Lecithin-modified nanoscale oxygen carriers (LENOX)

Abstract

The present invention relates to a novel type of composition for developing stable synthetic perfluoro-hydrocarbon-based oxygen carriers, the manufacturing thereof, and the use thereof as a synthetic blood and oxygen substitute carrier and as a volume replacement.

Description

[0001] Lecithin-modified nanoscale oxygen carriers (LENOX)

[0002] The present invention relates to novel, stable artificial oxygen carriers based on perfluorocarbons, their preparation and their use as synthetic blood substitutes.

[0003] Background of the invention

[0004] Perfluorocarbons (PFCs) are synthetically produced, perfluorinated carbon compounds (hydrocarbons whose hydrogen atoms are completely replaced by halogens, usually fluorine atoms). Due to the cavities between the individual molecules, they exhibit a high solubility for respiratory gases such as oxygen and carbon dioxide compared to water, depending on the partial pressure. They can therefore take over or support oxygen transport in the blood instead of, or even together with, erythrocytes. Oxygen uptake and release occurs twice as fast as in erythrocytes and is directly proportional to the oxygen partial pressure. More than 90% of the dissolved oxygen is released into the tissue, achieving three times the oxygen extraction rate (oxygen release to the tissue) of an erythrocyte.Due to their very high carbon-fluorine bond energy, perfluorocarbons are chemically and metabolically inert, meaning they do not react even with highly reactive compounds and do not form toxic degradation products. Regarding degradation, the perfluorodecalin (PFD) we use is excreted in the breath due to its high vapor pressure. High vapor pressure means that a liquid evaporates under mild conditions. In this case, this means that PFD reaches the lungs as a liquid and is then vaporized and exhaled.

[0005] Due to the insolubility of perfluorocarbons in both water and oil, either emulsification or encapsulation is required for the use of corresponding PFC-based oxygen carriers (PFOCs) as additives to a perfusion medium or transfusion medium. EP0282948B1 and EP0282949B1 describe aqueous emulsions of perfluorocarbons in which, among other things, phospholipids are used as emulsifiers. Emulsification aids may also be present, such as albumin, particularly bovine serum albumin (BSA). This can be present in the emulsion in an amount of 0.2–2% by weight as an "oncogenic agent."

[0006] CN111214459A (“Perfluorocarbon and albumin nanoparticles and application in production of tumor treating medicine thereof”) describes nanoparticles made of perfluorocarbon and albumin as a drug delivery system for phosphonic acid drugs. A manufacturing process is described in which the phosphonic acid drug is added to the perfluorocarbon / albumin nanoparticles in an emulsion, which may contain lecithin and cholesterol, among other ingredients, in order to produce biologically active double-membrane particles. This publication therefore relates to liposomes. Nanoparticles with a shell made of albumin and lecithin are not described. The application is not related to artificial oxygen carriers or blood substitute solutions, but to tumor therapy.

[0007] CN109908085A describes similar emulsions of BSA, lecithin, and perfluorocarbons. The BSA does not serve as an emulsifier, but rather as an example protein for the drug carrier system. However, nanoparticles with a shell of albumin and lecithin are not explicitly described in the application. The application is also not related to artificial oxygen carriers or blood substitute solutions, but rather to the treatment of heart failure.

[0008] EP0033402A1 describes a perfusate fluid consisting of a previously used crystalloid volume replacement solution (Ringer's solution) in which albumin is dissolved and which may additionally contain, among other things, a perfluorocarbon and an emulsifier. The emulsifier may be, among other things, a phospholipid. In this case, the albumin is not used as an emulsifier, but rather as an additive in the carrier solution.

