How to use iloprost for treating frostbite

Abstract

This disclosure generally relates to the treatment of frostbite by intravenous injection or infusion of iloprost or a pharmaceutically acceptable salt thereof.

Description

Cross - reference to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 514,244, filed on July 18, 2023, the disclosure of which is incorporated herein by reference in its entirety.

Technical Field

[0002] The present disclosure generally relates to the treatment of frostbite by intravenous injection or infusion of iloprost or a pharmaceutically acceptable salt thereof.

Background Art

[0003] Frostbite is a thermal injury that occurs when tissue is exposed to freezing temperatures for long enough for ice crystals to form in the affected tissue. The overall incidence of frostbite in the United States (US) is extremely low, at 0.83 cases / year per 100,000 people. In addition, since the majority of cases relate to superficial frostbite (64.8%) compared to severe frostbite (35.2%), severe (grade 3 and grade 4) frostbite is an extremely rare condition, estimated to be less than 1,000 cases per year in the US (Non - Patent Document 1).

[0004] Iloprost is a stable synthetic analogue of prostacyclin (prostaglandin I2; [PGI2]), exhibits anti - platelet activity, and reverses vasoconstriction in the peripheral and systemic arterial vascular beds (Non - Patent Documents 2 and 3). Injection of iloprost is accompanied by immediate systemic vasodilation, and the ratio of the in - vivo anti - platelet aggregation effect to the vasodilatory effect is about 2 - 7:1 (Non - Patent Documents 4 and 5).

[0005] Severe frostbite usually results in amputation of the affected limb. Traumatic finger amputations such as those due to frostbite have an adverse impact on daily life and work movements (Non - Patent Documents 6 and 7). Furthermore, since the hand is very prominent, finger amputations can lead to social withdrawal and a decline in quality of life (Non - Patent Document 8). Therefore, an effective treatment method for treating severe frostbite to prevent or reduce the risk of finger amputation is needed.

Prior Art Documents

Non-Patent Literature

[0006]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

Non-Patent Literature 4

Non-Patent Literature 5

Non-Patent Literature 6

Non-Patent Literature 7

Non-Patent Literature 8

Summary of the Invention

Means for Solving the Problems

[0007] This disclosure provides a method for treating frostbite in a subject, comprising administering a composition containing iloprost or a pharmaceutically acceptable salt thereof at a concentration ranging from about 0.8 μg (mcg) per 1 mL of pharmaceutically acceptable carrier to about 1.1 μg / mL.

[0008] In one embodiment of the method disclosed herein, the composition comprising iloprost or a pharmaceutically acceptable salt thereof has a concentration of about 0.9 μg / mL. In some embodiments, the composition comprising iloprost or a pharmaceutically acceptable salt thereof has a concentration of about 1 μg / mL.

[0009] In one embodiment of the method disclosed herein, administration is by intravenous injection or intravenous infusion. In some embodiments, administration is by continuous infusion.

[0010] In one embodiment of the method disclosed herein, administration is carried out in one or more doses ranging from about 0.2 ng / kg / min to about 3.0 ng / kg / min. In some embodiments, administration is carried out in one or more doses ranging from about 0.25 ng / kg / min to about 2.0 ng / kg / min.

[0011] In one embodiment of the method disclosed herein, administration includes a titration step on the first day of administration. In some embodiments, administration includes a titration step on the second day of administration. In some embodiments, administration includes a titration step on the third day of administration.

[0012] In one embodiment of the method disclosed herein, the titration step includes administering the composition at an initial dose of about 0.25 ng / kg / min, about 0.3 ng / kg / min, about 0.4 ng / kg / min, about 0.5 ng / kg / min, about 0.6 ng / kg / min, or about 0.7 ng / kg / min.

[0013] In one embodiment of the method disclosed herein, the titration step includes administering the composition at an initial dose of about 0.5 ng / kg / min. In some embodiments, the subject does not have pre-existing Child-Pugh class B or Child-Pugh class C hepatic impairment.

[0014] In one embodiment of the method disclosed herein, the target is 30 mL / min / m². 2 If a patient has renal impairment with an eGFR of less than 0.5 ng / kg / min and cannot tolerate the initial dose of approximately 0.5 ng / kg / min, the dose will be reduced to approximately 0.25 ng / kg / min at the start of the titration step.

[0015] In one embodiment of the method disclosed herein, the titration step includes administering the composition at an initial dose of about 0.25 ng / kg / min. In some embodiments, the subject has Child-Pugh class B or Child-Pugh class C hepatic impairment.

[0016] In one embodiment of the method disclosed herein, the titration step includes increasing the dose every 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes until the maximum dose is reached. In some embodiments, the dose is increased every 30 minutes.

[0017] In one embodiment of the method disclosed herein, the titration step includes increasing the dose by about 0.1 ng / kg / min, about 0.2 ng / kg / min, about 0.25 ng / kg / min, about 0.3 ng / kg / min, about 0.4 ng / kg / min, about 0.5 ng / kg / min, about 0.6 ng / kg / min, about 0.7 ng / kg / min, or about 0.8 ng / kg / min until the maximum dose is reached. In some embodiments, the dose is increased by about 0.5 ng / kg / min. In some embodiments, the dose is increased by about 0.25 ng / kg / min.

[0018] In one embodiment of the method disclosed herein, the maximum dose is predetermined. In some embodiments, the predetermined maximum dose is about 1.8 ng / kg / min, about 2.0 ng / kg / min, about 2.2 ng / kg / min, about 2.4 ng / kg / min, about 2.6 ng / kg / min, about 2.8 ng / kg / min, or about 3.0 ng / kg / min. In some embodiments, the predetermined maximum dose is about 2.0 ng / kg / min.

[0019] In one embodiment of the method disclosed herein, the maximum dose is about 2.0 ng / kg / min or the highest dose that the subject can tolerate between 0.25 ng / kg / min and about 2.0 ng / kg / min. In some embodiments, the maximum dose is about 2.0 ng / kg / min or the highest dose that the subject can tolerate between 0.5 ng / kg / min and about 2.0 ng / kg / min.

[0020] In one embodiment of the method disclosed herein, the titration step includes reducing the dose if the subject has a dose-limiting reaction. In some embodiments, the dose-limiting reaction is headache, flushing, jaw pain, muscle pain, nausea, or vomiting. In some embodiments, the dose is reduced by about 0.5 ng / kg / min every 30 minutes until the tolerated dose is reached.

[0021] In one embodiment of the method disclosed herein, the composition is administered continuously daily during the treatment period. In some embodiments, the continuous administration per day is about 4 hours, about 5 hours, about 6 hours, about 7 hours, or about 8 hours. In some embodiments, the continuous administration per day is about 6 hours. In some embodiments, the treatment period includes titration steps on the first day, the second day, and / or the third day.

[0022] In one embodiment of the method disclosed herein, once the maximum dose is determined in the titration step, that maximum dose is maintained for the remainder of the treatment period.

[0023] In one embodiment of the method disclosed herein, the composition is administered for 4, 5, 6, 7, 8, 9, or 10 consecutive days. In some embodiments, the composition is administered for up to 8 consecutive days.

[0024] In one embodiment of the method disclosed herein, the maximum dose found in the titration step (which is performed on day 1, day 2, or day 3) is administered from day 4 onward.

[0025] In one embodiment of the method disclosed herein, if the subject has a dose-limiting reaction before the end of the treatment period, administration is discontinued. In some embodiments, after the dose-limiting reaction of the subject has resolved, administration is resumed at the previously tolerated dose.

[0026] In one embodiment of the methods disclosed herein, the pharmaceutically acceptable carrier is an IV fluid. In some embodiments, the pharmaceutically acceptable carrier is a 0.9% sodium chloride solution, a 0.45% sodium chloride solution, a 0.33% sodium chloride solution, a 0.225% sodium chloride solution, a 5% glucose aqueous solution (5% dextrose in water: D5W), a 2.5% glucose aqueous solution (D2.5W), a 5% glucose-lactated Ringer's solution (5% dextrose in lactated Ringer's solution: D5LRS), Ringer's solution (RS), or sterile water. In some embodiments, the pharmaceutically acceptable carrier is a 0.9% sodium chloride solution.

[0027] In one embodiment of the method disclosed herein, the method further comprises diluting a 100 μg / mL concentrated solution of iloprost or a pharmaceutically acceptable salt thereof with a sodium chloride solution to provide a composition of iloprost or a pharmaceutically acceptable salt thereof having a concentration in the range of about 0.8 μg / mL to about 1.1 μg / mL. In some embodiments, 1 mL of the 100 μg / mL concentrated solution of iloprost or a pharmaceutically acceptable salt thereof is contained in a single-dose vial. In some embodiments, the single-dose vial is a glass vial.

[0028] In one embodiment of the method disclosed herein, the most common adverse events are headache, flushing, palpitations / tachycardia, nausea, vomiting, dizziness, or hypotension.

[0029] In one embodiment of the method disclosed herein, the safety profile of the method in a subject is consistent with the safety profile observed in a multicenter, double-blind, randomized, placebo-controlled trial of continuous iloprost infusion in subjects with systemic sclerosis experiencing a symptomatic digital ischemic episode.

[0030] In one embodiment of the method disclosed herein, the frostbite is stage 3 or stage 4 frostbite. In some embodiments, the frostbite is severe frostbite.

[0031] In one embodiment of the method disclosed herein, the method prevents or reduces the risk of finger amputation. [Brief explanation of the drawing]

[0032] [Figure 1] This shows the percentage of patients who underwent amputation after standard treatment for frostbite. [Figure 2] This shows the percentage of patients who underwent amputation after iloprost treatment for frostbite. [Figure 3]This shows finger amputation after treatment with iloprost alone, compared to standard treatment. [Figure 4] This shows finger amputation after treatment with iloprost alone and iloprost + tPA, compared to standard treatment. [Modes for carrying out the invention]

[0033] All publications, patents, and patent applications (including any drawings and appendices contained herein) are incorporated by reference in all purposes to the same extent as each individual publication, patent, or patent application, drawing, or appendix is ​​specifically and individually indicated to be incorporated by reference in all purposes.

[0034] definition The following terms are expected to be well understood by those skilled in the art, but the following definitions are provided to facilitate the explanation of the subject matter of this disclosure.

[0035] Throughout this specification, the terms “about” and / or “approximately” may be used in relation to numbers and / or ranges. The term “about” is understood to mean a value close to the stated value. Furthermore, the phrases “less than approximately [value]” or “greater than approximately [value]” should be understood in consideration of the definition of the term “about” provided herein. The terms “about” and “approximately” may be used interchangeably.

[0036] Throughout this specification, numerical ranges are provided for specific quantities. It should be understood that these ranges include all subranges within them. Thus, the range "50-80" includes all possible ranges within it (e.g., 51-79, 52-78, 53-77, 54-76, 55-75, 60-70, etc.). Furthermore, all values ​​within a given range can be endpoints of the ranges it encompasses (e.g., the range 50-80 includes ranges with endpoints such as 55-80, 50-75, etc.).

