, the additives included in the freeze-dried composition include alanine, arginine, cellulose, cellobiose, cyclodextrin, dextrose, erythritol, fructose, galactose, glucose, glycerol, glycine, histidine, inositol, lactose, maltose, mannitol, mannose, meglumine, raffinose, ribose, sorbitol, sucrose, trehalose, tromethamine, xylitol, xylose, or any combination thereof. In some embodiments, the additives include arginine, glycine, mannitol, meglumine, trehalose, tromethamine, or any combination thereof.

[0088] In some embodiments, the additives included in the freeze-dried composition include a first additive comprising arginine, meglumine, glycine, or any combination thereof; and an optional second additive comprising mannitol, trehalose, or both. In some embodiments, the mass ratio of the first additive to the second additive is about 10:1 to about 1:10, or about 10:1 to about 9:1, or about 9:1 to about 8:1, or about 8:1 to about 7:1, or about 7:1 to about 6:1, or about 6:1 to about 5:1, or about 5:1 to about 4:1, or about 4:1 to about 3:1, or about 3:1 to about 2:1, or about 2:1 to about 1:0, or about 1.8:1 to about 1.7:1, or about 1.7:1 to about 1.6:1, or about 1.6:1 to about 1.5:1, or about 1.5:1 to about 1.4:1, or about 1.4:1 to about 1.3:1, or about 1.3:1 to about 1.2:1, or about 1. 2:1 to about 1.1:1, or about 1:1.1 to about 1:1.2, or about 1:1 to about 1:2.5, or about 1:1.2 to about 1:1.3, or about 1:1.3 to about 1:1.4, or about 1:1.4 to about 1:1.5, or about 1:1.5 to about 1:1.6, or about 1:1.6 to about 1:1.8, or about 1:1.8 to about 1:1.9, or about 1:1.9 to about 1:2, or about 1:2 to about 1:3, or about 1:3 to about 1:4, or about 1:4 to about 1:5, or about 1:5 to about 1:6, or about 1:6 to about 1:7, or about 1:7 to about 1:8, or about 1:8 to about 1:9, or about 1:9 to about 1:10.

[0089] In some embodiments, the additives included in the freeze-dried composition include arginine, arginine-mannitol, arginine-trehalose, arginine-glucamine, glucamine, glucamine-glycine, or glucamine-mannitol. In some embodiments, the additive includes arginine-trehalose. In some embodiments, the mass ratio of arginine to trehalose is about 10:1 to about 1:10, or about 10:1 to about 9:1, or about 9:1 to about 8:1, or about 8:1 to about 7:1, or about 7:1 to about 6:1, or about 6:1 to about 5:1, or about 5:1 to about 4:1, or about 4:1 to about 3:1, or about 3:1 to about 2:1, or about 2:1 to about 1:0, or about 1.8:1 to about 1.7:1, or about 1.7:1 to about 1.6:1, or about 1.6:1 to about 1.5:1, or about 1.5:1 to about 1.4:1, or about 1.4:1 to about 1.3:1, or about 1.3:1 to about 1.2:1, or about 1.2: 1 to about 1.1:1, or about 1:1.1 to about 1:1.2, or about 1:1 to about 1:2.5, or about 1:1.2 to about 1:1.3, or about 1:1.3 to about 1:1.4, or about 1:1.4 to about 1:1.5, or about 1:1.5 to about 1:1.6, or about 1:1.6 to about 1:1.8, or about 1:1.8 to about 1:1.9, or about 1:1.9 to about 1:2, or about 1:2 to about 1:3, or about 1:3 to about 1:4, or about 1:4 to about 1:5, or about 1:5 to about 1:6, or about 1:6 to about 1:7, or about 1:7 to about 1:8, or about 1:8 to about 1:9, or about 1:9 to about 1:10.

[0090] In some embodiments, the mass ratio of peptides to additives in the lyophilized composition is from about 5:1 to about 1:5. In some embodiments, the mass ratio of peptides to additives in the lyophilized composition is from about 5:1 to about 4.5:1, or from about 4.5:1 to about 4.0:1, or from about 4.0:1 to about 3.5:1, or from about 3.5:1 to about 3.0:1, or from about 3.0:1 to about 2.5:1, or from about 2.5:1 to about 2.0:1, or from about 2.0:1 to about 1.5:1, or from about 1.5:1 to about 1.0:1, or from about 1... The ratio of the peptide to the additive is from about 0.0:1 to about 0.5:1, or about 1:0.5 to about 1:1.0, or about 1:1.0 to about 1:1.5, or about 1:1.5 to about 1:2.0, or about 1:2.0 to about 1:2.5, or about 1:2.5 to about 1:3.0, or about 1:3.0 to about 1:3.5, or about 1:3.5 to about 1:4.0, or about 1:4.0 to about 1:4.5, or about 1:4.5 to about 1:4.0. In other embodiments, the mass ratio of the peptide to the additive is from about 1.0 to about 1.5.

[0091] Another embodiment relates to a method of forming a lyophilized pharmaceutical composition by lyophilizing the aqueous composition disclosed herein. In some embodiments, lyophilization includes freezing the aqueous composition at a temperature of about -100°C to about -45°C to obtain a frozen solid composition. In some embodiments, the frozen solid composition is formed by freezing the aqueous composition at a temperature 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 other embodiments, lyophilization includes freezing the aqueous composition at a temperature of about -80°C to about -50°C to obtain a frozen solid composition.

[0092] In some embodiments, freeze drying further includes annealing the frozen solid composition at a temperature of about -40°C to about -20°C. In some embodiments, annealing occurs at a temperature 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.

[0093] In some embodiments, lyophilization further includes drying the frozen solid composition at a temperature of about -20°C to room temperature and a pressure of about 100 mTorr to about 300 mTorr to obtain a lyophilized composition. In some embodiments, the drying temperature is 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.

[0094] In some embodiments, drying includes: (i) pre-drying the frozen solid composition at a temperature of about -20°C to about 0°C and a preliminary pressure of about 100 mTorr to about 300 mTorr; and (ii) secondary drying the frozen solid composition at a temperature of about 0°C to room temperature and a secondary pressure of about 100 mTorr to about 300 mTorr.

[0095] Another embodiment involves a lyophilized pharmaceutical composition formed by the lyophilization method described herein.

[0096] Reconstituted aqueous drug compositions and preparation methods

[0097] Another embodiment relates to a reconstituted aqueous pharmaceutical composition comprising ALM-488 and its pharmaceutically acceptable salts, solvates, hydrates, isomers, tautomers, racemates, or isotopes, formed by combining the lyophilized composition described herein with an aqueous medium.

[0098] In some embodiments, the aqueous medium used to prepare the reconstituted aqueous composition includes sterile water, saline solution, aqueous buffer solution, or any combination thereof. In some embodiments, the aqueous medium includes sterile water, physiological saline, half-concentration physiological saline, or any combination thereof.

[0099] In some embodiments, the reconstituted aqueous composition is filtered. In other embodiments, the reconstituted aqueous composition is unfiltered. In some embodiments, the reconstituted composition is homogeneous.

[0100] In some embodiments, the peptide of formula (I) contained in the reconstituted aqueous composition (i.e., Bevonescein (ALM-488)) is in the form of a pharmaceutically acceptable salt as described above. For example, in some embodiments, the reconstituted aqueous pharmaceutical composition comprises an HCl salt of the neurotargeting peptide. Based on the total mass of the peptide in the reconstituted aqueous composition, the chloride content of the HCl salt may be 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).

[0101] In some embodiments, the molar ratio of the peptide to a pharmaceutically acceptable salt in the reconstituted aqueous composition is 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.

[0102] In some embodiments, the additives in the reconstituted aqueous composition include pH adjusters, buffers, buffer solutions, fillers, tension modifiers, preservatives, at least one solvent, and combinations thereof. Such additives may include, for example, pH adjusters, buffers, buffer solutions, fillers, tension modifiers, and preservatives as described above.

[0103] In some embodiments, the additives in the reconstituted aqueous composition include alanine, arginine, cellulose, cellobiose, cyclodextrin, dextrose, erythritol, fructose, galactose, glucose, glycerol, glycine, histidine, inositol, lactose, maltose, mannitol, mannose, meglumine, raffinose, ribose, sorbitol, sucrose, trehalose, tromethamine, xylitol, xylose, or any combination thereof. In some embodiments, the additives include arginine, glycine, mannitol, meglumine, trehalose, tromethamine, or any combination thereof.

[0104] In some embodiments, the additives in the reconstituted aqueous composition include a first additive comprising arginine, meglumine, glycine, or any combination thereof; and an optional second additive comprising mannitol, trehalose, or both. In some embodiments, the mass ratio of the first additive to the second additive is about 10:1 to about 1:10, or about 10:1 to about 9:1, or about 9:1 to about 8:1, or about 8:1 to about 7:1, or about 7:1 to about 6:1, or about 6:1 to about 5:1, or about 5:1 to about 4:1, or about 4:1 to about 3:1, or about 3:1 to about 2:1, or about 2:1 to about 1:0, or about 1.8:1 to about 1.7:1, or about 1.7:1 to about 1.6:1, or about 1.6:1 to about 1.5:1, or about 1.5:1 to about 1.4:1, or about 1.4:1 to about 1.3:1, or about 1.3:1 to about 1.2:1, or about 1. 2:1 to about 1.1:1, or about 1:1.1 to about 1:1.2, or about 1:1 to about 1:2.5, or about 1:1.2 to about 1:1.3, or about 1:1.3 to about 1:1.4, or about 1:1.4 to about 1:1.5, or about 1:1.5 to about 1:1.6, or about 1:1.6 to about 1:1.8, or about 1:1.8 to about 1:1.9, or about 1:1.9 to about 1:2, or about 1:2 to about 1:3, or about 1:3 to about 1:4, or about 1:4 to about 1:5, or about 1:5 to about 1:6, or about 1:6 to about 1:7, or about 1:7 to about 1:8, or about 1:8 to about 1:9, or about 1:9 to about 1:10.

[0105] In some embodiments, the additives in the reconstituted aqueous composition include arginine, arginine-mannitol, arginine-trehalose, arginine-glucamine, glucamine, glucamine-glycine, or glucamine-mannitol. In some embodiments, the additive includes arginine-trehalose. In some embodiments, the mass ratio of arginine to trehalose is about 10:1 to about 1:10, or about 10:1 to about 9:1, or about 9:1 to about 8:1, or about 8:1 to about 7:1, or about 7:1 to about 6:1, or about 6:1 to about 5:1, or about 5:1 to about 4:1, or about 4:1 to about 3:1, or about 3:1 to about 2:1, or about 2:1 to about 1:0, or about 1.8:1 to about 1.7:1, or about 1.7:1 to about 1.6:1, or about 1.6:1 to about 1.5:1, or about 1.5:1 to about 1.4:1, or about 1.4:1 to about 1.3:1, or about 1.3:1 to about 1.2:1, or about 1.2: 1 to about 1.1:1, or about 1:1.1 to about 1:1.2, or about 1:1 to about 1:2.5, or about 1:1.2 to about 1:1.3, or about 1:1.3 to about 1:1.4, or about 1:1.4 to about 1:1.5, or about 1:1.5 to about 1:1.6, or about 1:1.6 to about 1:1.8, or about 1:1.8 to about 1:1.9, or about 1:1.9 to about 1:2, or about 1:2 to about 1:3, or about 1:3 to about 1:4, or about 1:4 to about 1:5, or about 1:5 to about 1:6, or about 1:6 to about 1:7, or about 1:7 to about 1:8, or about 1:8 to about 1:9, or about 1:9 to about 1:10.