[0009] Jacoby C, et al. (in: Probing different perfluorocarbons for in vivo inflammation imaging by 19F MRI: image reconstruction, biological half-lives and sensitivity. NMR Biomed. 2014 Mar;27(3):261-71. doi: 10.1002 / nbm.3059. Epub 2013 Dec 19. PMID: 24353148) describe PFCE emulsions that were generated by high pressure homogenization and further contained 10% w / w crown ether and 4% w / w purified egg lecithin E 80 S (Lipoid GmbH, Ludwigshafen, Germany) in isotonic buffer. US Pat. No. 4,866,096 A describes a stable aqueous emulsion containing 10-59% of an oxygen-transferring saturated perfluorodecalin, triglycerides of fatty acids, and 0.5-7% of a surfactant phospholipid, as well as optionally albumin. The emulsion is prepared under cooling in a microfluidizer. It is further described that the emulsion is used as a synthetic blood substitute.

[0010] Jägers et al. (in: "Perfluorocarbon-based oxygen carriers: from physics to physiology", PFLÜGERS ARCHIV - EUROPEAN JOURNAL OF PHYSIOLOGY, Vol. 473, No. 2, November 3, 2020, pages 139-150) describe stable emulsions containing perfluorodecalin (PFD) together with lecithin. Albumin is explicitly mentioned in this publication as an emulsifier to increase emulsion stability. However, a combination of albumin, lecithin, and perfluorodecalin is not described.

[0011] US Pat. No. 4,186,253 A describes the non-refrigerated production of a perfluorocarbon emulsion using jet-type emulsifiers, the emulsion intended for use in organ transplantation. The emulsion also contains a modified Ringer's solution in which albumin is dissolved.

[0012] WO 94 / 18954 A1 describes microemulsions for use as blood substitutes containing albumin, lecithin and perfluorodecalin.

[0013] In previous in vivo research approaches, perfluorocarbons are used as artificial oxygen carriers only in the form of emulsions, whereby the instability of the emulsions and the short half-life, among other things, still pose difficulties (Lambert, E., Janjic, JM Quality by design approach identifies critical parameters driving oxygen delivery performance in vitro for perfluorocarbon based artificial oxygen carriers. Sei Rep 11, 5569 (2021). https: / / doi.org / 10.1038 / s41598-021-84076-l).

[0014] Although products such as Fluosol-DA®, Perftoran® and Oxycyte® have been tested in clinical trials, none of the preparations have yet been approved in Europe or the USA due to side effects (Ferenz KB, Steinbicker AU. Artificial Oxygen Carriers- Past, Present, and Future-a Review of the Most Innovative and Clinically Relevant Concepts. J Pharmacol Exp Ther. 2019 May;369(2):300-310. doi: 10.1124 / jpet.118.254664. Epub 2019 Mar 5. PMID: 30837280).

[0015] Newer preparations, e.g. Oxygent® (a 60% PFC emulsion with 58% perfluorooctyl bromide, 2% perfluorodecyl bromide and egg yolk phospholipids), work with a combination of the somewhat less stabilizing (but better tolerated) phospholipids, e.g. from egg yolk and high molecular weight PFCs such as perfluorodecyl bromide (CF3(CF2)10Br), perfluorotributylamine (N(CF2CF2CF2CF3)3) or perfluoromethylcyclohexylpiperidine (C12F22N) (Ferenz KB, 2015. Artificial oxygen carriers - how long do we have to wait? Hemotherapy 25, 27-36).

[0016] Despite certain advances in this field, the problem still exists that perfluorocarbons cannot be emulsified in aqueous solution sufficiently stably to allow them to be used effectively as an additive to a perfusion or transfusion medium. It is therefore an object of the present invention to provide correspondingly improved processes for the production of artificial oxygen carriers based on perfluorocarbons and correspondingly improved compositions. Further functions of artificial oxygen carriers will become apparent to those skilled in the art upon reading the "state of the art" below in a detailed description, illustrations, and examples.

[0017] In a first aspect of the present invention, the above object is achieved by a process for producing artificial oxygen carriers based on perfluorocarbons (PFOCs). The process comprises the steps of a) providing a suitable aqueous albumin solution and mixing it with at least one suitable artificial oxygen carrier based on perfluorocarbons, b) suitably adding lecithin, c) pre-emulsification by rapid stirring with suitable cooling, d) emulsification of the pre-emulsion from step c) under pressure via a high-pressure homogenizer with suitable cooling, and subsequent storage for at least 30 minutes, also with suitable cooling.