[0037] The terms “a” or “an” refer to one or more entities; for example, “therapeutic” refers to one or more therapeutics, or at least one therapeutic. Thus, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein. Furthermore, a reference to “a certain inhibitor” using the indefinite article “a” or “an” does not preclude the possibility of multiple inhibitors unless the context clearly requires the existence of one inhibitor or only one inhibitor.

[0038] As used herein, the verb “comprise” and its conjugations are used in their non-restrictive sense, meaning that the items following the word are included, but not excluded, items not specifically mentioned. The present invention may appropriately “include,” “consist of,” or “essentially consist of” the steps, elements, and / or reagents described in the claims.

[0039] Furthermore, it should be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as an antecedent for the use of exclusive terms such as "simply" or "only," or for the use of "negative" limitations, in relation to the enumeration of claim elements.

[0040] The term "pharmaceutically acceptable salt" includes both acid addition salts and base addition salts. Pharmaceutically acceptable salts include those obtained by reacting an active compound that functions as an acid with an inorganic or organic base to form a salt. Examples of organic bases, but not limited to, include monoethanolamine, diethanolamine, triethanolamine, trometamol, and meglumine. Those skilled in the art will further recognize that base addition salts can be prepared by reaction of a compound with a suitable inorganic or organic base using any of the various known methods.

[0041] The term “treating” means one or more of the actions of alleviating, reducing, delaying, decreasing, improving, or managing at least one symptom of a condition in a subject. The term “treating” may also mean one or more of the actions of preventing, delaying the onset of a condition (i.e., the period prior to the clinical signs of the condition) or reducing the risk of the condition progressing or worsening.

[0042] The compounds of the present invention or their pharmaceutically acceptable salts contain a chiral center and can therefore give rise to enantiomers, diastereomers, and other stereoisomeric forms, which can be defined as (R) or (S) in absolute stereochemistry. This disclosure is intended to include all such possible isomers, as well as their racemates and optically pure forms, whether or not they are specifically shown herein. Stereoiomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor, or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC). The compounds described herein also contain olefinic double bonds, and unless otherwise specified, the compounds are intended to include both E and Z geometric isomers.

[0043] "Stereoisomers" refer to compounds composed of the same atoms that are bonded together by the same bonds but have different three-dimensional structures, and these are not interchangeable. This disclosure intends to cover various stereoisomers and mixtures thereof, including diastereomers.

[0044] Pharmaceutically active ingredients This disclosure relates to the use of iloprost or a pharmaceutically acceptable salt thereof or a stereoisomer thereof for the treatment of frostbite. Iloprost has the following structure and can also be identified as (5E)-5-[(3aS,4R,5R,6aS)-5-hydroxy-4-[(E,3S)-3-hydroxy-4-methylocta-1-en-6-inyl]-3,3a,4,5,6,6a-hexahydro-1H-pentalene-2-ylidene]pentanoic acid. [ka]

[0045] In some embodiments, iloprost consists of a mixture of 4R and 4S diastereomers. In some embodiments, iloprost consists of a mixture of 4R and 4S diastereomers in a ratio of about 1:1. In some embodiments, iloprost consists of a mixture of 4R and 4S diastereomers in a ratio of about 53:47.

[0046] This disclosure also relates to pharmaceutical compositions comprising iloprost or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

[0047] International Publication Nos. 2022 / 032141 and International Publication Nos. 2022 / 036234 disclose iloprost compositions and treatments, each of which is incorporated herein by reference in whole for all purposes.

[0048] Iloprost composition This disclosure relates to the administration of a composition comprising iloprost or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the composition is a pharmaceutical composition.

[0049] In some embodiments, iloprost or its pharmaceutically acceptable salts or stereoisomers are formulated for injection. In some embodiments, iloprost or its pharmaceutically acceptable salts or stereoisomers are formulated for intravenous injection or intravenous infusion. In some embodiments, iloprost or its pharmaceutically acceptable salts or stereoisomers are formulated for continuous infusion.

[0050] In some embodiments, iloprost or a pharmaceutically acceptable salt or stereoisomer thereof is formulated as a sterile solution. In some embodiments, a composition comprising iloprost or a pharmaceutically acceptable salt or stereoisomer thereof further comprises a pharmaceutically acceptable carrier or excipient.

[0051] Pharmaceutically acceptable excipients include, but are not limited to, solubilizers, pH adjusters, isotonic agents, buffers, and / or solvents.

[0052] In some embodiments, the solubilizer is selected from pharmaceutically acceptable alcohols, glycols, esters, ethers, or silicones. In some embodiments, the solubilizer is ethanol.

[0053] In some embodiments, the pH adjuster is a pharmaceutically acceptable acid or base. In some embodiments, the pH adjuster is hydrochloric acid.

[0054] In some embodiments, the isotonic agent may include, but is not limited to, glycerin, lactose, mannitol, glucose (dextrose), sodium chloride, sodium sulfate, and sorbitol. In some embodiments, the isotonic agent is sodium chloride.

[0055] In some embodiments, the buffering agent may include, but is not limited to, citrate buffer, phosphate buffer, citrate phosphate buffer, bicarbonate buffer, tartaric acid buffer, acetate buffer, and trometamol (Tris) buffer. In some embodiments, the buffering agent is trometamol.

[0056] In some embodiments, the solvent is a sterile solvent. In some embodiments, the sterile solvent is an IV fluid. In some embodiments, the solvent includes, but is not limited to, water, glucose solution, dextrose solution, physiological saline, Ringer's solution, and lactated Ringer's solution. In some embodiments, physiological saline is a 0.9% sodium chloride solution, a 0.45% sodium chloride solution, a 0.33% sodium chloride solution, or a 0.225% sodium chloride solution. In some embodiments, the glucose solution is a 5% aqueous glucose solution (D5W), a 2.5% aqueous glucose solution (D2.5W), or a 5% glucose-added lactated Ringer's solution (D5LRS). In some embodiments, the solvent is water. In some embodiments, the solvent is a 0.9% sodium chloride solution.

[0057] In some embodiments, a composition comprising iloprost or a pharmaceutically acceptable salt or stereoisomer thereof further comprises trometamol, ethanol, sodium chloride, hydrochloric acid, and water.

[0058] In some embodiments, compositions comprising iloprost or its pharmaceutically acceptable salts or stereoisomers are free of preservatives.

[0059] In some embodiments, a composition comprising iloprost or a pharmaceutically acceptable salt or stereoisomer thereof is provided in a single-use vial. In some embodiments, a composition comprising iloprost or a pharmaceutically acceptable salt or stereoisomer thereof is provided in a single-dose vial. In some embodiments, the single-use or single-dose vial is a glass vial. In some embodiments, each vial contains about 100 mg of iloprost. In some embodiments, each vial contains about 100 μg (mcg) of iloprost per 1 mL of pharmaceutically acceptable carrier and / or excipient. In some embodiments, each single-use or single-dose vial has a composition comprising about 100 μg of iloprost or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, each single-use vial or single-dose vial contains a composition comprising about 100 μg (0.1 mg) iloprost or a pharmaceutically acceptable salt or stereoisomer thereof, about 8.1 mg of ethanol, about 0.242 mg of tromethamine, and about 0.9 mg of sodium chloride. In some embodiments, each single-use vial or single-dose vial has a pH of about 8.3. In some embodiments, each single-use vial or single-dose vial has a pH of about 8.3, and the pH is adjusted with hydrochloric acid and sodium hydroxide. In some embodiments, each single-use vial or single-dose vial has a volume of 1 mL, and after the above components are weighed, it is filled with water for injection to a total volume of 1 mL.

[0060] Glass vials containing 100 μg of iloprost per 1 mL of pharmaceutically acceptable carrier and / or excipient represent a significant advance in the pharmaceutical use of iloprost.

[0061] Prior to the approval of AURLUMYN®, all approved iloprost was sold worldwide in glass ampoules. Glass ampoules carry safety risks, including percutaneous injury to healthcare workers when crushing glass ampoules and accidental transfer of glass microparticles during pharmacy compounding. Accidental transfer of glass microparticles, which may result from healthcare workers not using appropriate filter needles when transferring ampoule contents, is particularly dangerous as it can lead to the injection of glass microparticles into the patient and cause serious adverse events. Adverse events associated with accidental injection of glass microparticles include pulmonary thrombosis, microemboli, infusion phlebitis, end-organ granuloma formation, and inflammation. These risks associated with the use of glass ampoules can be eliminated by providing therapeutic drugs in glass vials. In addition, single-use vials will further reduce potential errors when diluting therapeutic agents for injection compared to situations where it is necessary to combine the contents of two or more drug preparations (ampoules or vials) or when it is necessary to draw a set amount with a syringe from a suitable container containing a larger volume of therapeutic agent.

[0062] In some embodiments, single-use or single-dose vials containing iloprost are stored at room temperature (20°C to 25°C). In some embodiments, single-use or single-dose vials containing iloprost are stored at a temperature of approximately 20°C to approximately 25°C, with acceptable temperature deviations of 15°C to 30°C. In some embodiments, single-use or single-dose vials containing iloprost are stored at a refrigerated temperature (approximately 5°C). In some embodiments, single-use or single-dose vials containing iloprost are protected from light during storage. In some embodiments, single-use or single-dose vials contain 100 μg of iloprost per mL.

[0063] In some embodiments, a single-use or single-dose vial containing iloprost is stable at 25°C for at least 6 months. In some embodiments, a single-use or single-dose vial containing iloprost is stable at 25°C for at least 12 months. In some embodiments, a single-use or single-dose vial containing iloprost is stable at 25°C for at least 18 months.

[0064] In some embodiments, single-use or single-dose vials containing iloprost are stable for at least 6 months at 25°C and 60% relative humidity (RH). In some embodiments, single-use or single-dose vials containing iloprost are stable for at least 12 months at 25°C / 60%RH. In some embodiments, single-use or single-dose vials containing iloprost are stable for at least 18 months at 25°C / 60%RH.

[0065] In some embodiments, a single-use or single-dose vial containing iloprost is stable at 40°C for at least one month. In some embodiments, a single-use or single-dose vial containing iloprost is stable at 40°C for at least three months. In some embodiments, a single-use or single-dose vial containing iloprost is stable at 40°C for at least six months.

[0066] In some embodiments, single-use or single-dose vials containing iloprost are stable for at least one month at 40°C and 75% relative humidity (RH). In some embodiments, single-use or single-dose vials containing iloprost are stable for at least three months at 40°C / 75%RH. In some embodiments, single-use or single-dose vials containing iloprost are stable for at least six months at 40°C / 75%RH.

[0067] In some embodiments, a single-use or single-dose vial containing iloprost is stable at 5°C for at least 3 months. In some embodiments, a single-use or single-dose vial containing iloprost is stable at 5°C for at least 6 months. In some embodiments, a single-use or single-dose vial containing iloprost is stable at 5°C for at least 9 months.

[0068] In some embodiments, compositions comprising iloprost or a pharmaceutically acceptable salt or stereoisomer thereof have a pH of about 7.0 to about 9.0. In some embodiments, compositions comprising iloprost or a pharmaceutically acceptable salt or stereoisomer thereof have a pH of about 8.0 to about 9.0.