[0106] In some embodiments, the mass ratio of peptide to additive in the reconstituted aqueous composition is from about 5:1 to about 1:5. In some embodiments, the mass ratio of peptide to additive in the aqueous composition is from about 5:1 to about 4.5:1, or from about 4.5:1 to about 4.0:1, or from about 4.0:1 to about 3.5:1, or from about 3.5:1 to about 3.0:1, or from about 3.0:1 to about 2.5:1, or from about 2.5:1 to about 2.0:1, or from about 2.0:1 to about 1.5:1, or from about 1.5:1 to about 1.0:1, or from about 1... The ratio of the peptide to the additive is from about 0.0:1 to about 0.5:1, or about 1:0.5 to about 1:1.0, or about 1:1.0 to about 1:1.5, or about 1:1.5 to about 1:2.0, or about 1:2.0 to about 1:2.5, or about 1:2.5 to about 1:3.0, or about 1:3.0 to about 1:3.5, or about 1:3.5 to about 1:4.0, or about 1:4.0 to about 1:4.5, or about 1:4.5 to about 1:4.0. In other embodiments, the mass ratio of the peptide to the additive is from about 1.0 to about 1.5.

[0107] In some embodiments, the pH of the reconstituted aqueous composition is from about 5 to about 12. In some embodiments, the pH of the reconstituted aqueous composition is from about 5 to about 12, or from about 5 to about 6, or from about 6 to about 7, or from about 7.0 to about 7.1, or from about 7.1 to about 7.2, or from about 7.2 to about 7.3, or from about 7.3 to about 7.4, or from about 7.4 to about 7.5, or from about 7.5 to about 7.6, or from about 7.6 to about 7.7, or from about 7.7 to about 7.8, or from about 7.8 to about 7.9, or from about 7... pH values ​​of 0.9 to about 8.0, or about 8.0 to about 8.1, or about 8.1 to about 8.2, or about 8.2 to about 8.3, or about 8.3 to about 8.4, or about 8.4 to about 8.5, or about 8.5 to about 8.6, or about 8.6 to about 8.7, or about 8.7 to about 8.8, or about 8.8 to about 8.9, or about 8.9 to about 9.0, or about 9 to about 10, or about 10 to about 11, or about 11 to about 12. In some embodiments, the pH of the reconstituted aqueous composition is about 7.2 to about 7.8.

[0108] In some embodiments, the concentration of the peptide (i.e., Bevonescein (ALM-488)) in the reconstituted aqueous composition is from about 10 mg / mL to about 500 mg / mL, based on the total volume of the aqueous composition. In some embodiments, based on the total volume of the aqueous composition, the peptide concentration 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 140 mg / mL, or about 140 mg / mL to about 150 mg / mL, or about 150 mg / mL to about 160 mg / mL, or about 160 mg / mL to about 170 mg / mL. mg / mL, or about 170 mg / mL to about 180 mg / mL, or about 180 mg / mL to about 190 mg / mL or from about 190 mg / mL to about 200 mg / mL, or about 200 mg / mL to about 250 mg / mL, or about 250 mg / mL to about 300 mg / mL, or about 300 mg / mL to about 350 mg / mL, or about 350 mg / mL to about 400 mg / mL, or about 400 mg / mL to about 450 mg / mL. In some embodiments, the peptide concentration in the reconstituted aqueous composition is about 25 mg / mL to about 200 mg / mL based on the total volume of the aqueous composition. In other embodiments, the peptide concentration in the reconstituted aqueous composition is about 40 mg / mL to about 75 mg / mL based on the total volume of the aqueous composition.

[0109] In some embodiments, the density of the reconstituted aqueous composition is from about 0.6 g / mL to about 1.3 g / mL. In some embodiments, the density of the reconstituted aqueous composition is from about 0.6 g / mL to about 0.7 g / mL, or from about 0.7 g / mL to about 0.8 g / mL, or from about 0.8 g / mL to about 0.85 g / mL, or from about 0.85 g / mL to about 0.90 g / mL, or from about 0.90 g / mL to about 0.95 g / mL, or from about 0.95 g / mL to about 1.0 g / mL, or from about 1.0 g / mL to about 1.05 g / mL, or from about 1.05 g / mL to about 1.10 g / mL, or from about 1.10 g / mL to about 1.15 g / mL, or from about 1.15 g / mL to about 1.20 g / mL, or from about 1.20 g / mL to about 1.25 g / mL. In other embodiments, the density of the reconstituted aqueous composition is from about 1.0 g / mL to about 1.08 g / mL.

[0110] In some embodiments, the weight osmotic concentration of the reconstituted aqueous composition is from about 150 mOsm / kg to about 400 mOsm / kg. In some embodiments, the weight osmotic concentration of the reconstituted aqueous composition is from about 150 mOsm / kg to about 175 mOsm / kg, or from about 175 mOsm / kg to about 200 mOsm / kg, or from about 200 mOsm / kg to about 225 mOsm / kg, or from about 225 mOsm / kg to about 250 mOsm / kg, or from about 250 mOsm / kg to about 275 mOsm / kg, or from about 275 mOsm / kg to about 300 mOsm / kg, or from about 300 mOsm / kg to about 325 mOsm / kg, or from about 325 mOsm / kg to about 350 mOsm / kg. In other embodiments, the weight osmotic concentration of the reconstituted aqueous composition is from about 280 mOsm / kg to about 300 mOsm / kg.

[0111] In some embodiments, the volume of the reconstituted aqueous composition is from about 0.5 mL to about 5.0 mL. In some embodiments, the volume of the reconstituted aqueous composition is from about 0.5 mL to about 1.0 mL, or from about 1.0 mL to about 1.5 mL, or from about 1.5 mL to about 2.0 mL, or from about 2.0 mL to about 2.5 mL, or from about 2.5 mL to about 3.0 mL, or from about 3.0 mL to about 3.5 mL, or from about 3.5 mL to about 4.0 mL, or from about 4.0 mL to about 4.5 mL. In other embodiments, the volume of the reconstituted aqueous composition is from about 1.8 mL to about 2.2 mL.

[0112] In some embodiments, the reconstituted aqueous composition comprises at least 95% by mass of a peptide (i.e., Bevonescein (ALM-488)), as measured relative to the total mass of the reconstituted aqueous composition after storage at 40°C for 14 days. In some embodiments, the reconstituted aqueous composition comprises 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 a peptide, as measured relative to the total mass of the reconstituted aqueous composition after storage at 40°C for 14 days.

[0113] Another embodiment relates to a method for forming a reconstituted aqueous composition by combining the lyophilized composition disclosed herein with an aqueous medium.

[0114] In some embodiments, the method includes stirring or agitating a mixture of the lyophilized composition and an aqueous medium to obtain a reconstituted aqueous composition.

[0115] In some embodiments, the method includes heating the combined mixture.

[0116] In some embodiments, the lyophilized composition used to prepare the reconstituted aqueous composition is in the form of a well-formed lyophilized cake.

[0117] How to use

[0118] Another embodiment relates to a method of labeling or identifying a neuron or nerve by contacting it with a composition containing Bevonescein (ALM-488) disclosed herein. In some embodiments, the neuron or nerve contacts a free fluorescent portion of the composition. In some embodiments, the fluorescent portion comprises a fluorescent protein, a fluorescent peptide, a fluorophore, or any combination thereof. In some embodiments, the contact occurs in vivo. In some embodiments, the contact occurs in vitro. In some embodiments, the neuron or nerve is a human neuron or nerve.

[0119] In some embodiments, the neurotargeting peptide ALM-488 is administered in combination with a fluorescent moiety (e.g., a fluorescent moiety not conjugated to the neurotargeting peptide conjugate, or a “free” fluorescent moiety) (simultaneously, at the same time, or sequentially). In some embodiments, the fluorescent moiety is a fluorescein, such as carboxyfluorescein.

[0120] Another embodiment relates to a method of delivering at least one drug to a neuron or nerve by contacting the neuron or nerve with the ALM-488-containing composition disclosed herein. In some embodiments, the delivery of the at least one drug to the neuron or nerve is carried out by administering the composition to a subject in need. In some embodiments, the subject is a human subject. In some embodiments, the composition is administered to the subject prior to a surgical procedure. In some embodiments, the surgical procedure is a cancer surgical procedure, such as a prostate cancer surgical procedure.

[0121] In some embodiments of the methods described herein, the neural targeting peptide ALM-488 is administered to subjects, including humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In some embodiments, the subject is a mammal. In some embodiments, the subject is a primate. In some embodiments, the subject is a human. In some embodiments, the human subject is a pediatric subject (21 years of age and younger), an adult subject (22 to 65 years of age), or an elderly subject (65 years of age and older).

[0122] In some embodiments of the methods of use described herein (e.g., labeling or identifying neurons or nerves, delivering drugs to neurons or nerves), the procedure may be performed before or during surgery on the subject. In some embodiments, the surgery relates to traumatic injury to the subject. In some embodiments, the surgery relates to infection in the subject. In some embodiments, the surgery is orthopedic surgery on the subject, such as cosmetic or reconstructive surgery.

[0123] In some implementations, the surgery is a head, neck, skull base, spine, prostate, heart, kidney, hand, arm, foot, leg, lower abdomen, or gynecological surgery. In some implementations, the surgery is a head and neck surgery. In some implementations, the surgery is a gastrointestinal (GI) surgery or a genitourinary (GU) surgery.

[0124] In some implementations, the surgery is cancer surgery. In some implementations, the cancer is selected from 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 tumors, mesothelioma, and esophageal cancer. In some implementations, the cancer is prostate cancer.

[0125] In some embodiments, the method includes administering the ALM-488-containing composition disclosed herein to a subject who will undergo surgery. In some embodiments, the method includes administering it to a subject currently undergoing surgery. In some embodiments, the ALM-488-containing composition disclosed herein is administered systemically to the patient, for example, via intravenous injection. In some embodiments, the neurotargeting peptide conjugate disclosed herein is administered locally to the patient.

[0126] In some embodiments, the neurotargeting peptide ALM-488 is delivered to the subject via a drug delivery medium or carrier. In some embodiments, the delivery medium is made of natural or synthetic materials or both. In some embodiments, the delivery medium may include nanoparticles, microparticles, polymeric micelles, nanocapsules, dendritic polymers, large PEGs, nanogels, liposomes, fullerenes, nanostructured lipid carriers, nanoshells, quantum dots, protein-based nanocarriers (e.g., albumin, elastin, gliadin, ginseng, zein, soy protein, milk protein, whey nanocarriers), organic nanocarriers (e.g., gelatin, dextran, guar gum, chitosan, collagen), polysaccharide-based carriers (e.g., dextran, chitosan, pectin), lipid emulsions, or combinations thereof.

[0127] In some embodiments, the ALM-488-containing compositions disclosed herein are administered to the subject via any suitable route of administration, including but not limited to topical, oral, rectal, vaginal, nasal, inhalation, parenteral (intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, intravitreal, infusion) administration. In some embodiments, the pharmaceutical compositions disclosed herein are administered to the subject either locally or systemically. In embodiments, the pharmaceutical compositions disclosed herein are administered intravenously, for example, via systemic intravenous injection.

[0128] Formulations suitable for intramuscular, subcutaneous, or intravenous injection comprise physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders (e.g., the lyophilized compositions described herein) for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or mediators include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, Cremophor, etc.), suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters, such as ethyl oleate. Other commonly used surfactants, such as Tweens and Spans, and other emulsifiers or bioavailability enhancers, are typically used to prepare pharmaceutically acceptable solids, liquids, or other dosage forms for formulation purposes. Bioavailability enhancers may include penetration or permeation 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 flowability can be maintained, for example, by using a coating such as lecithin, in the case of dispersions, by maintaining the desired particle size, and by using surfactants. Formulations suitable for subcutaneous injection also contain optional additives, such as preservatives, wetting agents, emulsifiers, and dispersants, as known in the art. In embodiments, formulations suitable for intravenous injection can be prepared in aqueous solutions, such as saline buffers and other physiologically compatible buffers known in the art.

[0129] Parenteral injection optionally involves bolus or continuous infusion. The formulation for injection is optionally present in a unit dosage form, such as in an ampoule or a multi-dose container with added preservatives. In some embodiments, the ALM-488-containing compositions described herein are in a form suitable for parenteral injection (such as a sterile suspension, solution, or emulsion in an oily or aqueous medium) and contain formulations such as suspending agents, stabilizers, and / or dispersants. Pharmaceutical formulations for parenteral administration comprise aqueous solutions of the active agent in a water-soluble form. Additionally, optionally (e.g., from the lyophilized compositions disclosed herein), the suspension is prepared as a suitable oily injectable suspension.