[0018] Preferred is a method according to the present invention, wherein albumin in a concentration between 2-20%, preferably 5-10%, more preferably 5%, perfluorocarbon perfluorodecalin (PFD) in a concentration between 10-50%, preferably 17%, lecithin in a concentration between 1-16%, preferably 2%, are emulsified in a medium selected from water and electrolytes in a plasma-like composition, preferably Sterofundin® ISO, Ringerfundin®, Ringer's solution and hydroxyethyl starch, STEEN solution, OCS solution and other crystalloid and colloidal volume replacement solutions.

[0019] Further preferred is a process according to the present invention, wherein the artificial oxygen carrier is produced exclusively from the specified materials.

[0020] In a second aspect of the present invention, the above object is achieved by an artificial oxygen carrier produced by a process according to the present invention.

[0021] The artificial oxygen carrier according to the invention has a higher stability compared to non-lecithin-containing artificial oxygen carriers based on perfluorocarbons (PFOCs) and can be produced directly in the typical crystalloid and colloidal volume replacement solutions, such as Sterofundin® ISO.

[0022] In a third aspect of the present invention, the above object is achieved by the use of the artificial oxygen carrier according to the present invention as a perfusion or transfusion solution or as a synthetic blood substitute.

[0023] The present inventors have already developed artificial oxygen carriers based on perfluorocarbons that were emulsified with 5% albumin (albumin-derived perfluorocarbon-based artificial oxygen carrier, A-AOC) and which have already shown promising results (see, among others, Wrobeln A, et al. Albumin-derived perfluorocarbon-based artificial oxygen carriers: A physico-chemical characterization and first in vivo evaluation of biocompatibility. Eur J Pharm Biopharm. 2017 Jun; 115:52-64. https: / / doi.Org / 10.1016 / j.ejpb.2017.02.015. Epub 2017 Feb 20. PMID: 28232105).

[0024] To date, these oxygen carriers have been successfully used for tissue oxygenation in the blood exchange model (in rats) and for the prevention of decompression sickness (in rats) (Wrobeln A, Jägers J, Quinting T, Schreiber T, Kirsch M, Fandrey J, Ferenz KB. Albumin-derived perfluorocarbon-based artificial oxygen carriers can avoid hypoxic tissue damage in massive hemodilution. Sci Rep. 2020 Jul 20; 10(1): 11950. Doi: 10.1038 / s41598-020- 68701-z. PMID: 32686717; PMCID: PMC7371727, Mayer D, Guerrero F, Goanvec C, Hetzel L, Linders J, Ljubkovic M, Kreczy A, Mayer C, Kirsch M, Ferenz KB. Prevention of Decompression Sickness by Novel Artificial Oxygen Carriers. Med Sei Sports Exerc. 2020 Oct;52(10):2127-2135. Doi: 10.1249 / MSS.0000000000002354. PMID: 32251255.).In the field of transplantation medicine, the inventors were also able to demonstrate successful tissue oxygenation in extracorporeally perfused organs (organs outside the organism, such as an isolated heart or isolated kidney) (Jägers J, Wrobeln A, Ferenz KB. Perfluorocarbon-based oxygen carriers: from physics to physiology. Pflügers Arch. 2021 Feb;473(2):139-150. Doi: 10.1007 / s00424-020-02482-2. Epub 2020 Nov 3. PMID: 33141239; PMCID: PMC7607370).

[0025] DE102008045152A1 and EP 2 296 635 B1 concern artificial oxygen carriers and their use. However, these oxygen carriers are not yet stable in every carrier solution that is clinically desirable. In particular, stable emulsions could not be generated in typical crystalloid and colloidal volume replacement solutions such as Sterofundin® ISO, thus the oxygen carriers are not universally applicable.