[0069] In some embodiments, a composition comprising iloprost or a pharmaceutically acceptable salt or stereoisomer thereof is further diluted with a sterile solvent for injection or infusion. In some embodiments, the composition is further diluted with a 0.9% sodium chloride solution, a 0.45% sodium chloride solution, a 0.33% sodium chloride solution, a 0.225% sodium chloride solution, a 5% glucose aqueous solution (D5W), a 2.5% glucose aqueous solution (D2.5W), a 5% glucose-added Ringer's lactate solution (D5LRS), Ringer's solution (RS), or sterile water. In some embodiments, the composition is further diluted with physiological saline for injection or infusion. In some embodiments, the composition is further diluted with a 0.9% sodium chloride solution. In some embodiments, the composition is further diluted with a 0.9% sodium chloride injection solution (USP).

[0070] In some embodiments, a composition for injection or infusion containing iloprost or a pharmaceutically acceptable salt or stereoisomer thereof has a concentration of iloprost or a pharmaceutically acceptable salt thereof of about 0.8 μg (0.8 / mL) to about 1.1 μg / mL per 1 mL of pharmaceutically acceptable carrier. In some embodiments, the composition has a concentration of about 0.9 μg / mL. In some embodiments, the composition has a concentration of about 1 μg / mL (1,000 ng / mL).

[0071] In some embodiments, 1 mL of a composition containing 100 μg of iloprost or a pharmaceutically acceptable salt thereof per 1 mL is diluted with 99 mL of 0.9% sodium chloride. In some embodiments, 1 mL of 100 μg / mL iloprost is diluted with 99 mL of 0.9% sodium chloride injection (USP) to obtain an iloprost concentration of 1,000 ng / mL (1 μg / mL).

[0072] In some embodiments, 1 mL of a composition containing 100 μg of iloprost or a pharmaceutically acceptable salt thereof per 1 mL is added without concern for overfilling to an injection bag labeled to contain 100 mL of 0.9% sodium chloride injection (USP). In some embodiments, 1 mL of 100 μg / mL iloprost is diluted with 0.9% sodium chloride injection (USP) to obtain iloprost concentrations of approximately 0.8 μg / mL to approximately 1.1 μg / mL.

[0073] In some embodiments, the diluted composition is ready to use and can be used immediately, or can be stored at refrigerated temperatures (2°C to 8°C) for up to 8 days before use, or can be stored at room temperature (20°C to 25°C) for 4 hours before being administered as a 6-hour continuous infusion. In some embodiments, the continuous infusion is a continuous intravenous infusion.

[0074] In some embodiments, a composition containing iloprost or a pharmaceutically acceptable salt or stereoisomer thereof for injection or infusion has a concentration of approximately 25 μg of iloprost per mL. In some embodiments, a composition containing 25 μg of iloprost per mL is ready for immediate use (prepared) or can be stored at refrigerated temperatures (2°C to 8°C) for up to 8 days before use, or can be stored at room temperature (20°C to 25°C) for 4 hours before administration as a 6-hour continuous infusion. In some embodiments, the continuous infusion is a continuous subcutaneous infusion.

[0075] In some embodiments, ready-to-use compositions containing iloprost or its pharmaceutically acceptable salts or stereoisomers disclosed herein are safe, effective, and stable for up to 8 days at 2°C to 8°C. The 8-day stability of the ready-to-use compositions is crucial for ensuring that patients receive the correct dose of iloprost or its pharmaceutically acceptable salts or stereoisomers and for minimizing the risk of infection (sepsis or line-infection). In some embodiments, the 8-day stability of the ready-to-use compositions is important for enabling decentralized infusion in the patient's home environment or off-site outpatient infusion facilities, which reduces the patient's risk of hospital-acquired infection (infection at the hospital or infusion center) and improves patient convenience, thereby reducing the risk of medication non-compliance.

[0076] In some embodiments, continuous subcutaneous infusion uses a higher concentration of fully diluted prepared iloprost product (e.g., 25 μg / mL instead of 1 μg / mL for intravenous infusion), but the dose delivered (ng / kg / min) and delivery time (6 hours) are equivalent.

[0077] therapeutic use This disclosure relates to the administration of iloprost or a pharmaceutically acceptable salt thereof or its stereoisomer to patients with frostbite. In some embodiments, iloprost or a pharmaceutically acceptable salt thereof or its stereoisomer is administered by injection. In some embodiments, administration is by intravenous injection or subcutaneous injection. In some embodiments, administration is by intravenous injection or intravenous infusion. In some embodiments, administration is by continuous infusion. In some embodiments, administration by continuous infusion is by intravenous infusion or subcutaneous infusion. In some embodiments, administration is performed via a peripheral catheter system, a peripheral inserted central catheter (PICC), or a subcutaneous catheter in the abdomen. In some embodiments, administration is performed via a NovaCath Integrated IV Catheter System or a Poly Per-Q-Cath Catheter. In some embodiments, the same peripheral catheter system or peripheral inserted central catheter (PICC) is used for 1, 2, 3, 4, 5, 6, 7, or 8 days of treatment. In some embodiments, the same peripheral catheter system or peripherally inserted central venous catheter (PICC) is used for the entire duration of the procedure.

[0078] In embodiments of administering iloprost or a pharmaceutically acceptable salt thereof or its stereoisomer to a patient with frostbite, the administration is performed in a medical facility by a medically trained professional. In some embodiments, the administration is performed in a decentralized setting. In some embodiments, the administration is performed at the patient's home or in an outpatient infusion room. In embodiments of administration in a decentralized setting, the administration of iloprost or a pharmaceutically acceptable salt thereof or its stereoisomer is performed by a medically trained professional. In embodiments of administration in a decentralized setting, the physician is accessible via telehealth during the procedure to assess adverse events and vital signs.

[0079] In some embodiments, iloprost or a pharmaceutically acceptable salt thereof is administered to a subject at a concentration ranging from approximately 0.8 μg (approximately 0.8 μg / mL) to approximately 1.1 μg / mL per 1 mL of pharmaceutically acceptable carrier. In some embodiments, iloprost or a pharmaceutically acceptable salt thereof is administered to a subject at a concentration of approximately 0.8 μg / mL, approximately 0.9 μg / mL, approximately 1.0 μg / mL, or approximately 1.1 μg / mL.

[0080] In some embodiments, a concentrated solution of iloprost or a pharmaceutically acceptable salt thereof is diluted before therapeutic use. In some embodiments, dilution is performed using a suitable IV fluid or a suitable sterile injection solution. In some embodiments, dilution is performed using 0.9% sodium chloride injection (USP).

[0081] In some embodiments, 1 mL of a composition containing 100 μg of iloprost or a pharmaceutically acceptable salt thereof per mL is diluted with 99 mL of 0.9% sodium chloride solution. In some embodiments, 1 mL of 100 μg iloprost / mL is transferred to a 100 mL 0.9% sodium chloride solution infusion bag. In some embodiments, 1 mL of 100 μg iloprost / mL is transferred to a commercially available infusion bag labeled to contain 100 mL of 0.9% sodium chloride solution (this includes overfilling, and the actual volume is greater than 100 mL).

[0082] This disclosure also relates to methods for treating frostbite. In some embodiments, the frostbite is severe frostbite. In some embodiments, the frostbite is grade 3 or grade 4 frostbite.

[0083] In some embodiments, the frostbitten subject has at least one finger (or toe) affected by frostbite. In some embodiments, the frostbitten subject has at least one finger (or toe) having frostbite stage 3 (lesions extending slightly beyond the proximal phalanx) or stage 4 (lesions extending proximal to the metacarpal or metatarsal joint).

[0084] This disclosure also relates to a method for treating frostbite to reduce the risk of finger amputation.

[0085] This disclosure also relates to administering iloprost or a pharmaceutically acceptable salt thereof or a stereoisomer thereof to a frostbitten subject by intravenous injection, subcutaneous injection, intravenous infusion, or subcutaneous injection. In some embodiments, administration is by intravenous infusion or intravenous injection. In some embodiments, administration is by continuous intravenous infusion.

[0086] In some embodiments, the advantage of continuous infusion of iloprost or its pharmaceutically acceptable salts or stereoisomers is its higher bioavailability compared to other routes of administration. For example, iloprost (a synthetic analog of prostacyclin (PGI2)) has poor oral bioavailability and tolerability, making oral administration impractical.

[0087] In some embodiments, another advantage of continuous infusion of iloprost or its pharmaceutically acceptable salts or stereoisomers is that the iloprost compositions for infusion disclosed herein are stable and enable home infusion and dispersed infusion.

[0088] In some embodiments, another advantage of continuous infusion of iloprost or its pharmaceutically acceptable salts or stereoisomers is iloprost's ability to act as a potent prostacyclin (IP-) receptor agonist. In some embodiments, iloprost or its pharmaceutically acceptable salts or stereoisomers increase cyclic AMP concentration in the relevant cells, thereby having effects as a vasodilator, or as an anti-vasoconstrictor, anti-fibrotic, anti-platelet, and / or anti-inflammatory agent. In some embodiments, once iloprost or its pharmaceutically acceptable salts or stereoisomers reach the site of action (skin circulation in the fingers), the infusion therapy will inhibit platelet aggregation.

[0089] As used herein, the treatment period refers to the period during which a subject is administered iloprost or a pharmaceutically acceptable salt or stereoisomer thereof for the treatment of frostbite. In some embodiments, iloprost or a pharmaceutically acceptable salt or stereoisomer thereof is administered once daily for 3 to 10 consecutive days (i.e., the treatment period is 3 to 10 days). In some embodiments, iloprost or a pharmaceutically acceptable salt or stereoisomer thereof is administered once daily for 3 to 8 consecutive days. In some embodiments, iloprost or a pharmaceutically acceptable salt or stereoisomer thereof is administered once daily for up to 8 consecutive days.

[0090] In some embodiments, iloprost or a pharmaceutically acceptable salt or stereoisomer thereof is administered once daily by intravenous injection or infusion over a period of about 4 to 8 hours. In some embodiments, iloprost or a pharmaceutically acceptable salt or stereoisomer thereof is administered once daily by intravenous injection or infusion over a period of about 4, 5, 6, 7, or 8 hours. In some embodiments, iloprost or a pharmaceutically acceptable salt or stereoisomer thereof is administered once daily by intravenous injection or infusion over a period of about 6 hours.

[0091] In some embodiments, administration is continuous unless it is necessary to discontinue administration due to an adverse event. In some embodiments, the daily continuous administration of iloprost or its pharmaceutically acceptable salt or stereoisomer is about 4 hours, about 5 hours, about 6 hours, about 7 hours, or about 8 hours. In some embodiments, the daily continuous administration of iloprost or its pharmaceutically acceptable salt or stereoisomer is about 6 hours. In some embodiments, the daily continuous administration of iloprost or its pharmaceutically acceptable salt or stereoisomer is about 6 hours, and the treatment period is up to 8 consecutive days. In some embodiments, the continuous administration is by intravenous injection or infusion.