[0130] In some embodiments, the ALM-488-containing compositions disclosed herein can be administered orally. Dosage forms suitable for oral administration can be solid or liquid and can include, for example, pills, capsules, lozenges, tablets, capsules, gelcapsules, syrups, aqueous suspensions or solutions, chewable forms, swallowable forms, soluble forms, effervescent forms, granulated forms, and oral liquid solutions. In specific embodiments, the dosage form is a solid dosage form, and more specifically, includes tablets or capsules, or is administered orally via a medium or carrier as disclosed herein.

[0131] In some embodiments, the pharmaceutical compositions containing ALM-488 described herein are unit dosage forms suitable for a single, precise dose. In a unit dosage form, the formulation is divided into unit doses containing an appropriate amount of the active agent disclosed herein. In some embodiments, the unit dose is a packaged form containing discrete amounts of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. In some embodiments, aqueous suspension compositions are packaged in single-dose, non-resealable containers. Alternatively, multi-dose, resealable containers are used, in which case a preservative is typically included in the composition. By way of example only, formulations for parenteral injection are available in unit dosage forms, including but not limited to ampoules, or multi-dose containers with added preservatives.

[0132] As described above, certain embodiments of this disclosure relate to methods of targeted drug delivery. In some embodiments, the ALM-488-containing compositions disclosed herein deliver drugs to specific targets. In some embodiments, the compositions deliver drugs to neurons or nerves.

[0133] In some embodiments, the drug is an agent that relieves pain (the sensation of pain or the activity of a pain stimulus). In some embodiments, the drug is an anesthetic. In some embodiments, the drug is benzocaine; carticaine; cinchocaine; cyclomethycaine; lidocaine; prilocaine; propxycaine; proparacaine; tetracaine; tocainide; and trimecaine; or combinations thereof.

[0134] In some embodiments, the drug is an agent that modulates neuronal or neurogenesis (e.g., by apoptosis or necrosis). In some embodiments, the drug is a cytotoxic agent. In some embodiments, the drug is methotrexate (RHEUMATREX®); cyclophosphamide (CYTOXAN®); thalidomide (THALIDOMID®); paclitaxel; pemetrexed; pentostatin; pipebroman; pixantrone; plicamycin; procarbazine; proteasome inhibitors (e.g., bortezomib); raltitrexed; rebeccamycin; rubitecan; SN-38; salinosporamide. A); satraplatin; streptozotocin; swainsonine; tariquidar; taxane; tegafur-uracil; temozolomide; testosterone; tbioTEPA; thioguanine; topotecan; trabectedin; retinoic acid; triplatinum tetranitrate; tri(2-chloroethyl)amine; troxacitabine; uracil mustard; valrubicin; vinblastine; vincristine; vinorelbine; vorinostat; zosuquidar; or combinations thereof. In some embodiments, the drug is a pro-apoptotic agent. In some embodiments, the drug is an anti-apoptotic agent. In some embodiments, the drug is selected from minocycline; SB-203580 (4-(4-fluorophenyl)-2-(4-methanesulfonylphenyl)-5-(4-pyridyl)1H-imidazole); PD 169316 (4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole); SB 202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole); RWJ 67657 (4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridyl)-1H-imidazole-2-yl]-3-butyn-1-ol); SB220025 (5-(2-amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazol);D-JNKI-1 ((D)-hJIP175_i 57-DPrO-DPrO-(D)-HIV-TAT57-48); AM-111 (Auris); SP600125 (anthracite[1,9-cd]pyrazole-6(2H)-one); JNK inhibitor I ((L)-HIV-TAT48-57-PP-JBD20); JNK inhibitor III ((L)-HIV-TAT47-57-gaba-c-Junδ33-57); AS601245 (1,3-benzothiazolyl-2-yl(2-[[2-(3-pyridyl)ethyl]amino]-4-pyrimidinyl)acetonitrile); JNK inhibitor VI (H2N-RPKRPTTLNLF-NH2); JNK inhibitor VIII (N-(4-amino-5-cyano-6-ethoxypyridin-2-yl)-2-(2,5-dimethoxyphenyl)acetamide); JNK inhibitor IX (N-(3-cyano-4,5,6,7-tetrahydro-1-benzothiophene-2-yl)-1-naphthylcarboxamide); biscoumarin (3,3'-methylenebis(4-hydroxycoumarin)); SC-236 (4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide); CEP-1347 (Cephalon); CEP-11004 (Cephalon); artificial proteins containing at least a portion of the Bcl-2 polypeptide; recombinant FNK; V5 (Also known as Bax inhibitor peptide V5); Bax channel blocker ((±)-1-(3,6-dibromocarbazole-9-yl)-3-piperazin-1-yl-2-propanol); 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; FasM3 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); Act1 (NF-κB activator 1); anti-DcB antibody; acetyl-11-keto-β-boswellic acid; andrographolide; phenethyl caffeate (CAPE); mycotoxin; isohelenin; NEMO-binding domain-binding peptide (DRQIKIWFQNRRMKWKKTALDWSWLQTE);NF-κB activation inhibitor (6-amino-4-(4-phenoxyphenylethylamino)quinazoline); NF-κB activation inhibitor II (4-methyl-N1-(3-phenylpropyl)phenyl-1,2-diamine); NF-κB activation inhibitor III (3-chloro-4-nitro-N-(5-nitro-2-thiazolyl)-benzamide); NF-κB activation inhibitor IV ((E)-2-fluoro-4'-methoxystilbene); NF-κB activation inhibitor V (5-hydroxy-(2,6-diisopropylphenyl)-1H-isoindole-1,3-dione); NF-κB SN50 (AA VALLP A VLLALLAP VQRKRQKLMP); Oridonin; Parthenolide; PPM-18 (2-Benzoylamino-1,4-Naphthoquinone); Rol06-9920; Sulfasalazine; TIRAP inhibitor peptide (RQIKIWFNRRMKWKKLQLRD AAPGG AIVS); Withaferin A; Wogonin; BAY 11-7082 ((E)3-[(4-methylphenyl)sulfonyl]-2-acrylonitrile); BAY 11-7085 ((E)3-[(4-tert-butylphenyl)sulfonyl]-2-acrylonitrile); (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); IkB kinase inhibitor peptide; IKK-3 inhibitor LX; ARRY-797 (Array BioPharma); SB-220025 (5-(2-amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazol); SB-239063 (trans-4-[4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol); SB-202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazolium); JX-401 ([2-methoxy-4-(methylthio)benzoyl]-4-(benzyl)piperidine); PD-169316 (4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazolium); SKF-86002 (6-(4-fluorophenyl)-2,3-dihydro-5-(4-pyridyl)imidazolium[2,1-b]thiazole dihydrochloride);SB-200646 (N-(1-methyl-1H-indole-5-yl)-N'-3-pyridylurea); CMPD-I (2'-fluoro-N-(4-hydroxyphenyl)-[1,1'-biphenyl]-4-butanamide); EO-1428 ((2-methylphenyl)-[4-[(2-amino-4-bromophenyl)amino]-2-chlorophenyl] ketone); SB-253080 (4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine); SD-169 (1H-indole-5-carboxamide); SB-203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazolium); 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 (3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1-((2'-(1H-tetrazol-5-yl)(1,1'-biphenyl)-4-yl)methyl)-1H-1-benzozaza-3-yl)butyramide, (R)-); EP1572 (Aib-DTrp-DgT; -CHO); Diltiazem; Diltiazem metabolites; BRE (brain and reproductive organ expressed protein); Verapamil; Nimodipine; Diltiazem; Omega-conotoxin; GVIA; Amlodipine; Felodipine; Lacidipine; Mibefradil; NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid); Flunarizine; Erythropoietin; Piperine; Heme; 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-Aspartic(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-LEHD-FMK (Benzyloxycarbonyl-Leu-Glu-His-Asp-Fluoromethyl ketone); FAM-LETD-FMK (Benzyloxycarbonyl-Leu-Glu-Thr-Asp-Fluoromethyl ketone); Q-VD-OPH (Quinoline-Val-Asp-CH2-O-Ph); XIAP; cIAP-1; cIAP-2; ML-IAP; ILP-2; NAIP; Survivin; Brace; IAPL-3; fortilin; leupeptine; PD-150606 (3-(4-iodophenyl)-2-mercapto-(Z)-2-acrylic acid); MDL-28170 (Z-Val-Phe-CHO); calpeptin; acetylated calpain inhibitor; MG132 (N-[(benzyloxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucine); MYODUR; BN 82270 (Ipsen); BN 2204 (Ipsen);AHLi-11 (Quark Pharmaceuticals), MDM2 protein, pifithrin-α (1-(4-methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazolyl) ethyl ketone); trans-stilbene; cis-stilbene; resveratrol; piceatannol; rhapontin; deoxyrhein; butein; chalcon; isoliquirtigen; butein; 4,2',4'-trihydroxychalcon; 3,4,2',4',6'-pentahydroxychalcon; flavonoids; morin ; fisetin; luteolin; quercetin; kaempferol; apigenin; gossypetin; myricetin; 6-hydroxyapigenin; 5-hydroxyflavone; 5,7,3',4',5'-pentahydroxyflavone; 3,7,3',4',5'-pentahydroxyflavone; 3,6,3',4'-tetrahydroxyflavone; 7,3',4',5'-tetrahydroxyflavone; 3,6,2',4'-tetrahydroxyflavone; 7,4'-dihydroxyflavone; 7,8,3',4'-tetrahydroxyflavone Hydroxyflavonoids; 3,6,2',3'-Tetrahydroxyflavonoids; 4'-Hydroxyflavonoids; 5-Hydroxyflavonoids; 5,4'-Dihydroxyflavonoids; 5,7-Dihydroxyflavonoids; Daidzein; Genistein; Naringenin; Flavanones; 3,5,7,3',4'-Pentahydroxyflavanones; Chlorinated pelargonidin; Chlorinated cyanidin; Chlorinated delphinidin; (-)-Epicatechin (hydroxyl site: 3,5,7,3',4'); (-)- Catechins (hydroxyl sites: 3,5,7,3',4); (-)-gallic acid catechin (hydroxyl sites: 3,5,7,3',4',5); (+)-catechin (hydroxyl sites: 3,5,7,3',4'); (+)-epicatechin (hydroxyl sites: 3,5,7,3',4'); 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-thio-1H-imidazol-4-ethylammonium inner salt); phenyl caffeate; MCI-186 (3-Methyl-1-phenyl-2-pyrazolin-5-one); HBED (N,N'-di-(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid); H2O); Ambroxol (trans-4-(2-amino-3,5-dibromobenzylamino)-cyclohexane-HCl; and U-83836E ((-)-2-((4-(2,6-di-1-pyrrolyl-4-pyrimidinyl)-1-piperazinyl)-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'-deoxyfuran riboside; 3-1'-4,5-dimethylnicotinamide-2'-deoxyribose; 3-D-1'-4,5-dimethylnicotinamide-2'-deoxyfuran riboside; 1-naphthylPP1 (1-(1,1-dimethylethyl)-3-(1-naphthyl)-1H-pyrazolo[3,4-d]pyrimidine-4-amine); Lavendustin A (5-[[(2,5-dihydroxyphenyl)methyl][(2-hydroxyphenyl)methyl]amino]-2-hydroxybenzoic acid); MNS (3,4-methylenedioxy-β-nitrostyrene); PP1 (1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-4-amine); PP2 (3-(4-chlorophenyl)-1-(1,1-dimethylethyl)-1H-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-(1H-1,2,3-triazol-1-yl)ethoxy)quinazolin-4-amine); AP22408 (Ariad Pharmaceuticals); AP23236 (Ariad Pharmaceuticals); AP23451 (Ariad Pharmaceuticals); AP23464 (Ariad Pharmaceuticals); AZD0530 (Astra Zeneca); AZM475271 (M475271; AstraZeneca); Dasatinib (N-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)-piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide);GN963 (trans-4-(6,7-dimethoxy-quinoxalin-2-ylamino)cyclohexanol sulfate); Bosutinib (4-((2,4-dichloro-5-methoxyphenyl)-amino)-6-methoxy-7-(3-(4-methyl-1-piperazinyl)propoxy)-3-quinoline carboxynitrile); or combinations thereof. In some embodiments, the drug is an agent that reduces unwanted neurons or nerve impulses. In some embodiments, the drug relieves one or more symptoms of motor disorders or associated motor disorders. In some embodiments, the drug is carbamazepine, oxcarbazepine, phenytoin, valproic acid, sodium valproate, cinnarizine, flunarizine, or nimodipine, or combinations thereof.