[0026] In a first aspect of the present invention, as mentioned above, the above object is achieved by a process for producing artificial oxygen carriers based on perfluorocarbons (PFOCs). The process first comprises the step of providing a suitable aqueous albumin solution.

[0027] In principle, all aqueous carriers based on perfluorocarbons suitable for use as artificial oxygen carriers are suitable. Albumin is preferably dissolved in a suitable aqueous buffer, water, a balanced full electrolyte solution for infusion therapy, such as Sterofundin® ISO, Ringerfundin®, Ringer's solution, STEEN solution, OCS solution, and / or hydroxyethyl starch. Typical crystalloid and colloidal volume replacement solutions, such as Sterofundin® ISO, are particularly suitable.

[0028] In principle, the albumin used is not limited to one species. A preferred method according to the present invention is one in which the albumin is selected from the group consisting of mammalian albumin, human serum albumin (HSA), and bovine serum albumin (BSA), or suitable derivatives thereof, such as pegylated albumins.

[0029] The albumin solution is then mixed with at least one suitable artificial oxygen carrier based on perfluorocarbons. All artificial oxygen carriers based on perfluorocarbons can be used. A method according to the present invention is preferred, wherein the artificial oxygen carrier based on perfluorocarbons is selected from the group consisting of perfluorodecalin, perfluorodecyl bromide, perfluorotributylamine, perfluoromethylcyclohexylpiperidine, perfluorodichlorooctane, perfluorotertbutylcyclohexane, perfluoro-15-crown-5-ether, dodecafluoropentane, perfluorooctyl bromide, and perfluorocyclohexane. Also suitable are artificial oxygen carriers based on perfluorocarbons as published in Wesseler (Eugene P. Wesseler, Ronald Iltis, Leland C.Clark, The solubility of oxygen in highly fluorinated liquids, Journal of Fluorine Chemistry, Volume 9, Issue 2, 1977, Pages 137-146, ISSN 0022-1139, https: / / doi.org / 10.1016 / S0022-1139(00)82152-1), dort insbesondere in Tabelle 2.

[0030] Lecithin is then added in a suitable manner. Fat-free vegetable lecithin, for example from soy with a purity of >97%, is preferred. These new, stable oxygen carriers, which the inventors call "lecithin modified nanoscale oxygen carrier" (LENOX), differ from previous oxygen carriers, the precursors called albumin-derived artificial oxygen carriers (A-AOCs) and organ-life fluid (OLF), in that lecithin is now added during the synthesis. To improve the process, in addition to a higher operating pressure of the manufacturing machine and a multi-cycle synthesis, the phospholipid lecithin was also incorporated into the albumin shell. These changes have completely eliminated various stability problems of previously known emulsions. Lecithin is a mixture of phosphatidylcholine and other phospholipids and is a component of animal and plant cell membranes.

[0031] The present emulsion has the advantage of requiring no additional stabilizers besides the two emulsifiers, albumin and lecithin. Therefore, a process according to the present invention is preferred, wherein the artificial oxygen carrier is produced or consists exclusively of the specified materials. Both albumin and lecithin are already clinically approved for intravenous use. Typically, the components used to produce an emulsion are first premixed to form a coarsely dispersed preemulsion, which can also be referred to as a crude or preemulsion or premix. This is followed by homogenization, whereby the dispersed phase is reduced to droplets (fine emulsification). This shifts the droplet size spectrum of the crude or preemulsion significantly toward smaller droplets.O / W emulsions for parenteral use are usually prepared by first premixing an oil phase and a water phase to form a preemulsion using a rotor-stator stirrer.

[0032] Thus, according to the invention, the subsequent pre-emulsification of the mixture prepared above (which is usually still present in phases) is carried out by rapid stirring with suitable cooling. A preferred process according to the present invention is one in which the pre-emulsification is carried out by rapid stirring with a homogenizer at between 8,000 and 12,000 revolutions / min, such as with an Ultra-Turrax® at 9,500 revolutions / min.