[0092] In some embodiments, the continuous administration of iloprost or a pharmaceutically acceptable salt or stereoisomer thereof is once daily for about 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours. In some embodiments, the continuous administration of iloprost or a pharmaceutically acceptable salt or stereoisomer thereof is once daily for about 6 hours. In some embodiments, the continuous administration of iloprost or a pharmaceutically acceptable salt or stereoisomer thereof is once daily for about 6 hours, with a treatment period of up to 8 consecutive days.

[0093] In some embodiments, iloprost or a pharmaceutically acceptable salt or stereoisomer thereof is administered once daily by a continuous infusion over 6 hours. In some embodiments, iloprost or a pharmaceutically acceptable salt or stereoisomer thereof is administered once daily by a continuous infusion over 6 hours for up to 8 consecutive days.

[0094] In some embodiments, iloprost or its pharmaceutically acceptable salt or stereoisomer is administered at a rate or dose ranging from about 0.2 ng iloprost / kg body weight / min (ng / kg / min) to about 3.0 ng / kg / min. In some embodiments, iloprost or its pharmaceutically acceptable salt or stereoisomer is administered at a rate or dose ranging from about 0.25 ng / kg / min to about 2.0 ng / kg / min. In some embodiments, the rate or dose of injection or infusion is adjusted according to the patient's tolerance within the range of 0.25 to about 2.0 ng / kg / min. In some embodiments, the rate or dose of injection or infusion is adjusted according to the patient's tolerance within the range of 0.5 to about 2.0 ng / kg / min.

[0095] In some embodiments, the administration rate or dose is titrated on the first day of treatment (day 1). In some embodiments, the titration step is repeated on day 2 of treatment. In some embodiments, the titration step is repeated on day 3 of treatment. In some embodiments, treatment on day 4 is started at the highest tolerated dose from day 3. During treatment with iloprost, the dose or rate may be adjusted based on the patient's tolerance.

[0096] In some embodiments, on day 1 of treatment, administration of iloprost or a pharmaceutically acceptable salt or stereoisomer thereof is initiated at a rate or dose of approximately 0.25 ng / kg / min, approximately 0.3 ng / kg / min, approximately 0.4 ng / kg / min, approximately 0.5 ng / kg / min, approximately 0.6 ng / kg / min, or approximately 0.7 ng / kg / min (initial dose). In some embodiments, the initial dose is 0.5 ng / kg / min.

[0097] In some embodiments, if the subject does not have pre-existing Child-Pugh classification B or Child-Pugh classification C liver dysfunction, the starting dose is 0.5 ng / kg / min. In some embodiments, the starting dose is 0.5 ng / kg / min, but the subject has a 30 mL / min / m² dose. 2If a patient has renal impairment with an eGFR of less than 0.5 ng / kg / min and cannot tolerate the initial dose of approximately 0.5 ng / kg / min, the dose will be reduced to approximately 0.25 ng / kg / min at the start of the titration step.

[0098] In some embodiments, the starting dose is 0.25 ng / kg / min. In some embodiments, if the subject has Child-Pugh class B or Child-Pugh class C hepatic impairment, the starting dose is 0.25 ng / kg / min.

[0099] In some embodiments, the rate or dose of administration is gradually increased during titration at intervals of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes until the maximum dose is reached. In some embodiments, the rate or dose of administration is gradually increased at intervals of 30 minutes during titration until the maximum dose is reached.

[0100] In some embodiments, the rate or dose of administration is gradually increased during titration by approximately 0.1 ng / kg / min, 0.2 ng / kg / min, 0.25 ng / kg / min, 0.3 ng / kg / min, 0.4 ng / kg / min, 0.5 ng / kg / min, 0.6 ng / kg / min, 0.7 ng / kg / min, or 0.8 ng / kg / min until the maximum dose is reached. In some embodiments, the rate or dose of administration is gradually increased during titration by approximately 0.5 ng / kg / min. In some embodiments, the rate or dose of administration is gradually increased during titration by approximately 0.25 ng / kg / min.

[0101] In some embodiments, the maximum dose is predetermined. In some embodiments, the predetermined maximum dose is approximately 1.8 ng / kg / min, approximately 2.0 ng / kg / min, approximately 2.2 ng / kg / min, approximately 2.4 ng / kg / min, approximately 2.6 ng / kg / min, approximately 2.8 ng / kg / min, or approximately 3.0 ng / kg / min. In some embodiments, the predetermined maximum dose is approximately 2.0 ng / kg / min.

[0102] In some embodiments, the maximum dose is approximately 2.0 ng / kg / min, or the highest dose tolerable by the subject between 0.25 ng / kg / min and 2.0 ng / kg / min.

[0103] In some embodiments, once the maximum dose is reached on the titration days (i.e., day 1, day 2, and / or day 3), the maximum dose is maintained for the remainder of the treatment on that day.

[0104] In some embodiments, the rate or dose of administration is increased by 0.5 ng / kg / min (in increments of 0.5 ng / kg / min) approximately every 30 minutes up to 2.0 ng / kg / min in order to determine the maximum dose on the first day of treatment (Day 1). In some embodiments, the titration step of Day 1 is repeated on Days 2 and 3 of treatment. In some embodiments, from Day 4 onward, treatment is started at the maximum dose tolerated by the subject on Day 3. In some embodiments, the rate or dose of injection or infusion can be adjusted by the administering healthcare professional at any point during the treatment.

[0105] In some embodiments, titration includes dose reduction if the subject has a dose-limiting reaction. In some embodiments, the dose-limiting reaction is headache, flushing, jaw pain, muscle pain, nausea, or vomiting.

[0106] In some embodiments, titration includes reducing the dose if the subject experiences an adverse event. In some embodiments, the adverse event is headache, flushing, palpitations / tachycardia, nausea, vomiting, dizziness, or hypotension.

[0107] In some embodiments, if administration of iloprost or a pharmaceutically acceptable salt thereof or its stereoisomer is discontinued due to a dose-limiting adverse event, administration may be resumed at a previously tolerated rate or dose once the adverse event or dose-limiting reaction has resolved.

[0108] In embodiments in which iloprost or a pharmaceutically acceptable salt thereof or its stereoisomer is administered to a patient with frostbite, administration is temporarily interrupted if the patient experiences symptomatic hypotension, systolic blood pressure below 80 mmHg, an unbearable adverse event (e.g., vomiting), or a drop in systolic blood pressure of more than 10 mmHg from the patient's pre-infusion measurement. In some embodiments, if the patient's systolic blood pressure drops more than 10 mmHg from the patient's pre-infusion measurement, the physician must decide whether to resume infusion after correcting the hypotension. In some embodiments, symptomatic hypotension is a drop in blood pressure accompanied by symptoms (e.g., dizziness, lightheadedness, fainting). In embodiments in which the treatment is temporarily interrupted, the physician decides whether to resume the treatment after correcting the hypotension.

[0109] In some embodiments, iloprost or a pharmaceutically acceptable salt thereof or its stereoisomer is administered to a frostbitten patient via a peripheral line or peripherally inserted central venous catheter using an infusion pump (or infusion pump). In the infusion administration embodiment, an in-line 0.22-micron or 0.2-micron filter is used. In the infusion administration embodiment, the infusion pump should be capable of delivering fluid at a rate of 0.1 to 99.99 mL / hour. In the infusion administration embodiment, the infusion pump should be able to adjust the infusion rate in increments of 0.1 mL / hour. In the infusion administration embodiment, the infusion pump should have an accuracy of within ±5.0% of the programmed rate. In the infusion administration embodiment, the infusion pump should be positive-pressure driven (continuous or pulsating).In some embodiments, the reservoir or injection line set is made of polytetrafluoroethylene, fluorinated ethylene propylene, polyvinylidene fluoride, polyether urethane, polycarbonate urethane, urethane, polyurethane, polyolefin, polyethylene, polypropylene, ethylene polymer, ethylene vinyl acetate, ethylene / acrylic acid copolymer, ethylene / vinyl alcohol copolymer. Polyimide, polyether ether ketone, polyaryl ether ketone, polysulfone, parylene, parilast, polyethylene terephthalate, polyethylene oxide, silicone, polyester; polyolefin, polyamide, polycaprolactam, polyvinyl chloride, polyacrylate, polymethacrylate; polyurea, polyvinylhalide, polyvinylidenehalide, polyvinyl ether, polyvinylaromatics, polyvinyl ester, alkyd resin, polysiloxane, epoxy resin, polyvinyl methyl ether, polyvinyl alcohol, acrylic polymers and copolymers, polyacrylonitrile, polystyrene copolymers of vinyl monomers with olefins and vinyl monomers The materials include olefins, styrene-acrylonitrile copolymers, ethylene methyl methacrylate copolymers, ethylene vinyl acetate, polyethers, rayons, cellulosines, cellulose acetate; cellulose nitrate, cellulose propionate, or any derivatives, analogs, homologues, salts, copolymers, or combinations thereof. In some embodiments, the reservoir or injection line set may be made from polyvinyl chloride, polypropylene, silicone, ethyl vinyl acetate, copolyester ethers, polyolefins, or combinations thereof. In some embodiments, the reservoir and injection line set may be made from polyvinyl chloride.

[0110] In some embodiments, the dose infusion rate (including approximately 0.8 μg / mL to approximately 1.1 μg / mL) when administering approximately 1 μg / mL of iloprost or a pharmaceutically acceptable salt thereof can be calculated as follows:

number

[0111] In some embodiments, the method of the present invention reduces the risk of amputation by at least 10% compared to standard of care treatment for frostbite. In some embodiments, the method of the present invention reduces the risk of amputation by at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% compared to standard of care treatment for frostbite. In some embodiments, the method of the present invention reduces the risk of amputation by at least 30% compared to standard of care treatment for frostbite.

[0112] In some embodiments, the standard treatment for frostbite includes rewarming as the first-line treatment. In some embodiments, rewarming is performed in conjunction with the administration of aspirin and / or ibuprofen. In some embodiments, the standard treatment for frostbite includes the administration of thrombolytic therapy. In some embodiments, the standard treatment for frostbite includes the administration of tissue plasminogen activator (tPA). In some embodiments, the standard treatment for frostbite includes the administration of buflomedil.

[0113] In some embodiments, the method of the present invention provides a safety profile in subjects consistent with the safety profile observed in multicenter, double-blind, randomized, placebo-controlled studies of continuous iloprost infusion in subjects with systemic sclerosis (systemic sclerosis) experiencing symptomatic finger ischemic episodes. In some embodiments, the method of the present invention provides a safety profile in subjects consistent with the safety profile observed in Example 2.