[0135] In some embodiments, the drug is an agent that promotes the regeneration of neurons or neural tissue. In some embodiments, the drug is a growth factor. In some embodiments, the drug is selected from brain-derived neurotrophic factor (BDNF); ciliary neurotrophic factor (CNTF); glial cell line-derived neurotrophic factor (GDNF); neurotrophic factor-3; neurotrophic factor-4; fibroblast growth factor (FGF) receptor; insulin-like growth factor (IGF); or combinations thereof.

[0136] In some embodiments, this document discloses a method for delivering a photosensitizer to a human neuron or nerve, comprising contacting the human neuron or nerve with the ALM-488-containing composition disclosed herein. In some embodiments, the method further comprises exposing the human neuron or nerve to a light source activating the photosensitizer contained in ALM-488, wherein the activated photosensitizer induces ablation or killing of the human neuron or nerve. When exposed to light of a specific wavelength, the photosensitizer reacts with molecular oxygen to produce cytotoxic singlet oxygen.

[0137] In some embodiments, the disclosed ALM-488-containing compositions can be used to treat prostate cancer in a subject. Autonomic innervation may contribute to prostate cancer growth and metastasis through photoinduced local autonomic nerve ablation. Therefore, local autonomic nerves may be a viable target for prostate cancer treatment. In some embodiments, renal vascular hypertension in a subject can be treated by photoinduced sympathetic nerve ablation in renal vessels. In some embodiments, the disclosed ALM-488-containing compositions can be used to treat excessive sweating. In some embodiments, the disclosed ALM-488-containing compositions can be used to treat arrhythmias. In some embodiments, the disclosed ALM-488-containing compositions can be used to treat pathological muscle spasms (e.g., Meige syndrome, hemifacial spasm, torticollis).

[0138] In some embodiments, the compositions containing ALM-488 disclosed herein may be administered in combination with a second neurotargeting peptide conjugate (simultaneously, at the same time, or sequentially).

[0139] Another embodiment relates to the use of the ALM-488-containing compositions disclosed herein in the preparation of a medicament for labeling or identifying neurons or nerves of a subject. Another embodiment relates to the use of the ALM-488-containing compositions disclosed herein in the preparation of a medicament for delivering at least one medicament to neurons or nerves of a subject. Another embodiment relates to the ALM-488-containing compositions disclosed herein in the preparation of a medicament for delivering at least one medicament to neurons or nerves of a subject.

[0140] While aspects of this disclosure have been shown and described throughout this disclosure, it will be apparent to those skilled in the art that such aspects are provided merely by way of example. Many variations, alterations, and substitutions can be made by those skilled in the art without departing from this disclosure. It should be understood that various alternatives to the aspects of this disclosure described throughout this disclosure can be used in the practice of this disclosure. The following claims are intended to define the scope of this disclosure and to cover the methods and structures within the scope of these claims and their equivalents.

[0141] The various embodiments described above can be combined to provide several embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in and / or listed in the application data sheets are incorporated herein by reference in their entirety. If necessary, aspects of the embodiments can be modified to incorporate the concepts of various patents, applications, and publications to provide further embodiments.

[0142] Example

[0143] Abbreviations

[0144] 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-fluorenylmethoxycarbonyl; FDM: Freeze-drying microscopy technique; HBTU: N,N,N',N' -Tetramethyl- O -(1 H -benzotriazol-1-yl)urea hexafluorophosphate; HNS: Half-concentration physiological saline; HOBT: Hydroxybenzotriazole; HPLC: High Performance Liquid Chromatography; MBHA: 4-Methyldiphenylamine; MeOH: Methanol; NS: physiological saline; Pip: Piperidine; PVDF: Polyvinylidene fluoride; RP: Inverted; SPPS: Solid-phase peptide synthesis; TFA: Trifluoroacetic acid; TIS: Triisopropylsilane; UPLC: Ultra-high performance liquid chromatography; instrument The instruments used in this paper are listed in Table 1.

[0145] Table 1 The instruments used

[0146] Material

[0147] The materials used in this paper are shown in Table 2 below.

[0148] Table 2 Materials used

[0149] HPLC conditions

[0150] The HPLC conditions used to analyze the purity of ALM-488 are summarized in Table 3 below.

[0151] Table 3 HPLC method conditions

[0152] Figure 2 The HPLC chromatogram of a 0.25 mg / mL ALM-488 solution (HCl saline solution) is shown using the HPLC conditions in Table 3. Figure 3 Showing Figure 2A magnified view of the main peak in the HPLC chromatogram. Under the HPLC conditions shown in Table 3 (e.g.) Figure 2 and Figure 3 As shown), ALM-488 has a retention time of ~10 minutes, with major impurities eluted at retention times of ~0.96 (0.15 area%) and ~1.06 (0.11 area%).

[0153] Example 1 Preparation of crude ALM-488

[0154] A. Overview of the peptide synthesis process

[0155] In an embodiment of Example 1, the ALM-488 peptide was assembled from the C-terminus to the N-terminus of the peptide sequence using a solid support, MBHA resin. Figure 4 A flowchart for the synthesis of the solid support used to prepare ALM-488 is provided.

[0156] Rink amide linkers are coupled to MBHA resin, followed by Fmoc-Gly-OH. 5-FAM is then coupled to the resulting resin. To couple the next Fmoc amino acid in the sequence, the Fmoc protecting group of the previous amino acid (from the peptide bound to the resin) is removed by deprotection using 20% ​​piperidine / DMF. The resulting amino acid is then coupled to the N-terminal group of the previous amino acid. Two coupling methods are used in the manufacturing process. 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 the re-coupling of the amino acids, if desired.

[0157] B. Resin swelling and washing

[0158] Before coupling to the Rink amide linker, the starting resin was swollen and washed. The resin was then dispensed into a reaction vessel, and DMF was added to affect the swelling (DMF to starting resin ratio of 7 mL / g to 12 mL / g). The reaction was then stirred for at least 2 hours and drained.

[0159] Then add additional DMF to the container, followed by stirring and adding 5% DIEA. After stirring for 10 to 20 minutes, drain the reaction vessel and add additional DMF. Then stir the reaction vessel for at least 2 minutes, followed by draining the solvent. Wash the resin twice more by repeating the above washing steps. Perform a ninhydrin test to confirm that there are no residual amines and / or amino acids in the resulting resin.

[0160] C. Fmoc-Rink amide linker loading

[0161] The molar ratio of Fmoc-Rink amide linker / HOBt (or HOBt-A) / DIC / synthetic scale is 1.5:1.5:1.5:1. Dissolve the required amounts of Fmoc-Rink amide linker and HOBt in an amino acid (AA) mixing vessel containing DMF. After the Fmoc-Rink amide linker / HOBt solution is completely dissolved, transfer it to the reaction vessel. Then stir the reaction vessel and slowly add DIC (the permissible range for DIC is ±2.0 mL). Next, stir the reaction mixture for 2 to 72 hours, and perform a first ninhydrin test at least 2 hours after the start of stirring. If the first ninhydrin test is negative, the procedure continues to the additional DMF washing step described below. If the first ninhydrin test is positive, extend the reaction time and perform a second ninhydrin test at least 1 hour after the first ninhydrin test. Perform consecutive ninhydrin tests until the test is negative.

[0162] After obtaining a negative result in the ninhydrin test, the resin was washed three times with DMF, stirring for at least 2 minutes after each wash. After each DMF wash, the solvent was drained from the reaction vessel. Then, additional DMF was added to the resin, and the resulting mixture was stirred. While stirring, 5% DIEA and 5% Ac2O were added, and the reaction mixture was stirred for 15 to 25 minutes. The resin was then washed four times with DMF, stirring for at least 2 minutes each time, and the solvent was drained from the reaction vessel after each wash.

[0163] D. Temporarily remove protection and clean.

[0164] Remove the Fmoc protecting groups 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 to 10 minutes. After this stirring time, drain the solvent from the reaction vessel and add another deprotection solution. Stir the resulting suspension for another 20 to 30 minutes, then drain the solvent from the vessel. Wash the resin a total of 8 times with DMF, stirring for at least 2 minutes each time. After each of the 8 washes, drain the solvent from the reaction vessel. Perform a ninhydrin test for each AA cycle, stopping the step after obtaining a negative ninhydrin test.

[0165] E. Amino acid coupling and washing (DIC method)

[0166] The molar ratio of Fmoc-amino acid / HOBt (or HOBt-A) / DIC / synthetic scale is 3:3:3:1, except for AA cycle 1 with 5-Fam, which uses a molar ratio of 1.5:1.5:1.5:1. Dissolve the required amounts of Fmoc-AA-OH and HOBt in an AA mixing vessel containing DMF. Once the Fmoc-AA / HOBt solution is completely dissolved, transfer it to a reaction vessel and stir for 2 to 72 hours while slowly adding DIC. Perform a ninhydrin test at least 2 hours after the start of the reaction. If a negative ninhydrin result is obtained, this step can be continued to the next cycle or the final wash after DMF washing (only for cycle 1). If a positive ninhydrin test is obtained, extend the reaction time and perform a second ninhydrin test at least 1 hour after the first test. For DMF washing, wash the resin with DMF a total of 3 times, stirring for at least 2 minutes, and 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 re-coupling step is performed.

[0167] F. Amino acid recoupling and washing

[0168] As described above, after a positive result in the ninhydrin test during amino acid coupling, the HBTU / DIEA recoupling method is followed. The required amounts of amino acids and reagents (the permissible range for AA, 5-FAM, and HBTU is ±2.0 g) are dissolved in an AA mixing vessel containing DMF. After the amino acid and reagent solutions are completely dissolved, the resulting mixture is transferred to a reaction vessel and stirred for 1 to 3 hours while slowly adding DIEA. The ninhydrin test is performed at least 1 hour after the start. If the ninhydrin test is positive, the reaction time is extended, and the ninhydrin test is performed again at least 1 hour after the first test. When the ninhydrin test is negative, this step can be performed to proceed to the next cycle after DMF washing or to the final wash. For DMF washing, the resin is washed 3 times with DMF while stirring for at least 2 minutes. The solvent is drained from the reaction vessel after each wash. If the ninhydrin test is negative, the next AA cycle is continued using the same procedure. If the ninhydrin test shows consecutive positive results, perform the capping step as described below to deactivate the uncoupled amino groups (except for AA cycle 1).

[0169] G. Add a hat

[0170] A capping step is performed to treat consecutive positive results of the ninhydrin test during the recoupling step described above, thereby deactivating the uncoupled amino groups. For this capping, DMF is added to the resin and the resulting mixture is stirred. While stirring, 5% DIEA and 5% Ac2O are added, and the mixture is stirred for a total of 15 to 25 minutes. After the stirring time is complete, the resin is washed four times with DMF, with stirring for at least 2 minutes during each wash.

[0171] H. Peptide preparation, synthesis, and cleavage steps

[0172] After adding the final unit (i.e., the 5-FAM moiety), the resulting resin was washed five times with MeOH, stirring for at least 2 minutes during each wash. For the final wash, the solvent was drained from the reaction vessel after each of the five washes. The peptide resin was washed with DMF, followed by MeOH, and the resulting resin was dried under vacuum. The weight loss during drying was used to determine the completion of the drying step.

[0173] Once 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 yield the desired peptide. In Fmoc SPPS, this step is performed by treating the peptide resin with a TFA containing a scavenging agent, namely EDT / TIS / water. The precipitated cleaved mixture is then washed with ether and dried to obtain the crude ALM-488 peptide.

[0174] Example 2 Purification of crude ALM-488

[0175] 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 outlined in Table 3 above.

[0176] A. Separation by HPLC

[0177] Crude ALM-488 was dissolved in ACN and distilled water. If necessary, a small amount of acetic acid / ammonium hydroxide could be added to completely dissolve the crude ALM-488 in the ACN / water. The resulting homogeneous mixture was then purified using RP-HPLC with different buffer systems. The buffer system consisted of an aqueous solution (buffer A) paired with an organic phase modifier (composed of 100% acetonitrile (buffer B)).

[0178] HPLC separation was performed using the following steps: 1) Add purified water or aqueous buffer solution to dilute the peptide solution; 2) Equilibrate the RP-HPLC column with at least two column volumes of aqueous buffer A and organic buffer B (acetonitrile); 3) Load the diluted peptide solution into an 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) Run the HPLC gradient, collecting the eluent for peptide elution when absorption increases or reaches the corresponding %B; stop collecting when absorption decreases to near its starting point; and 6) Wash the HPLC column with a high percentage of organic buffer B and aqueous buffer A for at least two column volumes.

[0179] In each purification step, the eluted fractions were collected, and their purity was analyzed by UPLC / HPLC. Fractions meeting the first-cut and second-cut purity standards were combined, while fractions below the second-cut purity standard were discarded. The combined first-cut fractions proceeded to the next purification step, and the second-cut fractions were combined for further HPLC purification.

[0180] B. Ion exchange of ALM-488 purified by HPLC

[0181] The first fraction of the combined HPLC-purified ALM-488 was then subjected to ion exchange using the following procedure to produce ALM-488 chloride salt.

[0182] Dilute the HPLC-purified ALM-488 with purified water or aqueous buffer. Then, equilibrate the RP-HPLC column with at least three column volumes of aqueous buffer A (1% triethylamine phosphate / water) and organic buffer B (acetonitrile). Next, add the diluted peptide solution to the RP-HPLC column.

[0183] Then, the RP-HPLC column was equilibrated with the following: (i) at least three column volumes of aqueous buffer A (1% triethylamine phosphate / water) and organic buffer B (acetonitrile), (ii) followed by at least three column volumes of aqueous buffer A (50 mM ammonium acetate / water) and organic buffer B (acetonitrile), (iii) followed by at least three column volumes of aqueous buffer A (1% acetic acid / water) and organic buffer B (acetonitrile), and (iv) followed by at least three column volumes of aqueous buffer A (0.01 N hydrochloric acid / water) and organic buffer B (acetonitrile).

[0184] Then, an HPLC gradient was performed, and eluent was collected for peptide elution as absorption increased or at the corresponding %B point, and collection was stopped when absorption decreased to near its starting point. Finally, the HPLC column was washed with a high percentage of organic buffer B and aqueous buffer A for at least two column volumes.

[0185] C. Lyophilized ALM-488 chloride salt

[0186] After subjecting the combined first fraction to the aforementioned ion exchange procedure, the combined first fraction is lyophilized using a wide-mouth flask lyophilization method. If multiple batches of lyophilization are required to lyophilize the combined first fraction, these individual lyophilizations should be performed for at least 12 hours, followed by a final (combined) lyophilization for at least 72 hours. The individual lyophilized products can be stored at ≤-15°C before the final (combined) lyophilization.

[0187] Example 3 Analysis of purified ALM-488

[0188] The above procedure provides a yellow-orange solid ALM-488 chloride salt. In other embodiments, the ion exchange process can be modified so that the ALM-488 product is in the form of different salts, such as acetate.

[0189] A. HPLC purity of purified ALM-488

[0190] like Figure 2 and Figure 3 As shown, under the HPLC methods in Table 3, ALM-488 chloride salt eluted with a retention time of ~10 minutes. At the ~0.14 and ~0.11% area levels, the major impurities were detected with relative retention times of ~0.96 and ~1.06, respectively (see Table 3). Figure 3 Based on HPLC analysis, the purity of ALM-488 chloride salt is 98.95%.

[0191] B. Chloride content of purified ALM-488

[0192] The chloride content of ALM488 chloride salt was measured to be 3.57% w / w (3.67% w / w, as HCl).

[0193] C. Thermogravimetric analysis of purified ALM-488

[0194] When the sample was heated to 100°C using a Perkin Elmer TGA 7 thermogravimetric analyzer, the thermogravimetric analysis (TGA) of the ALM-488 chloride salt showed a weight loss of 3.96%. Figure 5 Show the thermogravimetric analysis (TGA) curves of ALM-488 chloride salt. For example... Figure 5 As shown, further weight loss is gradual before the sharp decrease in weight, likely due to degradation starting at ~200°C to 250°C.

[0195] D. Water content of purified ALM-488

[0196] Karl Fischer titration of ALM-488 chloride salt was performed using a Mettler Toledo Stromboli automatic oven sample changer system. The instrument was standardized with Hydranal ® water standard (Fluka). Solid ALM-488 samples were weighed into sample vials and heated to 100 °C in an oven. The water evaporated from the samples 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.

[0197] E. Peptide content of purified ALM-488

[0198] The measured contents of chloride and water in the ALM-488 chloride salt were corrected, and the peptide content was calculated to be 91.28%. In the following disclosure, all concentrations and amounts of ALM-488 are expressed as the pure peptide without counterions and water. For the purpose of weighing ALM-488 for experiments and product manufacturing, a correction multiplier factor of 1.096 was applied to the ALM-488 chloride salt to determine the amount of free peptide.

[0199] F. Linear relationship between the amount of ALM-488 and the HPLC peak area

[0200] Standard solutions of ALM-488 at different concentrations and different 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 . For mass loadings outside the Figure 6 plotted range (>5 μg of ALM-488), the increase in peak area was lower than the proportional increase in the loading amount. All samples were appropriately diluted to remain within the indicated linear range. Repeated injections of the standard solutions gave acceptable precision. For example, the relative standard deviation of the average peak area for six 4 μL injections of a ~1 mg / mL aqueous solution of ALM-488 was 0.35%.

[0201] Example 4 : Study on pH adjustment of ALM-488 salt solubility

[0202] Studies were performed using purified ALM-488 chloride and acetate salts to understand the relationship between pH and solubility.

[0203] A. Solubility of ALM-488HCl salt based on pH

[0204] Initial visual assessment showed that the solubility of ALM-488 HCl salt was >180 mg / mL at a solution pH of <pH 2.

[0205] Titration experiments were performed using ALM-488 HCl salt to determine whether the nominal solubility of 40 mg / mL to 50 mg / mL or higher could be maintained by increasing the pH through the stepwise addition of NaOH. An aqueous solution of ALM-488 HCl salt with an initial concentration of approximately 55 mg / mL (approximately 24 mM for a pure peptide) had a solution pH of 1.7. Figure 7 The graph shows the measured pH as a function of the increased concentration of NaOH added to the mixture. At solution pH < 4, the peptide maintained a concentration > 50 mg / mL in solution. Above pH 4, the peptide precipitated. In the pH range of approximately 4.5 to 5.5, the degree of precipitation increased, resulting in an opaque dispersion. With further addition of NaOH, as the pH increased to approximately 6.5, the peptide dissolved, causing the mixture to become clear, and complete dissolution was observed at pH > 7. At pH > 7, the peptide concentration decreased to approximately 47 mg / mL due to dilution of the mixture caused by the addition of alkali. Figure 7 The shaded areas in the figure indicate that the ALM-488 peptide exhibits sufficient solubility over a wide pH range. As shown in the titration curves, resistance to pH changes with added alkali was observed at approximately pH 4.5 and approximately 6.5, which may be consistent with the pKa values ​​of glutamate and 5-FAM.

[0206] B. pH-based solubility of ALM-488 chloride and acetate salts

[0207] Figure 8 It is a superposition of the titration curves of ALM-488 hydrochloride and ALM-488 acetate. For example... Figure 8 As shown, the initial pH of the ALM-488 acetate mixture (approximately 50 mg / mL) was significantly higher than that of the hydrochloride (approximately 1.8, pH approximately 4). At the initial pH of approximately 4, the ALM-488 peptide acetate did not completely dissolve and formed an opaque orange dispersion. Increasing the pH of the acetate mixture by adding NaOH resulted in the dissolution of the precipitate and the acquisition of a clear solution at pH > 7. Figure 8 The shaded areas (pH>7) indicate that the chloride and acetate salts of ALM-488 are expected to exhibit sufficient solubility within a given pH range. Figure 8 As shown, the amount of NaOH required to adjust the pH of the ALM-488 acetate solution to approximately 7 (to obtain sufficient solubility and an acceptable pH for intravenous preparations) is less than the amount of NaOH required to adjust the pH of the ALM-488 hydrochloride solution to approximately 7.

[0208] Example 5 Effect of pH on the stability of ALM-488

[0209] To understand the pH-stability relationship, ALM-488 HCl salt solutions were prepared at approximately 1 mg / mL in 50 mM sodium citrate (pH 4–6) or sodium phosphate (pH 7–8) buffer solutions. As shown in Table 4 below, ALM-488 dissolves rapidly and completely in water (pH 3.5) and phosphate buffer (pH 7–8). However, in citrate buffer (pH 4–6), the solubility of ALM-488 is <1 mg / mL, and incomplete dissolution results in an excess of undissolved solids. Before storage, all mixtures were filtered through a 0.45 μm PVDF syringe filter to remove undissolved solids. The pH of the filtered solutions 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 pH values ​​of the solutions.

[0210] Table 4 ALM-488 concentration and solution pH measured in different buffer media

[0211] As shown in Table 4, the solubility of ALM-488 decreased significantly as the solution pH approached the isoelectric pH, with a solubility of approximately 0.2 mg / mL (50 nM citrate buffer, pH=6.01) at pH 5. The solutions were then transferred to HPLC vials and stored at 5°C, room temperature (22°C to 23°C), and 40°C.

[0212] Table 5 below summarizes the measured concentrations of the intact ALM-488 peptide relative to the standard solution, as well as the areas of the major peptide peaks measured using the HPLC analysis methods in Table 3. Figure 9 This is a superposition of representative chromatograms of ALM-488 solutions at different pH values ​​after storage at 40°C for 24 hours. As the superposition shows, impurities with RRT~1.4 increase to approximately 0.5 area in solutions with lower pH values—most notably in water (pH 3.46). At higher pH values, impurities with RRT~0.96 appear to increase during storage.

[0213] Table 5 ALM-488 concentrations in solutions prepared at different pH values ​​initially and after storage at 40°C for 24 hours.

[0214] The results of Example 5 show that ALM-488 has sufficient solution stability at neutral pH and can be further developed into a lyophilized product.

[0215] Example 6The effect of storage temperature on the stability of ALM-488

[0216] To evaluate whether ALM-488 HCl salt could be provided as a concentrated frozen solution, a 50 mg / mL solution of the HCl salt was prepared in water, and its storage stability at different temperatures was evaluated. Solutions with a pH of 1.87 were prepared in sterile water for injection. These solutions were stored at different temperatures (-20°C, 5°C, and 40°C) and analyzed at different time points using the HPLC methods listed in Table 3. Table 6 lists the chromatographic data of these sample solutions. Figure 10 The image shows a superimposed chromatogram of the initial ALM-488 aqueous solution (50 mg / mL) and the analysis after 7 days at different storage temperatures.

[0217] Table 6 Chromatographic data of ALM-488 50 mg / mL solution in sterile water stored at -20°C, 5°C, and 40°C for 7 days.

[0218] As shown in Table 6 and Figure 10 As shown, solutions stored at 40°C exhibited significant degradation, leading to an increase in impurities primarily at RRTs of ~1.06 and 1.38. The data also indicate that levels of impurities at RRTs of ~0.97 (which form under higher pH conditions) decreased in a temperature-dependent manner in acidic solutions. Less degradation was detected in samples stored at reduced temperatures (-20°C and 5°C).