[0114] This disclosure also relates to administering iloprost or a pharmaceutically acceptable salt thereof or its stereoisomer to a patient with frostbite, and the following steps: i) Transferring approximately 1 mL of a concentrated solution of iloprost or a pharmaceutically acceptable salt thereof at approximately 100 μg / mL to approximately 100 mL of a 0.9% sodium chloride solution to provide an infusion solution of iloprost or a pharmaceutically acceptable salt thereof; and ii) The step of administering the injection solution to a frostbite patient by injection. This includes iloprost or a pharmaceutically acceptable salt thereof. In some embodiments, a concentrated solution of iloprost or a pharmaceutically acceptable salt thereof is contained in a single-dose or single-use vial in an amount of about 1 mL. In some embodiments, the single-dose or single-use vial is a glass vial. [Examples]

[0115] While the present disclosure has been outlined herein, it will be more readily understood by referring to the following examples. These examples are intended solely to illustrate specific aspects and embodiments of the present invention and are not intended to limit the invention.

[0116] Example 1. Efficacy of iloprost in the treatment of frostbite Frostbite is damage caused by exposure of tissue to freezing temperatures. Severe frostbite can lead to amputation and a significant morbidity rate. The objective was to identify and integrate published evidence on historical amputation rates at the patient level in adults with severe frostbite treated with iloprost and without iloprost, through a systematic literature review and meta-analysis (MA).

[0117] Methods: A literature review was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. This identified English-language publications from January 1, 2000 to July 31, 2002, reporting amputation rates associated with iloprost and standard of care (SoC) treatment in severe frostbite. Study designs included clinical trials (randomized and single-arm), observational studies, and case series involving at least 10 patients. A meta-analysis (MA) was performed on the proportion of patients with amputation. The published studies considered for the MA included:

[0118] A retrospective review of 199 patients with severe (grade 3-4) frostbite who were admitted to a Level 1 trauma center in Colorado between 2015 and 2020 reported an amputation rate of 49.8% in the overall population (n=199) and 59.8% in patients who did not receive thrombolytic therapy (n=127) (Carmichael et al., 2022).

[0119] A prospective observational study of 311 patients with frostbite at a Level 1 trauma center in Minnesota between 2014 and 2019 reported that 30.3% of 165 patients with severe frostbite, defined as post-rewarming perfusion impairment on imaging, required amputation (Lacey et al., 2021). Thrombolytic therapy (tPA) was used in 75% of these patients. In the subset that did not receive tPA, the salvage rate was 53.4%.

[0120] A retrospective chart review study was conducted on 70 patients admitted to Chamonix Hospital in France between 1985 and 1999 due to severe frostbite. The calculated risk of amputation in these patients was reported to be 31%–67% for grade 3 frostbite (based on the extent of lesions to the middle or proximal phalanges) and 98%–100% for grade 4 frostbite (Cauchy et al., 2001).

[0121] A retrospective review of 10 patients with deep frostbite treated at a certified burn center in Chicago, Illinois between November 2013 and March 2014 found that 9 patients (90%) required amputation, including partial or complete finger amputation or more proximal limb amputation (Shenaq et al., 2019).

[0122] A retrospective review identified nine patients who suffered severe frostbite and received non-thrombolytic management at the University of Michigan Health System (Ann Arbor, Michigan) between 2000 and 2017 (Patel et al., 2017). In this group, all patients (100%) had at least one finger amputated (the mean number of amputated fingers per patient was 8.6; range: minimum 4 to maximum 14).

[0123] A prospectively maintained database from a single urban burn and trauma center in Minneapolis, Minnesota, identified 73 patients with severe frostbite injuries between 2006 and 2014 (Nygaard et al., 2017). Overall, 32 patients (44%) required amputation. In the group receiving SoC treatment without thrombolytic therapy, 16 out of 28 patients (50%) required amputation.

[0124] Between 1995 and 2006, 26 patients with severe frostbite were treated at a single burn center in Utah (Bruen et al., 2007). Bruen et al. reported that 41% of the affected fingers (97 out of 243 injured fingers) required amputation.

[0125] In a historical control group (based on past data) at a burn center in Minnesota (1985-1989), Twomey et al., 2005, reported that half (50%) of patients treated with standard care (8 out of 16 patients) required amputation.

[0126] Determining Amputation Risk: Table 1 discloses historical amputation rates in patients with severe frostbite. The studies listed in Table 1 formed the basis for a meta-analysis (MA) to determine historical amputation risk. Significant variability was observed in the treatments included as standard of care (SoC). In the United States, SoC treatment for severe frostbite is most likely to be best represented by a group of studies with “Severe,” “Deep,” or “Grade 3-4” (or equivalent clear definitions) frostbite, with the exception of studies using alprostadil. Therefore, only such studies (n=8) were included in the meta-analysis as the “base case” scenario.

[0127] The two studies with the largest outliers, both having below-average amputation rates, were Cauchy 2000 (23%) and Rogers 2022 (29%). Cauchy 2000 was likely affected by the unknown proportion of patients who received iloprost and / or tPA, the use of a different frostbite severity assessment system (compared to other studies), and the unknown proportion of grade 3 and 4 frostbite. For Rogers 2022, the low rate may be partly attributable to the extent of thrombolytic therapy, with nearly 80% of patients receiving tPA and the time to thrombolytic therapy in the final year of patient enrollment being 6.6 hours.

[0128] [Table 1] TIFF2026524951000005.tif210170TIFF2026524951000006.tif139169

[0129] A literature review identified 17 major studies; of which 16 were deemed eligible for a proportion meta-analysis (MA). Results from pooled MA arm-level data for standard of care (SoC) estimated a 55% risk (95% confidence interval [CI]: 38%–71%; Figure 1; RE = random effects) of patients undergoing at least one amputation. Tests for heterogeneity were performed. 2 The results showed a high degree of heterogeneity, with a percentage of 93.8% and a p-value of <0.0001.

[0130] Efficacy - Amputation rate: A systematic literature review (SLR) identified two studies reporting the amputation rate after iloprost (+SoC) treatment for frostbite ((Cauchy et al., 2011; Cheguillaume, B. (2011). Controlled trial of iloprost and iloprost and rt-PA in the treatment of severe frostbite. Presented for the Award of Doctoral Degree in Medicine (Saint-Martin-d'Heres, France); Poole et al., (2021). Management of severe frostbite with iloprost, alteplase and heparin. A Yukon case series. CMAJ open 9, E585-E591)). One of these studies reported amputation in 0 out of 16 patients treated with iloprost (+SoC) and in 3 out of 16 patients (19%) treated with iloprost + rtPA (Cauchy et al., 2011; Cheguillaume, 2011), while the other reported amputation in 4 out of 12 patients (33%) treated with iloprost (+SoC, with or without alteplase and heparin) (Poole et al., 2021).

[0131] The sensitivity analysis reflects several methods for deriving data (amputation rates for grade 3-4 frostbite) from the supplementary table in Cauchy 2011 (Cauchy et al., 2011). The first consideration was whether to include or exclude patients treated with iloprost + rtPA who were predicted to require amputation based on bone scans but were lost to follow-up. The second consideration was whether to combine the iloprost + rtPA group with the iloprost monotherapy group, or to consider iloprost monotherapy only.

[0132] Results from pooled arm-level data for iloprost analysis showed that the proportion of amputees was 15% (95% CI: 0-45%), indicating high heterogeneity (I 2 =78.1%; Qp value = 0.0325; Figure 2; RE = Random Effects) was observed. All other scenarios also provided similar results for the estimated proportion of amputees (15.4% to 18.0% for different scenarios), with a large error bar for this estimate and high heterogeneity (I 2 The percentages for different scenarios ranged from 63.1% to 78.1%.

[0133] Efficacy - Proportion of fingers with amputation: Data from two studies reporting the proportion of fingers with amputation after iloprost or standard of care (SoC) for grade 3 / 4 frostbite were included in the meta-analysis. Cauchy 2011 reported 0 amputations in 78 fingers treated with iloprost and 40 amputations in 75 fingers treated with SoC (53.3%; Cauchy et al., 2011; Cheguillaume, 2011). When data from cases treated with iloprost alone and iloprost + tPA were combined in Cauchy 2011 (n=177), the number of finger amputations increased from 0 to 3 (1.3%).

[0134] Crooks 2022 reported amputations in 62 out of 198 fingers (31.1%) treated with iloprost, compared to 104 out of 185 fingers (56.2%) treated with SoC (Crooks et al., 2022). The exact number of fingers treated with tPA is not specified, but it has been reported that only a small number of patients who received iloprost (5 out of 26) also received tPA, and subgroup analysis showed that these patients were more likely to experience amputations compared to patients who received iloprost alone (p<0.05). As mentioned above, there was a disparity between the two groups in the proportion of fingers with grade 3 versus grade 4 frostbite; of grade 3-4 frostbite, grade 4 frostbite occurred in only 23% (43 / 185) of fingers treated with SoC compared to 48% (96 / 198) of those treated with iloprost. To address this imbalance, stratified odds ratios and proportions were calculated for each study.

[0135] When the two studies were meta-analyzed, the odds ratio (OR) in the base case was 0.047 (95% CI: 0.002~0.966; p=0.047; Figure 3; RE = random effects). The results were similar when iloprost + tPA patients from Cauchy 2011 (Cauchy et al., 2011; Cheguillaume, 2011) were added (OR: 0.034, 95% CI: 0.001, 1.12; p=0.057; Figure 4; RE = random effects). Heterogeneity was extremely high in both analyses: for the base case, I 2 =82.4%, and for both studies, regarding the analysis including patients who received iloprost + tPA, I 2=90.3%. This reflects the significant differences in odds ratios between studies. While the individual odds ratios in each study demonstrate a statistically significantly higher risk of finger amputation with SoC compared to iloprost, the high between-study variance and large within-study variance for Cauchy 2011 contribute to a broad 95% CI. When these large variances are combined, the pooled analysis does not show the strong significance between treatments found in the individual studies. It should also be noted that the models employed make the general assumption that they follow a symmetric and normally distributed distribution as long as fundamental heterogeneity exists. These 95% confidence intervals are based on such normality assumptions and should therefore be interpreted cautiously in the presence of such heterogeneity.

[0136] In the meta-analysis, unadjusted (naive) indirect comparisons consistently showed iloprost to be superior to SoC by 36%–49% across various scenarios, and a 39% superiority was observed in the base case results (including studies where there was confidence that all patients had severe frostbite and were not administered alprostadil) [95% CI: 6–73%, p=0.02]. In other words, the comparison between iloprost and SoC showed a 39% reduction in amputation rates with iloprost.

[0137] Three iloprost studies reported safety data. Safety events associated with iloprost were generally mild and included headache, hot flashes / flushing, palpitations / tachycardia, and nausea and vomiting. These were managed symptomatically or by reducing the iloprost dose and rarely led to discontinuation of iloprost.

[0138] Conclusion: In our literature review of the treatment of severe frostbite with iloprost or standard treatment (SoC), we found that SoC resulted in a significantly higher amputation rate, while iloprost treatment showed a significantly lower amputation rate.

[0139] Example 2. Evaluation of intravenous iloprost in subjects with symptomatic Raynaud's phenomenon secondary to systemic sclerosis (SSc): A multicenter, double-blind, randomized, placebo-controlled trial - Safety evaluation of iloprost This was a multicenter, double-blind, randomized, placebo-controlled trial to provide an initial evaluation of the effect of iloprost on symptom relief of RP seizures in subjects with SSc.