[0219] Example 7 Study on the solubility of organic amine compounds in ALM-488 salt

[0220] To evaluate formulations suitable for preparing ALM-488 samples of approximately 50 mg / vial with a concentration of approximately 50 mg / mL ALM-488, formulations with different organic amine compounds were investigated. Each of these samples comprised (i) ALM-488 hydrochloride, (ii) a suitable base for lyophilization of the formulation (to adjust the solution pH to an optimal 7 to 8, as described in Examples 4 and 5), and (iii) at least one organic amine compound acting as a pH adjuster, filler, and / or tonicant. Three organic amine compounds were tested: tromethamine (Tris, pKa 8.06), N-methylglucosamine (glucosamine, pKa 9.6), and L-arginine (pKa 12.5). ALM-488 solutions of 55 mg / mL to 60 mg / mL (pH 1.8 to 1.9) were titrated by stepwise addition of 750 mM arginine, 1 M Tris, or 1 M glucosamine. Visual observation results were compared with... Figure 7Those similarities. For example... Figure 11 , Figure 12 and Figure 13 As shown, increasing the pH initially causes precipitation of the ALM-488 peptide, which then dissolves upon further addition of alkali at pH > 7. In each case, the ALM-488 concentration in the final solution at pH 7 to 8 is > 50 mg / mL.

[0221] Figure 11 The pH titration curve shows how pH affects the solubility of ALM-488HCl salt as a function of added tromethamine (Tris). The total ALM-488 peptide concentration in the sample was calculated to be 55 mg / mL (23.8 mM) (without Tris (pH 1.89)) to 49.2 mg / mL (21.3 mM) (with 107 mm Tris (pH 7.3)). Figure 11 The shaded areas indicate the pH range from which a clear solution was obtained. Precipitation occurs near the isoelectric pH, and the solubility is <50 mg / mL at pH approximately 4 to 7.

[0222] Figure 12 This is a pH titration curve, showing how pH affects the solubility of ALM-488HCl salt as a function of added meglumine (N-methylglucosamine). The calculated total ALM-488 peptide concentration in 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)). Figure 12 The shaded areas indicate the pH range from which a clear solution was obtained. Precipitation occurs near the isoelectric pH, and the solubility is <50 mg / mL at pH approximately 4 to 7.

[0223] Figure 13 This is a pH titration curve showing how pH affects the solubility of the HCl salt of ALM-488 as a function of added L-arginine (N-methylglucosamine). The calculated total ALM-488 peptide concentration in 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)). Figure 13 The shaded areas indicate the pH range from which a clear solution was obtained. Precipitation occurs near the isoelectric pH, and the solubility is <50 mg / mL at pH approximately 4 to 6.6.

[0224] from Figures 11 to 13The titration curves show that an organic base concentration of approximately 110 mM produces a near-neutral solution pH and sufficient drug solubility.

[0225] The base studied has been used in commercially available pharmaceutical products. Arginine is included in significantly higher amounts in several approved IV products compared to the amount expected in the ALM-488 formulation studied in Example 7. For example, Azactam... ® The maximum injectable dose of arginine is 6.2 g / day, Caldolor. ® The maximum injectable dose of arginine is 3.2 g / day. If tromethamine is used as a base with ALM-488, the required amount exceeds the usual amount when tromethamine is used as a buffer. However, the maximum daily dose of Ketorolac tromethamine (120 mg) administered via IV is equivalent to approximately 38 mg of tromethamine. Tham ® (Trebutanol injection) is a 300 mM (3.6%) tromethorphanol solution used for metabolic acidosis and administered via slow infusion at a much higher dose. Baxdela ® The total daily dose of (delafloxacin meglumine injection, 300 mg twice daily) is equivalent to a dose of 384 mg meglumine.

[0226] When using tromethamine (Tris), meglumine (N-methylglucosamine), and L-arginine corresponding to the preparation of 50 mg ALM-488 formulation, the expected dose is anticipated to be significantly lower than the amounts of these bases contained in the aforementioned approved pharmaceutical products.

[0227] Example 8 ALM-488 composition used for stability studies

[0228] A series of ALM-488 sample formulations were prepared using different organic amine additives and combinations thereof in a pH range of 7 to 8. As outlined in Table 7, formulations were prepared using L-arginine, meglumine, glycine, mannitol, and trehalose dihydrate, and combinations thereof. The formulations were designed to obtain a 25 mg / mL ALM-488 solution, with the pH adjusted to 7 to 8 using sufficient appropriate bases. Each vial was filled with 2 mL of solution to obtain an ALM-488 peptide concentration of 50 mg / vial. The lyophilized ALM-488 formulation was intended to be reconstituted into a 1 mL volume solution to obtain a 50 mg / mL ALM-488 peptide solution. The sample formulations in Table 7 were designed to achieve a weight osmotic concentration of approximately 300 mOsm / kg for the final reconstituted 50 mg / mL solution.

[0229] Table 7 ALM-488 composition prepared for evaluating solution stability

[0230] As shown in Table 7, all sample formulations had a pH of approximately 7.5. Samples 8-1 and 8-2, containing only a single pH adjuster (L-arginine and meglumine, respectively), had a solution weight osmotic concentration of approximately 70 mOsm / kg. However, in Samples 8-3 through 8-7, the use of combinations of pH adjusters resulted in significantly higher solution weight osmotic concentrations (148 mOsm / kg to 151 mOsm / kg). When used in combination, the concentration of the pH adjuster was calculated to produce a weight osmotic concentration value of 140 mOsm / kg to 150 mOsm / kg in the pre-lyophilized solution (25 mg / mL ALM-488). When the resulting lyophilized product was reconstituted with water to a 50 mg / mL ALM-488 solution, the weight osmotic concentration was expected to be in the desired range of approximately 280 mOsm / kg to 300 mOsm / kg.

[0231] The sample solutions were filtered through a 0.22-micron PVDF syringe filter, and aliquots of these filtered solutions were filled into USP Type 1 amber glass vials, which were then sealed with lyophilized rubber stoppers. The vials were stored at 5°C and 40°C. After 7 and 14 days of storage, the sample solutions were diluted 100-fold with water and analyzed using the HPLC methods described in Table 3.

[0232] Tables 8 to 11 summarize the chromatographic data of samples 8-1 to 8-7. Figure 14 It is a superposition of the magnified HPLC chromatograms of sample 8-4 after initial preparation, storage at 40℃ for 7 days, and storage at 40℃ for 14 days. Figure 15 It is a superposition of amplified HPLC chromatograms of samples 8-1 to 8-7 of Example 8 after being stored at 40°C for 14 days.

[0233] Table 8 Chromatographic data of samples 8-1 and 8-3 before and after storage at 5°C or 40°C for 7 days or 14 days.

[0234] Table 9 Chromatographic data of samples 8-4 and 8-7 before and after storage at 5°C or 40°C for 7 days or 14 days.

[0235] Table 10 Chromatographic data of samples 8-2 and 8-5 before and after storage at 5°C or 40°C for 7 days or 14 days.

[0236] Table 11Chromatographic data of samples 8-6 before and after storage at 5°C or 40°C for 7 or 14 days.

[0237] As shown in Tables 8 to 11, the main common impurities are found in samples with an RRT of ~0.97. Impurities with an RRT of ~1.20 appear to be more prevalent in samples 8-3 containing arginine and glycine, see [reference needed]. Figure 15 Except for the arginine-glycine solution (sample 8-3), which appeared to lose about 2% of intact ALM-488, all other sample formulations lost only about 1% of intact ALM-488 after 14 days at 40°C, expressed as the area of ​​the main peak.

[0238] Example 9 ALM-488 composition used for stability studies

[0239] Sub-environmental DSC experiments were performed on sample formulations 8-1 to 8-7. Solution samples (approximately 25 μL) were transferred to DSC sample dishes, sealed, and subjected to a cooling-heating cycle. In a typical run, the samples were cooled to -50 °C and held isothermally to ensure maximum freezing and formation of a frozen concentrated phase. Subsequently, the samples were heated at 2 °C per minute to temperatures exceeding the ice-melting event. Nitrogen purging at 40 mL / min was maintained in the DSC sample chamber. Although sharp exothermic ice crystallization was observed during the cooling of the sample formulations, no solute crystallization event was observed.

[0240] The freezing and heating curves of arginine-containing formulations (samples 8-1, 8-3, 8-4, and 8-7) are superimposed on... Figure 16 In the middle, and the freezing solution heating curves of the formulations containing meglumine (samples 8-2, 8-5 and 8-6) were superimposed on Figure 17 middle.

[0241] like Figure 16 As shown, the ALM488-arginine cryogenic formulation (8-1) exhibits a weak glass transition (Tg') starting at approximately -16°C. When mannitol (8-4) or glycine (8-3) is included as an additional solute, their crystallization appears to be inhibited. The retention of mannitol (8-4) or glycine (8-3) in the cryogenically concentrated amorphous phase leads to a decrease in Tg', starting at approximately -33°C and approximately -34°C, respectively. The arginine-trehalose formulation (8-7) also remains amorphous during solution freezing and also results in a decrease in Tg' (starting at approximately -28°C).

[0242] like Figure 17As shown, the ALM488-glucamine cryogenic formulation (8-2) did not exhibit a strong glass transition temperature. Crystallization of the glucamine-mannitol formulation (8-6) and the glucamine-glycine formulation (8-5) also appeared to be inhibited during freezing. This resulted in single, broad glass transitions with onset temperatures of approximately -36°C and approximately -37°C, respectively.

[0243] In DSC, the freezing solution at -15°C increased the Tg' initial temperature by about -2°C, indicating that the coexisting solutes ALM488 and arginine / glucamine essentially inhibited crystallization.

[0244] Table 12 below summarizes the DSC and FDM (freeze-drying microscopy) data of samples 8-1 to 8-7.

[0245] The Lyostat 2 is a fully integrated freeze-drying microscope capable of determining key parameters of formulations to be freeze-dried. Using this method, a liquid film of 2 μL sample solution is placed between a quartz slide and a glass coverslip, and then sealed within the freeze-drying microscope's stage. In a typical experiment, the sample is frozen to approximately -50°C using liquid nitrogen and then held for 30 minutes to ensure maximum freezing of the solution. Once the frozen film is obtained, ice sublimation is induced by reducing the chamber pressure to approximately 150 mTorr. This causes the film to dry from the periphery towards the center. When the freeze-dried peripheral region can be visualized in the images, the sample stage is heated at an appropriate rate, and images are captured at regular intervals to visualize the thermal events.

[0246] For each of Samples 8-1 to 8-7, the first visual evidence of the collapse event (due to increased fluidity of the frozen concentrate) and its glass transition onset temperature are listed in Table 12. In each case, the collapse event was visually observed at a temperature much higher than the onset temperature of the broadened glass transition event.

[0247] Table 12 Glass transition onset temperature of 25 mg / mL ALM-488 solution as measured by DSC and collapse temperature as measured by FDM.

[0248] Given that arginine is already used in several approved parenteral products, the arginine sample formulation (8-1) was selected for the lyophilization studies described below, and the arginine-containing formulation (8-1) showed a slightly higher glass transition and collapse temperature compared to other sample formulations. The arginine-glycine formulation (8-3) was not selected for further studies due to its slightly inferior solution stability compared to other solutions.

[0249] Example 10 Initial freeze-drying study

[0250] Sample ALM-488 formulations containing arginine (10⁻¹), arginine-mannitol (10⁻²), and arginine-trehalose (10⁻³) were prepared in amber glass vials for initial lyophilization studies, as outlined in Table 13 below. In 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 5 mL amber glass vials (20 mm opening, USP Type I glass tube) with a 20 mm lyophilization stopper (FluoroTech V10-F5974432 / 50, WestPharmaceutical Services) and a 20 mm fold-over aluminum cap.