[0140] Participants were randomly assigned in a 1:1 ratio to receive either iloprost injection (IV administration) or placebo (randomization). Randomization was stratified based on the use of phosphodiesterase inhibitors at the time of screening. Administration of the study drug was initiated on day 1 and administered intravenously (IV) over 6 hours per day via a peripheral line (NovaCath® Integrated IV Catheter System) or a peripherally inserted central venous catheter using an infusion pump for 5 consecutive days (e.g., Monday to Friday).

[0141] Subjects must have a systolic blood pressure of ≥85 mmHg (sitting) before administration of the investigational drug on each administration day. On day 1, the investigational drug was started at an initial dose of 0.5 ng / kg / min and increased by 0.5 ng / kg / min every 30 minutes (±5 minutes) until the subject reached 2.0 ng / kg / min or their individual tolerated dose. If a subject experienced an unacceptable dose-limiting adverse event (e.g., headache, flushing, jaw pain, muscle pain, nausea, or vomiting) or symptomatic hypotension, the dose was gradually reduced by 0.5 ng / kg / min every 30 minutes (±5 minutes) until a tolerated dose was determined, or the infusion was interrupted until the symptoms resolved, at which point the investigational drug was resumed at the previous tolerated dose. If symptomatic hypotension or a dose-limiting adverse event occurred during administration of iloprost at the initial dose (i.e., 0.5 ng / kg / min), the infusion was reduced to 0.25 ng / kg / min. If a dose of 0.25 ng / kg / min was unacceptable due to symptomatic hypotension, or if a dose-limiting adverse event occurred, the investigational drug could be discontinued, and an attempt could be made to resume infusion after the event resolved or was treated. Blood pressure and heart rate were monitored 15 minutes (±5 minutes) before and after all dose changes. The maximum tolerated dose was maintained for the remainder of the daily 6-hour infusion period. Infusion was stopped at the end of the 6-hour infusion period. Subjects were monitored up to 1 hour after completion of the investigational drug infusion (i.e., vital signs were measured 15 minutes [±5 minutes] and 1 hour [±15 minutes] after completion of the infusion).

[0142] From day 2 to day 5, infusions were initiated using the highest infusion rate tolerated the previous day, without dose increases or decreases (up-titration or down-titration), unless the subject was unable to tolerate the infusion or an adverse event occurred requiring dose reduction due to the subject's intolerance. Vital signs were measured before administration of the investigational drug and 15 minutes (±5 minutes) before and after all dose changes during the infusion. In addition, vital signs were monitored 15 minutes (±5 minutes) and 1 hour (±15 minutes) after the completion of the 6-hour infusion.

[0143] During the treatment period (days 1-5), the electronic patient-reported outcome (ePRO) diary was not completed while the subjects were receiving the investigational drug. To allow subjects to rest after the 5-day infusion and return to their normal daily living activity schedule, no study evaluations were conducted for two days after the end of the treatment (days 6 and 7 [i.e., Saturday and Sunday]).

[0144] On day 8, the subject was contacted by phone to confirm that they had resumed filling out their daily ePRO diary; the subject kept the ePRO diary from day 8 to day 21. On day 22, the subject returned to the clinic for post-treatment evaluation. A follow-up visit was conducted 30 days (day 35) after the last dose of the investigational drug.

[0145] Participants who discontinued the investigational drug early (unless they withdrew their consent) remained in the trial and completed daily ePRO diaries from day 8 to day 21, which included clinical laboratory evaluations for the remaining missing infusion days and post-treatment trial evaluations.

[0146] The total duration of the study per participant was approximately 9 weeks at most.

[0147] Number of subjects Randomization - 34 subjects; Completion - 34 subjects

[0148] Diagnostic and primary inclusion criteria The population for this study includes male and female subjects aged 18 years or older who meet the following criteria: having a diagnosis of systemic sclerosis (SSc) as defined by the 2013 American College of Rheumatology / European League Against Rheumatism criteria; having a self-reported or physician-reported diagnosis or history of Raynaud's phenomenon (RP), with at least two steps of color change in the fingers, pallor, cyanosis, and / or reactive hyperemia, in response to cold exposure or emotions; and having at least 10 symptomatic RP attacks recorded in an ePRO diary, occurring on at least three separate days during a 5-day eligibility period.

[0149] Information on the investigational drug and the control drug Iloprost injection (for intravenous administration) and the corresponding placebo were supplied in vials packaged in blinded investigational drug kits (10 vials per kit). The iloprost vials and placebo vials were identical except that 100 μg of iloprost was added to the active investigational drug vial. Before use, the drugs were diluted with 0.9% sodium chloride in a drug reservoir (IV bag).

[0150] Safety parameters included adverse events, physical examination findings, vital signs (heart rate and blood pressure), 12-lead electrocardiogram (ECG) findings, and standard clinical laboratory measurements (chemical and hematological tests).

[0151] Summary of Safety Results : In this study, no subjects experienced serious adverse events (SAEs), adverse events of special interest (AESIs), treatment-emergent adverse events (TEAEs) that led to discontinuation of the investigational drug, or TEAEs that resulted in death.

[0152] Overall, 31 subjects (91.2%) experienced a therapeutic adverse event (TEAE) while receiving the investigational drug: 14 subjects (82.4%) in the placebo group and 17 subjects (100.0%) in the iloprost group. The majority of TEAEs were judged to be mild to moderate in severity. Two subjects (11.8%) in the iloprost group experienced a TEAE that was judged to be severe by the principal investigator.

[0153] The most frequently reported TEAEs (based on the total number of subjects who experienced these TEAEs) were headache (21 subjects in total [61.8%]: 5 subjects in the placebo group [29.4%] and 16 subjects in the iloprost group [94.1%]), nausea (14 subjects in total [41.2%]: 3 subjects in the placebo group [17.6%] and 11 subjects in the iloprost group [64.7%]), abdominal pain (5 subjects in total [14.7%]: 1 subject in the placebo group [5.9%] and 4 subjects in the iloprost group [23.5%]), and flushing (5 subjects in total [14.7%]: 0 subjects in the placebo group [0.0%] and 5 subjects in the iloprost group [29.4%]).

[0154] During the study, there were no clinically significant changes in chemical or blood test values.

[0155] In this study, there were no TEAEs related to vital signs, physical examination findings, or ECG.

[0156] No hypertensive or hypotensive events were reported in this study. No bradycardia or tachycardia were reported in this study.

[0157] conclusion : The results confirmed that the iloprost dosing titration algorithm could be safely used in subjects. All investigational drug-related adverse events assessed by the principal investigator were expected and consistent with the known safety profile of iloprost. There were no deaths, SAEs, AESIs, or TEAEs leading to discontinuation of the investigational drug during this study.

[0158] Example 3. Solution stability of fully diluted ready-to-use iloprost Iloprost 100 μg / mL injection is administered to the patient over 6 hours using an intravenous (IV) infusion bag. The fully diluted and prepared iloprost is prepared by trained and qualified staff at the pharmacy according to sterile preparation standards (USP Chapter 19). <797> The preparation is carried out in a pharmacy cleanroom using ). Fully diluted prepared drugs can be prepared several hours before administration by storing them under ambient conditions, or several days before administration by storing them under refrigerated conditions (2-8°C). IV bags are prepared by filling an empty sterile IV bag with 99 mL of 0.9% NaCl and 1.0 mL of iloprost 100 μg / mL injection. The bag is then gently mixed by hand to obtain a homogeneous solution. The IV bags for administration were prepared and stored under several conditions, with periodic sampling to test their stability: i) 25°C / 60% relative humidity (RH), ii) 25°C / 60% RH after 5 days of storage at 5°C, and iii) 25°C / 60% RH after 8 days of storage at 5°C.

[0159] IV bag sample preparation Two 50 mL syringes with 18 gauge needles were assembled. One 1 mL syringe with 18 gauge needle was assembled. Using one of the 50 mL syringes, 50 mL of 0.9% NaCl was transferred to an empty IV bag (sterile, ICU Medical) using the infusion port. Using the second 50 mL syringe, 49 mL of 0.9% NaCl was transferred to the same IV bag using the infusion port, obtaining a total of 99 mL of solution. Using the 1 mL syringe, 1.0 mL of iloprost 100 μg / mL injection was transferred to the IV bag using the infusion port. The needles were removed and the ports were securely closed. The IV bag was gently inverted several times to mix. During inversion, the infusion port area was occasionally squeezed to ensure that the liquid was rinsed into the area several times.

[0160] Sampling procedure: For sampling, an 18-gauge needle was attached to a 3 mL syringe. Using the 3 mL syringe, 2 mL of the IV bag solution was withdrawn from the injection port. 1 mL was transferred to an HPLC vial for neat (stock) injection, and the remaining 1 mL was transferred to a backup HPLC vial. The HPLC vials were stored under refrigerated conditions (2-8°C).

[0161] Sample Storage and Sampling: For each test condition (i-iv above), 12 IV bag samples were prepared (6 IV bags for condition ia - see below for sampling times). For condition i), all 12 IV bags were placed in a stable chamber at 25°C / 60%RH, ensuring that each bag was placed with its maximum surface area side down. After the first time point, the IV bags were removed from the chamber and mixed by inverting each IV bag several times. Samples were taken from each IV bag for HPLC analysis. After sampling, the IV bags were returned to the stable chamber at 25°C / 60%RH, ensuring that each bag remained with its maximum surface area side down until the next sampling time. At each sampling time, the IV bags were removed from the chamber, mixed by inverting each IV bag several times, and then samples were taken.

[0162] For conditions ii) and iii), all 12 IV bags were placed in a stability chamber at 2–8°C, with each bag facing downwards towards its maximum surface area. After 5 days for condition ii), or 8 days for condition iii), the IV bags were removed from the chamber and allowed to equilibrate at room temperature. Each IV bag was mixed by inverting it several times. Samples were taken from each IV bag for HPLC analysis. Subsequently, the IV bags were placed in a stability chamber at 25°C / 60%RH, and samples were taken at the specific time points discussed herein. Sampling times for condition ia): 2 hours (h), 4 hours, 6 hours, 8 hours, and 24 hours Sampling times for conditions ib): 8 hours, 12 hours, 48 ​​hours, and 96 hours Sampling time for condition ii): 5 days (at the end of storage in a stable chamber at 2°C to 8°C), 5 days and 8 hours, and 5 days and 12 hours. Sampling times for condition iii): 8 days (at the end of storage in a stable chamber at 2°C to 8°C), 8 days and 8 hours, and 8 days and 12 hours.

[0163] Sample analysis The samples were analyzed by HPLC using a validated method (validation protocol not shown). TIFF2026524951000007.tif123169

[0164] The concentration of each sample is given by the following formula:

number

[0165] The initial percentage (percentage of initial concentration) of each stored sample was determined according to the following formula:

number

[0166] Table 1A shows the results of the IV bag stability test under condition ia (25°C / 60%RH stability; sampling times: 2 hours (h), 4 hours, 6 hours, 8 hours, and 24 hours). The % initial (percentage of initial concentration (%)) results for all IV bag preparations ranged from 94.2% to 94.9%. These results were as expected given the nature of the IV bag preparations, which were designed to mimic clinical procedures. After adjusting the IV bag volume to the target weight, the drug was spiked (added) using a disposable sterile 1 mL syringe with a disposable sterile 18 gauge needle. The combination of variability in IV bag weight and variability in syringe and needle volume accuracy was expected to affect the accuracy of the spike procedure. Each sample met the specification criteria of 90.0% to 110.0%, and each stored sample was also within 90.0% to 110.0% of the initial preparation results.