[0251] Table 13 ALM-488 composition for initial freeze-drying studies

[0252] 1 Chloride content (3.57% w / w; 3.67% w / w as HCl) and water content -4.08%, peptide purity = 98.95% by KFT; 50 mg ALM-488 = (50 × 100) / (0.9895)× (100-4.08-3.67) = 54.78 mg drug.

[0253] 2 The densities of the compositions prior to 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 filling volume for each vial was calculated as shown in the table. Water was removed during processing.

[0254] A. Solution mixing and vial filling

[0255] For each sample, the ALM-488 peptide was first weighed in a suitable container to prepare a solution. Approximately 80% of the estimated batch water volume was then slowly added to the peptide, and the mixture was stirred to dissolve the drug. The batch arginine volume was then added to the peptide solution while the mixture formed a vortex. The addition of arginine initially caused drug precipitation, but a clear orange solution was obtained upon mixing. For compositions containing mannitol or trehalose, additional reagents were added to the solution and stirred to produce a clear solution. The total solution weight was then made up 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 in vials with a lyophilized fill weight tolerance of ±2%.

[0256] B. Freeze-drying

[0257] The process parameters for the three compositions are summarized in Table 14, and the corresponding freeze-drying process diagrams are shown in Table 14. Figures 18 to 20 middle.

[0258] Table 14 Comparison of process parameters used for experimental freeze-drying

[0259] All samples 10-1 to 10-3 produced well-shaped freeze-dried cakes without any signs of collapse. Figure 21 and Figure 22 These are images of the lyophilized products from ALM-488 sample formulations 10-1 and 10-2. Figure 21 Showing a side view of the lyophilized cake of ALM-488 sample formulation 10-2. Figure 22 Showing a top view of the freeze-dried cake after detachment.

[0260] C. Moisture content of the freeze-dried sample

[0261] The water content of lyophilized samples ranging from 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 changing system. The instrument was operated using a Hydranal system. ® Standardized calibration was performed using water standards (Fluka). Solid lyophilized samples were weighed into sample vials and heated to 100°C in an oven. Water evaporated from the sample was transferred to the titrator cell by purging gas. The measured water content of the lyophilized batches is listed in Table 15 below. The water content was low, confirming the suitability of the lyophilization process. The mannitol-containing lyophilized product (10⁻²) had a higher residual water content than the other two batches.

[0262] Table 15 Water content in lyophilized ALM-488 formulation samples

[0263] D. Powder X-ray diffraction

[0264] X-ray diffraction patterns of lyophilized solid products of ALM-488 formulation samples at 10⁻¹, 10⁻², and 10⁻³ were recorded using a wide-angle X-ray diffractometer (Bruker D8 Advance). Diffraction patterns were recorded within a 2θ angle range of 5° to 45°. Scans were recorded using a step scan mode with a step size of 0.005° 2θ and a counting time of 0.25 seconds per step.

[0265] like Figure 23 and Figure 25 As shown, the PXRD patterns of the lyophilized samples 10⁻¹ (arginine) and 10⁻³ (arginine-trehalose) exhibit amorphous diffuse peaks (Halo). In contrast, as... Figure 24 As shown, the PXRD pattern of the lyophilized sample 10-2 (arginine-mannitol) includes characteristic peaks of crystalline mannitol. There is clear evidence of the presence of mannitol hemihydrate and peaks characteristic of the δ- and β-anhydrous forms of mannitol. The presence of mannitol hemihydrate can partially explain the higher residual water content in the lyophilized sample 10-2 in Table 15.

[0266] Figure 24 The PXRD pattern of sample 10-2 indicates that mannitol partially crystallized during cooling and / or subsequent heat treatment. However, the mannitol hemihydrate was completely dehydrated during secondary drying and retained in the final lyophilized product. It has been reported that a higher temperature drying step is required to dehydrate the mannitol hemihydrate formed during freeze-drying.

[0267] E. Drug product trials

[0268] 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 vial of the lyophilized product to achieve complete dissolution of the vial 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 volumetric flask, which was then brought to a volume of 200 mL with water. The resulting solution (approximately 0.25 mg / mL ALM488) was injected into the HPLC system and analyzed as described above. The lyophilization process appeared to cause a slight increase in some impurity levels. As shown in Table 16, impurities previously observed to form in solution (see RRT ~0.73, RRT ~1.16, and RRT ~1.20 in Tables 6 and 8 to 11) were also found in the lyophilized products of samples 10⁻¹ through 10⁻³.

[0269] Table 16 HPLC data of lyophilized samples from 10⁻¹ to 10⁻³ ppm compared to HPLC data of the starting ALM-488 peptide.

[0270] F. Reconstituted solution composition

[0271] Then, reconstituted solution compositions were prepared using samples 10-1, 10-2, and 10-3, wherein the target reconstituted solution concentration was 50 mg / mL, obtained by reconstituted the contents of the lyophilized sample to a final volume of 1 mL. The composition of the reconstituted solutions is summarized in Table 17 below. Solutions of these compositions were prepared using a volumetric method, and their weights were 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 thus an ALM-488 concentration of 50 mg / mL.

[0272] Table 17 Composition of ALM488 reconstituted solution

[0273] 1 Dehydration and amorphization of trehalose dihydrate by freeze-drying will result in a theoretical amount of 50.4 mg of anhydrous trehalose in the final freeze-dried vial. The original solution was prepared with 56.4 mg of trehalose dihydrate per vial.

[0274] 2 The amount of water is based on the theoretical amount required to obtain 1 mL of the final reconstituted solution volume.

[0275] G. Reconstituted lyophilized sample

[0276] The lyophilized solids of Samples 10-1, 10-2, and 10-3 were reconstituted with a fixed amount of reconstitution fluid (as outlined in Table 18). The lyophilized compositions used for Samples 10-2 and 10-3 contained mannitol or trehalose in amounts calculated to produce a weight osmotic concentration similar to that of human plasma after reconstitution with water. The lyophilized composition used for Sample 10-1 did not contain tension modifiers and was reconstituted with physiological saline (NS; 0.9% NaCl) or half-concentration physiological saline (HNS; 0.45% NaCl). As expected, acceptable weight osmotic concentrations were obtained when reconstituted with 0.45% NaCl based on the weight osmotic concentration of the compositions before lyophilization (see Table 7, Samples 8-1, 8-4, and 8-7).

[0277] Table 18 Reconstitution behavior of lyophilized solids and properties of reconstituted solutions

[0278] For all samples 10-1, 10-2, and 10-3, the lyophilized solids dissolved immediately within 1 minute of the addition of the rehydration fluid, producing a clear solution.

[0279] H. HPLC analysis and stability of reconstituted samples

[0280] 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 assess impurity levels. Aliquots of these samples were simultaneously 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 immediately upon reconstitution and after storage of the lyophilized products of Samples 10-1, 10-2, and 10-3. As shown in Tables 19A, 19B, and 19C, no significant increase in impurities was detected during the short-term storage of the reconstituted samples. Table 19A Initial HPLC analysis of reconstituted sample 10-1 and HPLC analysis after storage at 5°C and 25°C for 24 hours.

[0281] Table 19B Initial HPLC analysis of reconstituted sample 10⁻² and HPLC analysis after storage at 5 °C and 25 °C for 24 hours.

[0282] Table 19C Initial HPLC analysis of reconstituted sample 10⁻³ and HPLC analysis after storage at 5 °C and 25 °C for 24 hours.

[0283] Example 11 Batch of formulations containing ALM-488 / arginine-trehalose

[0284] A. Preparation of ALM-488 / arginine-trehalose lyophilized product (sample 11-1)

[0285] A series of batch formulations containing arginine-trehalose ALM-488 HCl salt (prepared under the conditions of Examples 1 and 2) are outlined in Table 20. The amounts in Table 20 represent the amount of ALM-488 product per 50 mg / vial, and the concentration of ALM-488 in the solution before lyophilization is 25 mg / mL. This batch formulation is collectively referred to as Sample 11-1. The chloride content of the ALM-488 HCl salt is 3.67% (expressed as HCl). After correction for water, the peptide:HCl molar ratio is 1:2.5.

[0286] The batch solution was prepared as described above, filtered through a 0.22-micron PVDF filter, and then filled into a series of amber glass 5 mL vials (2 mL / vial), followed by freeze-drying. The freeze-drying process used for sample 10-3 was used to prepare lyophilized sample 11-1, except that the initial drying plate temperature was maintained at -10°C for 17 hours, followed by a 30-minute warm-up to reach a secondary drying temperature of 25°C, and then a secondary drying at +25°C for 3.5 hours. During both the initial and secondary drying stages, the chamber pressure was maintained at approximately 150 mTorr. Figure 26 A process diagram of the freeze-drying process for sample 11-1 is shown.

[0287] Table 20 Lyophilized product of ALM-488 / arginine-trehalose formulation

[0288] 1 Chloride content (3.57% w / w; 3.67% w / w as HCl) and water content -4.08%, peptide purity -98.95% by KFT, 50 mg ALM488 = (50×100) / (0.9895)×(100-4.08-3.67) = 54.78 mg of pharmaceutical raw material.

[0289] 2 The density of the composition prior to lyophilization was determined to be 1.02 g / cc. Using this density, the total solution filling volume for each vial listed in the table was calculated. Water was removed during the process.

[0290] B. Reconstitution of ALM-488 / arginine-trehalose preparation (sample 11-2)

[0291] A 1 mL volume (50 mg / mL) ALM-488 / arginine-trehalose reconstituted formulation was prepared from Sample 11-1 and named Sample 11-2. 920 μL of sterile water was added to each vial of Sample 11-1 (50 mg / vial) to obtain a reconstituted formulation with the composition and properties shown in Table 21, wherein the ALM-488 peptide concentration was 50 mg / mL.

[0292] Table 21 Reconstituted ALM-488 / arginine-trehalose preparation

[0293] 1Chloride content (3.57% w / w; 3.67% w / w as HCl) and water content 4.08%, peptide purity -98.95% by KFT, 50mg ALM488 = (50×100) / (0.9895)×(100-4.08-3.67) = 54.78 mg of drug raw material.

[0294] 2 Trehalose dihydrate was dehydrated by freeze-drying, yielding 44 mg of theoretical anhydrous trehalose in a final freeze-dried vial.

[0295] 3 Based on the measured density, the water volume in the 50 mg / mL solution is 920 mg (920 μL) / mL. Theoretically, this amount of water added to the lyophilized vial should produce 1 mL of solution and a 50 mg / mL ALM488 concentration.

[0296] Example 12 Filtration suitability study using ALM-488 / arginine-trehalose

[0297] To evaluate whether filtration of the ALM-488 / arginine-trehalose formulation before lyophilization was necessary or advantageous, filtered and unfiltered samples of ALM-488 / arginine-trehalose before lyophilization were prepared in the same manner as described in Example 11. The unfiltered sample before lyophilization was designated Sample 12-1, and the filtered sample before lyophilization was designated Sample 12-2. After visual inspection to ensure complete dissolution of the sample formulation, Sample 12-2 was passed through a 0.22-micron Millipak Durapore filter. ® Capsule filter unit filtration.

[0298] The drug content and purity of samples 12-1 and 12-2 were analyzed by HPLC using the HPLC method conditions outlined in Table 3. Table 22 lists the main peak, the level (area %) of key drug-related impurities, and the calculated concentration of the drug in solution. As shown in Table 22, there was no significant difference in impurity levels or drug content (last row) between the unfiltered (12-1) and filtered (12-2) solutions. This data indicates that the drug underwent complete dissolution during formulation and was found to be within the 0.22 μm Millipak Durapore range. ® The filter materials in the capsule filter unit do not exhibit significant overall incompatibility.

[0299] Table 22 Chromatographic data of samples 12-1 (unfiltered) and 12-2 (filtered)

[0300] Example 13Long-term stability of ALM-488 lyophilized products

[0301] Lyophilized ALM-488 samples 13-1 (arginine), 13-2 (arginine-mannitol), and 13-3 (arginine-trehalose) were prepared in the same manner as the corresponding samples 10-1 (arginine), 10-2 (arginine-mannitol), and 10-3 (arginine-trehalose) of Example 10 and stored at different temperatures and relative humidityes for up to 3 months. The resulting lyophilized samples 13-1, 13-2, and 13-3 were then reconstituted (hydrated) and their purity was determined using the HPLC methods listed in Table 3. The water content of the lyophilized ALM-488 / arginine-trehalose sample 13-3 was also determined using Karl Fischer titration and analyzed by PXRD.

[0302] A. Effect of long-term storage on the HPLC purity of lyophilized ALM-488 / arginine (sample 13-1)

[0303] The lyophilized ALM-488 / arginine sample 13-1 (corresponding to sample 10-1 in Example 10) was aliquoted into three HPLC vials and stored at (a) 40°C and 75% RH for three months (sample 13-1-a), (b) 25°C and 60% RH for three months (sample 13-1-b), and (c) 5°C for three months (sample 13-1-c). The purity of each sample was then determined using the HPLC method conditions in Table 3. Table 23 below summarizes the HPLC purity data of 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 degradation of all stored samples was very low (<0.6% loss of intact drug after 3 months at 40°C / 75% RH).

[0304] Table 23 Chromatographic data of ALM-488 / arginine samples stored at different temperatures and relative humidity for 3 months.

[0305] B. Effect of long-term storage on the HPLC purity of lyophilized ALM-488 / arginine-mannitol (sample 13-2)

[0306] The lyophilized ALM-488 / arginine-mannitol sample 13-2 (corresponding to sample 10-2 in Example 10) was aliquoted into three HPLC vials and stored at (a) 40°C and 75% RH for three months (sample 13-2-a), (b) 25°C and 60% RH for three months (sample 13-2-b), and (c) 5°C for three months (sample 13-2-c). The purity of each sample was then tested using the HPLC method conditions in Table 3. Table 24 below summarizes the HPLC purity data of 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 measured ALM-488 proportion appears to increase after storage in all samples. However, the presence of mannitol hemihydrate in sample 10-2 is considered a potential risk of dehydration during extended storage, thus affecting stability.

[0307] Table 24 Chromatographic data of ALM-488 / arginine-mannitol sample stored at different temperatures and relative humidity for 3 months.

[0308] C. Effect of long-term storage on the HPLC purity of lyophilized ALM-488 / arginine-trehalose (sample 13-3)

[0309] The lyophilized ALM-488 / arginine-trehalose sample 13-3 (corresponding to sample 10-3 in Example 10) was aliquoted into three HPLC vials and stored at (a) 25°C and 60% RH for one month (sample 13-3-a), (b) 25°C and 60% RH for two months (sample 13-3-b), (c) 25°C and 60% RH for three months (sample 13-3-c), (d) 5°C for two months (sample 13-2-d), (e) 5°C for three months (sample 13-2-e), (f) 40°C and 75% RH for one month (sample 13-3-f), (g) 40°C and 75% RH for two months (sample 13-3-g), and (h) 40°C and 75% RH for three months (sample 13-3-h). The purity of each sample was then tested using the HPLC method conditions in Table 3. Table 25 below summarizes the HPLC purity data of 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.

[0310] Table 25 Chromatographic data of ALM-488 / arginine-trehalose samples stored at different temperatures and relative humidity for 1 to 3 months.

[0311] Table 26 below summarizes the HPLC purity data of samples 13-3-f, 13-3-g, and 13-3-h compared to the initial (pre-storage) sample 13-3.

[0312] Table 26 Chromatographic data of ALM-488 / arginine-trehalose sample stored at 40℃ / 75% RH for 3 months.

[0313] As shown in Tables 25 and 26, the overall degradation of all ALM-488 / arginine-trehalose lyophilized products was very low (loss of intact drug <0.6% after 3 months at 40°C / 75% RH). Major degradation products appeared as shoulder peaks on the main peak (RRT ~0.97) and increased in the samples, especially under the conditions of 40°C / 75% RH and 25°C / 60% RH.

[0314] Figure 27 and Figure 28 The images are superimposed HPLC chromatograms of sample 13-3 (ALM-488 / arginine-trehalose) lyophilized product at the initial stage and after 1, 2, and 3 months of storage at 40°C / 75%RH. The chromatograms show the growth of the main impurity as a shoulder peak (RRT ~ 0.97) during storage.

[0315] Comparing the long-term storage results of samples 13-1 (arginine), 13-2 (arginine-mannitol), and 13-3 (arginine-trehalose), the conclusion is that arginine-trehalose sample 13-3 is the best candidate for further development. Sample 13-3 is superior to sample 13-1 because the use of the tension modifier (trehalose) allows for fine-tuning of the pH and solubility characteristics of the formulation before lyophilization and reconstitution. Although sample 13-2 showed better long-term stability over three months, sample 13-3 is superior to sample 13-2 because the mannitol hemihydrate present in sample 13-2 is considered to pose a potential risk of dehydration during extended storage, thus affecting stability.

[0316] D. Effect of long-term storage on the water content of sample 13-3

[0317] ALM-488 / arginine-trehalose lyophilized sample 13-3 was stored at 40°C and 75% relative humidity for three months, and then tested using the Karl Fischer titration method described in Example 10 above. After long-term storage, the water content of sample 10-3 increased from an initial value of about 0.7% w / w to about 1.8% w / w. This increase is likely due to a combination of water transfer from the rubber stopper to the amorphous dried pharmaceutical product and water vapor transport from the humid external environment through the stopper.

[0318] E. Effects of long-term storage on powder X-ray diffraction of sample 10⁻³

[0319] ALM-488 / arginine-trehalose lyophilized sample 13-3 was stored at 40°C and 75% relative humidity for three months, and then tested using the PXRD method described in Example 10 above. Figure 29 As shown, the lyophilized sample 10⁻³ retained its amorphous properties after storage at elevated temperature and humidity. Furthermore, upon adding water to the sample vial, the resulting lyophilized sample rapidly dissolved into a clear solution within 1 minute.

[0320] Based on the detailed description above, these and other changes may be made to the embodiments. Generally, the terminology used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed in the specification, and the claims should be construed as encompassing all possible embodiments together with the full scope of their equivalents. Therefore, the claims are not limited by this disclosure. This application claims priority to U.S. Provisional Application No. 63 / 501,593, filed May 11, 2023, with the U.S. Patent and Trademark Office, the entire contents and disclosure of which are incorporated herein by reference.

Claims

1. An aqueous pharmaceutical composition comprising: (a) A fluorescein-labeled neural-targeting peptide of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, isomer, tautomer, racemate, or isotope thereof: (I) (b) Additives, including pH adjusters, buffers, fillers, tension modifiers, preservatives, or any combination thereof; and (c) Aqueous media; in: The pH of the composition is from about 7 to about 9; and Based on the total volume of the aqueous composition, the concentration of the peptide in the aqueous composition is from about 25 mg / mL to about 200 mg / mL.

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

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

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

5. The aqueous composition according to any one of claims 2 to 4, wherein the molar ratio of the peptide to the pharmaceutically acceptable salt is 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 filler, a tension modifier, 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, glycerol, 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. The aqueous composition according to any one of claims 1 to 8, wherein the additive comprises: The first additive includes arginine, meglumine, glycine, or any combination thereof; and The optional second additive may include mannitol, trehalose, or both mannitol and trehalose.

10. The aqueous composition according to claim 9, wherein the mass ratio of the first additive to the second additive is from 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-glucamine, glucamine, glucamine-glycine, or glucamine-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 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 the peptide to the additive is from 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-concentration 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 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 from 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 from 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 weight permeation concentration of the aqueous composition is from 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 from about 1.8 mL to about 2.2 mL.

22. The 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 unfiltered.

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, as measured relative to the total mass of the aqueous composition.

25. A lyophilized pharmaceutical composition formed by lyophilizing an aqueous composition according to any one of claims 1 to 24.

26. A lyophilized pharmaceutical composition comprising: (a) A fluorescein-labeled neural-targeting peptide of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, isomer, tautomer, racemate, or isotope thereof: (I) and (b) Additives, including pH adjusters, buffers, fillers, tension modifiers, preservatives, or any combination thereof. The water content of the freeze-dried composition is from about 0.2% (w / w) to about 2.0% (w / w) relative to the total mass of the freeze-dried composition.

27. The lyophilized composition of claim 26, wherein the lyophilized composition, when combined with an aqueous medium at ambient temperature, forms a homogeneous aqueous composition having a peptide concentration of 200 mg / mL or lower.

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

29. The lyophilized composition according to any one of claims 26 to 28, wherein after the lyophilized composition is stored at 40°C and 75% relative humidity for 3 months, the lyophilized composition contains at least 98.5% by mass of the peptide.

30. The lyophilized composition according to any one of claims 26 to 29, comprising a pharmaceutically acceptable salt of the peptide.

31. The lyophilized composition according to any one of claims 26 to 30, comprising an HCl salt of the peptide.

32. The lyophilized composition of claim 31, wherein the chloride content of the HCl salt is from about 3.0% (w / w) to about 4.0% (w / w) based on the total mass of the peptide.

33. The lyophilized composition according to claim 31 or 32, wherein the molar ratio of the peptide to the pharmaceutically acceptable salt is from 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 filler, a tension adjuster, or any combination thereof.

35. The 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, glycerol, glycine, histidine, inositol, lactose, maltose, mannitol, mannose, meglumine, raffinose, ribose, sorbitol, sucrose, trehalose, tromethamine, xylitol, xylose, or any combination thereof.

36. The 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. The freeze-dried composition according to any one of claims 26 to 36, wherein the additive comprises a first additive and an optional second additive, the first additive comprising arginine, meglumine, or glycine, and the second additive comprising 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 from about 1:1 to about 1:

5.

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

40. The 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 about 1:2 to about 1:

3.

42. The lyophilized composition according to any one of claims 26 to 41, wherein the mass ratio of the peptide to the additive is about 1.0 to about 1.

5.

43. A reconstituted aqueous pharmaceutical composition formed by combining a lyophilized composition according to any one 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-concentration 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 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 from 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 from 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 weight permeation concentration of the reconstituted composition is from about 200 mOsm / kg to about 350 mOsm / kg.

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

52. The 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. The reconstituted aqueous composition according to any one of claims 43 to 53, which, after being stored at 40°C for 14 days, contains at least 98.5% by mass of the peptide, as measured relative to the total mass of the reconstituted composition.

55. A method for forming a lyophilized pharmaceutical composition, the method comprising lyophilizing an aqueous composition according to at least one of claims 1 to 24 to obtain a lyophilized product.

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

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

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

59. The method of claim 58, wherein the drying comprises: The frozen solid composition is subjected to preliminary drying at a temperature of about -20°C to about 0°C and an initial pressure of about 100 mTorr to about 300 mTorr; and The frozen solid composition is subjected to secondary drying at a temperature of about 0°C to room temperature and a secondary pressure of about 100 mTorr to about 300 mTorr.

60. A lyophilized pharmaceutical composition formed by the method according to any one of claims 55 to 59.

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

62. The method of claim 61, further comprising filtering the reconstituted aqueous composition.

63. The method of claim 61, wherein the reconstituted aqueous composition is unfiltered.

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-concentration 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 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 from 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 from about 0.85 g / mL to about 1.15 g / mL.

70. The method according to any one of claims 61 to 69, wherein the weight permeation concentration of the reconstituted aqueous composition is from 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 from 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 the peptide after being stored at 40°C for 14 days, as measured relative to the total mass of the reconstituted aqueous composition.

73. A method for identifying a human neuron or nerve, the method comprising contacting the human neuron or nerve with a composition according to any one of claims 1 to 54.

74. The method of claim 73, wherein the human neuron or nerve is in contact with the free fluorescent portion, and the free fluorescent portion is combined with the composition.

75. A method of delivering a drug to a human neuron or nerve, the method comprising contacting the human neuron or nerve with a composition according to any one of claims 1 to 54.

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

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

78. The method according to any one of claims 73 to 77, comprising administering the composition prior to surgical procedures.

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

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

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