[0167] [Table 1A]

[0168] The results of the IV bag stability test under condition ib (25°C / 60%RH stability; sampling times: 8 hours, 12 hours, 48 ​​hours, and 96 hours) are shown in Tables 1B and 1C. Each sample met the specification criterion of 90.0–110.0%, and each stored sample was within 90.0–110.0% of the initial preparation result.

[0169] [Table 1B]

[0170] [Table 1C]

[0171] The results of the IV bag stability test under condition ii (5 days at 5°C followed by 25°C / 60%RH) are shown in Tables 1D and 1E. Each sample met the specification criteria of 90.0–110.0%, and each stored sample was within 90.0–110.0% of the initial preparation result.

[0172] [Table 1D]

[0173] [Table 1E]

[0174] The results of the IV bag stability test under condition iii (8 days at 5°C followed by 25°C / 60%RH) are shown in Tables 1F and 1G. Each sample met the specification criteria of 90.0–110.0%, and each stored sample was within 90.0–110.0% of the initial preparation result.

[0175] [Table 1F]

[0176] [Table 1G]

[0177] During the test, three unknown peaks were observed in the chromatogram; however, all of these unknown peaks were present in the control or stressed blank IV bag samples, indicating that all unknown peaks are related to the IV bag or saline solution and not caused by the iloprost drug. The unknown peaks did not interfere with the quantification of the iloprost peak, and therefore, the administration stability test is not considered to be affected by the presence of these peaks.

[0178] These results indicate that iloprost drugs exhibit adequate stability within IV bags, supporting the use of IV bags for administration and dosing designs.

[0179] Example 4. Study of the microbiological properties of an iloprost 100 μg / mL injectable composition. The purpose of this stability test is to provide data reflecting the microbiological quality of iloprost 100 μg / mL injectable composition and iloprost placebo injectable composition after preparation and after storage under controlled storage conditions for a specific period. The preparation and storage of these compositions simulate the preparation and storage conditions in a dispensing pharmacy prior to patient administration.

[0180] Samples of iloprost 100 μg / mL injectable and iloprost placebo injectable compositions were prepared (diluted in a saline IV bag), stored under controlled conditions, and tested at specific time points (day 0, day 10, and day 16). The prepared samples were evaluated in parallel with prepared samples (inoculated products) inoculated with a low level of bioburden (10–100 CFU / mL). This low level of bioburden is intended to simulate microbial contamination when a needle is inserted into the container closure. This microbial challenge test helps determine whether the diluted prepared products possess growth-promoting properties. The growth-promoting properties of these two pharmaceuticals are subject to USP standards. <51> It will be evaluated according to the guidance.

[0181] The microbial count of the inoculated product at 0 hours is compared to the microbial counts at 10 and 16 days. If the microbial counts at 10 and 16 days do not increase by more than 0.5 log 10 units compared to the baseline, the drug is considered non-growth-promoting, and its storage conditions (time and temperature) are considered appropriate to ensure the safety of the diluted prepared drug.

[0182] In-use stability studies are designed and conducted according to the following guidelines: Current United States Pharmacopeia General Chapter (USP) <51> "Antibacterial efficacy test" CDER guidance on subject matter by Metcalfe John W. (2009) CDER Microbiological Issues: A deep Dive by Candace Gomez-Broughton, “Aseptic Processing of Biological Products: Current Regulatory Issues”, August 2018 World Health Organization (WHO) Guidance on the Subject: WHO Technical Report Series, No. 863, 1996, Annex 5

[0183] The procedure outlined in this protocol Scope: This protocol applies to microbiological testing to confirm the microbiological stability of diluted, prepared iloprost 100 μg / mL injectable compositions and iloprost placebo injectable compositions. The stability of these two pharmaceuticals is evaluated after penetration of the container and closure system used for formulation preparation and storage under controlled conditions. The preparation and storage conditions of the two pharmaceuticals simulate preparation and storage prior to patient administration. This protocol describes a study design and bioburden test using representative samples of iloprost 100 μg / mL injectable compositions and iloprost placebo injectable compositions.

[0184] Interpretation of Results: The initial microbial count (CFU / mL) of each inoculated sample is defined as the microbial count of that sample at time zero and is determined by the filtration test method. The growth or absence of microbial growth for a specific sample / microbial combination at a given time is evaluated relative to time zero. The microbial count at time zero is determined for each microbial, and its logarithm (log10) is calculated. The microbial count of each test sample is measured at a specific time point, its logarithm (log10) is calculated, and it is compared to the logarithm (log10) value of that microbial count at time zero. If the difference between the two log10 values ​​is 0.5 log10 units or less, the sample does not show an increase in microbial growth.

[0185] Preparation of Iloprost 100 μg / mL Injectable Composition: Assemble two 50 mL syringes with 18 gauge needles. Using one 50 mL syringe, transfer 49 mL of 0.9% NaCl to an empty IV bag using the infusion port. Using the second 50 mL syringe, transfer 50 mL of 0.9% NaCl to the same IV bag using the infusion port. Using the other 1 mL syringe, transfer 1.0 mL of Iloprost 100 μg / mL Injectable Composition to an IV bag using the infusion port. Remove the needle and securely close the port. Gently invert the IV bag several times to mix. During inversion, occasionally interrupt and squeeze the infusion port area to allow the liquid to rinse into the area several times. Prepare a total of seven IV bags in this manner.

[0186] Microbial inoculation: Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Pseudomonas aeruginosa (P. aeruginosa), Candida albicans (C. albicans), Aspergillus brasiliensis (A. brasiliensis), and Escherichia coli (E. coli) are used. Frozen cultures from Patheon, freshly collected suspensions, or lyophilized microbial preparations with measured bacterial counts are used. Microorganisms from a master seed lot no more than five passages are used.

[0187] Test sample: Inoculate each IV bag and each IV bag of the positive control with the appropriate microbial suspension using an appropriately sized syringe and 18-gauge needle, so that each IV bag contains 5–10 CFU / mL (500–1000 CFU / IV bag total) after inoculation (inject through the infusion port). Gently invert each infusion bag several times to mix. Stop occasionally during inversion and squeeze the infusion port area to allow the liquid to rinse the area several times.

[0188] Inoculation verification (performed twice for each microorganism at time zero): Add 1 mL from each positive control sample to each of the two 100 × 15 mm petri dishes. Pour approximately 25 mL of Letheen agar medium, cooled to ≤45°C, into each of the two petri dishes and swirl to mix. Allow the agar to solidify, then invert the plates and incubate. Incubate bacterial plates at 30–35°C for 3–5 days. Incubate Candia albicans plates at 20–25°C for 3–5 days, and Aspergillus brasiliensis plates at 20–25°C for 3–7 days.

[0189] Sampling: On day 0, collect a sample immediately after preparing the IV bag. After storing the inoculated bags at 2-8°C for 10 and 16 days, collect the test sample. At the designated time points, assemble a 50 mL syringe fitted with an 18 gauge needle. Using the 50 mL syringe, withdraw 25 mL from each inoculated IV bag through the infusion port and transfer it to a sterile test tube of appropriate size with a cap. After collecting the test sample, leave the inoculated IV bags at 2-8°C until the next sampling time.

[0190] Test samples: Test the inoculated product sample, positive control, product negative control (iloprost and diluent), and diluent negative control. Test each sample in duplicate (using two filters). Pre-wash each of the two sterile filter membranes with 100 mL of diluent D. Add a 10 mL aliquot of the test sample (inoculated product, product negative control, positive control, or diluent negative control) to each filter unit and filter. Wash each filter three times (3 x 100 mL) with a 100 mL aliquot of diluent D. Aseptically transfer the two filters to two separate Letheen agar plates. Allow the agar to solidify and incubate as follows: a. Inoculated product samples and positive control samples containing bacteria are incubated at 30-35°C for 3-5 days. b. Inoculated product samples and positive control samples containing Candida albicans were incubated at 20-25°C for 3-5 days. c. Inoculated product samples and positive control samples containing Aspergillus brasiliensis are incubated at 20-25°C for 3-7 days. d. Product negative control and diluent negative control: From each set, one plate should be incubated at 30–35°C and the other plate at 20–25°C for the same duration as the test sample but not exceeding 7 days.

[0191] Rinse Fluid Negative Control: Add 100 mL of Diluent D to each of the two sterile filter units and filter. Aseptically transfer the two filters to two Letheen agar plates. Incubate one plate at 30–35°C and the other at 20–25°C for the same duration as the test sample but not exceeding 7 days.

[0192] Agar negative control: Incubate one Letheen agar plate at 30-35°C and the other plate at 20-25°C for the same duration as the test sample but not exceeding 7 days.

[0193] Analysis: Following SOP-QC-280, count the colony-forming units (CFUs) on each plate and calculate the arithmetic mean for each set of two plates. Calculate the recovery rate by dividing the average count (CFU) of the product plate by the average count (CFU) of the positive control plate. Multiply by 100 and round to the nearest integer.

number

[0194] Acceptance Criteria: The recovery rate of the product test plates should be at least 50% of the mean of the positive control at time zero. If a lower recovery rate (less than 50%) is observed, modify the method to overcome the inhibition by utilizing one or more of the following: Incorporate the neutralizing agent / dispersant into the plating medium or rinsing solution. Using different culture media

[0195] Each inoculated bacterial validation plate must contain at least 1 CFU and no more than 100 CFU (≥1 CFU and ≤100 CFU).

[0196] Growth should not occur on the agar medium or rinse solution negative control. If there is a problem with the medium or rinse solution negative control, an investigation is required to evaluate its effect on the tests described herein.

[0197] result : Suitability of the Microbial Enumeration Test Method. The suitability of the microbial enumeration test method was evaluated by comparing the microbial recovery rate from the diluted prepared product at time zero with the microbial recovery rate from the dilution (positive control). This method was considered suitable if it met the following criteria: 1. Each inoculated bacterial verification plate must have a concentration of ≥1 CFU and ≤100 CFU. 2. No growth should occur in the product-negative control, agar-negative control, or rinse solution-negative control. 3. The microbial recovery rate from the diluted prepared product is at least 50% of the recovery rate from the diluted solution (positive control).

[0198] Each inoculum validation plate (Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Pseudomonas aeruginosa (P. aeruginosa), Candida albicans (C. albicans), Aspergillus brasiliensis (A. brasiliensis), and Escherichia coli (E. coli)) count met the acceptable criteria: ≥1 CFU and ≤100 CFU. None of the media, dilutions, or dilution D negative controls showed growth. None of the product negative controls showed growth.

[0199] The recovery rates for Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Pseudomonas aeruginosa (P. aeruginosa), Candida albicans (C. albicans), Aspergillus brasiliensis (A. brasiliensis), and Escherichia coli (E. coli) from iloprost 100 μg / mL injection composition and iloprost placebo injection composition samples were all over 50% of the recovery rate of the positive control.

[0200] Evaluation of the growth-promoting properties of iloprost 100 μg / mL injectable composition and iloprost placebo injectable composition samples. The number of microorganisms was measured for each sample / microorganism combination at each time point, and the logarithm (log10) of the mean number of microorganisms was calculated. The difference between the logarithmic (log10) values ​​(time X and time zero) was used to evaluate whether the product was growth-promoting or not. When evaluated by this test, a product was considered non-growth-promoting if it met the following criteria: 1. USP <51> In accordance with the regulations, for each microorganism tested, the increase in the number of microorganisms compared to the number of microorganisms at time zero should not exceed 0.5log10. 2. No growth should occur in the product-negative control, agar-negative control, or rinse solution-negative control.

[0201] The results for each microorganism are summarized in Tables 2A to 2F. Logarithmic difference = Logarithmic (log10) value at time X - Logarithmic (log10) value at time 0

[0202] For each of the following organisms, the logarithmic difference (log10 difference) at each time point was not greater than 0.5 log10 units from time zero: Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Pseudomonas aeruginosa (P. aeruginosa), Candida albicans (C. albicans), Aspergillus brasiliensis (A. brasiliensis), and Escherichia coli (E. coli). The prepared iloprost 100 μg / mL injectable composition and the iloprost placebo injectable composition did not promote the growth of Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Pseudomonas aeruginosa (P. aeruginosa), Candida albicans (C. albicans), Aspergillus brasiliensis (A. brasiliensis), and Escherichia coli (E. coli) when stored at 2-80°C for up to 16 days.

[0203] The results shown in Tables 2A-2F indicate that the tested prepared iloprost 100 μg / mL injectable formulations and iloprost placebo injectable formulations showed no growth-promoting properties whatsoever against the tested microorganisms (Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Pseudomonas aeruginosa (P. aeruginosa), Candida albicans (C. albicans), Aspergillus brasiliensis (A. brasiliensis), and Escherichia coli (E. coli)). This test meets the acceptance criteria for evaluating the growth-promoting properties of both the iloprost 100 μg / mL injectable formulations and the iloprost placebo injectable formulations.

[0204] [Table 2A]

[0205] [Table 2B]

[0206] [Table 2C]

[0207] [Table 2D]

[0208] [Table 2E]

[0209] [Table 2F]

[0210] All publications, patents, patent applications, and disclosures of published patent applications referenced herein are incorporated herein by reference in their entirety.

[0211] In the event of any conflict between the cited references and this specification, this specification shall prevail. Certain terms are used for clarity when describing embodiments of this application. However, the present invention is not intended to be limited to such selected terms. Nothing in this specification should be considered to limit the scope of the present invention. All embodiments presented are representative and not limiting. The above embodiments may be modified or altered without departing from the invention as understood by those skilled in the art in light of the above teachings. Accordingly, it should be understood that within the scope of the claims and their equivalents, the present invention may be carried out in ways other than those specifically described.

Claims

1. A method for treating frostbite in a subject, comprising administering a composition containing iloprost or a pharmaceutically acceptable salt thereof at a concentration ranging from about 0.8 μg to about 1.1 μg / mL per 1 mL of pharmaceutically acceptable carrier.

2. The method according to claim 1, wherein the composition comprising iloprost or a pharmaceutically acceptable salt thereof has a concentration of about 0.9 μg / mL.

3. The method according to claim 1, wherein the composition comprising iloprost or a pharmaceutically acceptable salt thereof has a concentration of about 1 μg / mL.

4. The method according to any one of claims 1 to 3, wherein the administration is by intravenous injection or intravenous infusion.

5. The method according to any one of claims 1 to 4, wherein the administration is by continuous infusion.

6. The method according to any one of claims 1 to 5, wherein the administration is performed in one or more doses between approximately 0.2 ng / kg / min and approximately 3.0 ng / kg / min.

7. The method according to any one of claims 1 to 6, wherein the administration is performed in one or more doses between approximately 0.25 ng / kg / min and approximately 2.0 ng / kg / min.

8. The method according to any one of claims 1 to 7, wherein the administration includes a titration step on the first day of administration.

9. The method according to claim 8, wherein the administration includes a titration step on the second day of administration.

10. The method according to claim 9, wherein the administration includes a titration step on the third day of administration.

11. The method according to any one of claims 8 to 10, wherein the titration step comprises administering the composition at an initial dose of about 0.25 ng / kg / min, about 0.3 ng / kg / min, about 0.4 ng / kg / min, about 0.5 ng / kg / min, about 0.6 ng / kg / min, or about 0.7 ng / kg / min.

12. The method according to any one of claims 8 to 11, wherein the titration step comprises administering the composition at a starting dose of about 0.5 ng / kg / min.

13. The method according to claim 12, wherein the subject does not have existing Child-Pugh classification B or Child-Pugh classification C liver dysfunction.

14. The target is 30 mL / min / m². ２ The method according to claim 12, wherein if the patient has renal impairment with an eGFR of less than 0.5 ng / kg / min and cannot tolerate an initial dose of about 0.5 ng / kg / min, the dose is reduced to about 0.25 ng / kg / min at the start of the titration step.

15. The method according to any one of claims 8 to 11, wherein the titration step comprises administering the composition at a starting dose of about 0.25 ng / kg / min.

16. The method according to claim 15, wherein the subject has liver dysfunction classified as Child-Pugh classification B or Child-Pugh classification C.

17. The method according to any one of claims 8 to 16, wherein the titration step includes increasing the dose every 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes until the maximum dose is reached.

18. The method according to claim 17, wherein the dose is increased every 30 minutes.

19. The method according to any one of claims 8 to 18, wherein the titration step includes increasing the dose by about 0.1 ng / kg / min, about 0.2 ng / kg / min, about 0.25 ng / kg / min, about 0.3 ng / kg / min, about 0.4 ng / kg / min, about 0.5 ng / kg / min, about 0.6 ng / kg / min, about 0.7 ng / kg / min, or about 0.8 ng / kg / min until the maximum dose is reached.

20. The method according to claim 19, wherein the dose is increased by approximately 0.5 ng / kg / min.

21. The method according to claim 19, wherein the dose is increased by approximately 0.25 ng / kg / min.

22. The method according to any one of claims 17 to 21, wherein the maximum dose is predetermined.

23. The method according to claim 22, wherein the predetermined maximum dose is approximately 1.8 ng / kg / min, approximately 2.0 ng / kg / min, approximately 2.2 ng / kg / min, approximately 2.4 ng / kg / min, approximately 2.6 ng / kg / min, approximately 2.8 ng / kg / min, or approximately 3.0 ng / kg / min.

24. The method according to claim 22 or 23, wherein the predetermined maximum dose is approximately 2.0 ng / kg / min.

25. The method according to any one of claims 17 to 21, wherein the maximum dose is approximately 2.0 ng / kg / min, or the highest dose tolerable by the subject between 0.25 ng / kg / min and 2.0 ng / kg / min.

26. The method according to any one of claims 17 to 21, wherein the maximum dose is approximately 2.0 ng / kg / min, or the highest dose tolerable by the subject between 0.5 ng / kg / min and 2.0 ng / kg / min.

27. The method according to any one of claims 8 to 26, wherein the titration step includes reducing the dose if the subject has a dose-limiting response.

28. The method according to claim 27, wherein the dose-limiting reaction is headache, flushing, jaw pain, muscle pain, nausea, or vomiting.

29. The method according to claim 27 or 28, wherein the dose is reduced by approximately 0.5 ng / kg / min every 30 minutes until a tolerable dose is reached.

30. The method according to any one of claims 1 to 29, wherein the composition is administered continuously daily during the treatment period.

31. The method according to claim 30, wherein the continuous administration per day is approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, or approximately 8 hours.

32. The method according to claim 30 or 31, wherein the continuous administration per day is approximately 6 hours.

33. The method according to any one of claims 30 to 32, wherein the treatment period includes a titration step on day 1, day 2, and / or day 3.

34. The method according to claim 33, wherein the maximum dose is maintained for the remainder of the treatment period after the titration step is performed.

35. The method according to any one of claims 1 to 34, wherein the composition is administered for 4, 5, 6, 7, 8, 9, or 10 consecutive days.

36. The method according to claim 35, wherein the composition is administered for a maximum of eight consecutive days.

37. The method according to any one of claims 17 to 36, wherein the drug is administered at the maximum dose found in the titration step from the fourth day onward.

38. The method according to any one of claims 1 to 29, wherein if the subject has a dose-limiting reaction before the end of the treatment period, administration is discontinued.

39. The method according to claim 38, wherein administration is resumed at a previously tolerated dose after the target dose-limiting reaction has been resolved.

40. The method according to any one of claims 1 to 39, wherein the pharmaceutically acceptable carrier is an IV fluid.

41. The method according to any one of claims 1 to 40, wherein the pharmaceutically acceptable carrier is a 0.9% sodium chloride solution, a 0.45% sodium chloride solution, a 0.33% sodium chloride solution, a 0.225% sodium chloride solution, a 5% glucose aqueous solution (D5W), a 2.5% glucose aqueous solution (D2.5W), a 5% glucose-added lactated Ringer's solution (D5LRS), Ringer's solution (RS), or sterile water.

42. The method according to any one of claims 1 to 41, wherein the pharmaceutically acceptable carrier is a 0.9% sodium chloride solution.

43. The method according to any one of claims 1 to 42, further comprising diluting a 100 μg / mL concentrated solution of iloprost or a pharmaceutically acceptable salt thereof with a sodium chloride solution to provide a composition of iloprost or a pharmaceutically acceptable salt thereof having a concentration in the range of about 0.8 μg / mL to about 1.1 μg / mL.

44. The method according to any one of claims 1 to 43, wherein the frostbite is stage 3 or stage 4 frostbite.

45. The method according to any one of claims 1 to 44, wherein the frostbite is severe frostbite.

46. The method according to any one of claims 1 to 45, for preventing or reducing the risk of finger amputation.

47. The method according to claim 43, wherein the concentrated solution of iloprost or a pharmaceutically acceptable salt thereof is contained in a single-dose vial in an amount of about 1 mL.

48. The method according to claim 47, wherein the single-dose vial is a glass vial.

49. The method according to any one of claims 1 to 48, wherein the most frequent adverse event is headache, flushing, palpitations / tachycardia, nausea, vomiting, dizziness, or hypotension.

50. The method according to any one of claims 1 to 49, wherein the safety profile of the method in the subjects is consistent with the safety profile observed in a multicenter, double-blind, randomized, placebo-controlled trial of continuous iloprost infusion in subjects with systemic sclerosis experiencing a symptomatic finger ischemic episode.

51. The method according to any one of claims 1 to 50, which reduces the risk of amputation by at least 15%, at least 20%, at least 25%, or at least 30% compared to standard treatment for frostbite.

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