[0033] As a final step, the pre-emulsion is emulsified as above under (high) pressure using a high-pressure homogenizer, e.g., a counter-jet disperser (see, e.g., DE 102018205 493 A1), with suitable cooling, and then stored for at least 30 minutes, also with suitable cooling. A process according to the present invention is preferred, wherein the emulsification of the pre-emulsion takes place at a pressure between 10,000 PSI (approx. 689 bar) and 40,000 PSI (approx. 2758 bar), preferably at 30,000 PSI (2068 bar), and is optionally repeated several times, preferably between 1 and 12 times (more preferably between 5 and 10, optimally repeating 8 times).

[0034] The steps of the process, particularly those concerning homogenization and emulsification, are carried out under suitable cooling at < 10 °C or lower, preferably at 4 °C. Freezing should, of course, be avoided.

[0035] The inventors have now succeeded for the first time in synthesizing artificial oxygen carriers based on perfluorocarbons (PFOCs) that have a high level of stability with only the three components albumin, lecithin and PFD without any further additives (such as triblock copolymers such as Pluronic). They can also be produced stably directly in a clinically approved crystalloid or colloidal volume replacement solution (preferably Sterofundin® ISO) and other carrier solutions, which was not possible before.

[0036] Particularly preferred is a method according to the present invention, wherein albumin in a concentration between 2-20%, preferably 5-10%, more preferably about 5%, perfluorodecalin (PFD) in a concentration between about 10-50%, preferably about 17%, lecithin in a concentration between about 1-16%, preferably about 2%, are emulsified in water.

[0037] The albumin content used leads to a physiological colloid osmotic pressure, with an optimum surprisingly found at around 5%. The combination of PFOC + albumin + lecithin in a full electrolyte / volume replacement solution therefore results in a stable formulation and a ready-to-use product. However, artificial oxygen carriers (AOCs) with other albumin contents (between 2-20%, 4-10%, 4-6%) are also advantageous for a stable formulation.

[0038] In the context of the present invention, “about” means a deviation of ± 10%, unless otherwise stated.

[0039] Particularly preferred is a process according to the present invention wherein the emulsion is prepared at 20,000 PSI with BSA in water, and the particle diameter in the emulsion is between 50 nm and 400 nm, more preferably between 60 nm and 300 nm, optimally between 70 nm and 250 nm. See in particular Synthesis 5 below.

[0040] Particularly preferred is a process according to the present invention, wherein the mean particle diameter in the emulsion prepared with BSA is 92.5 nm ± 8.5 nm and the oxygen release is 3.99 pmol / mL ± 0.20 pmol / mL.

[0041] Particularly preferred is a process according to the present invention wherein the artificial oxygen carrier is produced exclusively from the specified materials, or albumin and lecithin are present as the only carriers and / or emulsifiers. A further aspect of the present invention relates to an artificial oxygen carrier produced by a process according to the present invention.

[0042] Preferred is an artificial oxygen carrier according to the present invention, consisting of about 5% albumin, perfluorodecalin (PFD) in a concentration of about 17% and lecithin in a concentration of about 2% in a medium selected from water, Sterofundin® ISO, Ringerfundin®, STEEN solution, OCS solution, Ringer solution and hydroxyethyl starch.

[0043] The artificial oxygen carrier according to the present invention is characterized by its higher stability compared to non-lecithin-containing artificial oxygen carriers based on perfluorocarbons (PFOCs). This can be determined in particular by measuring the viscosity, particle size, and particle distribution of the artificial oxygen carrier (see examples).

[0044] Another aspect of the present invention relates to a synthetic blood substitute comprising the artificial oxygen carrier according to the present invention.

[0045] A further aspect of the present invention relates to the use of the artificial oxygen carrier according to the present invention as a perfusion or transfusion solution or as a synthetic blood substitute.

[0046] A further aspect of the present invention relates to the artificial oxygen carrier according to the present invention for use in the treatment and prevention of oxygen deficiency and / or blood deficiency in an organ or patient, in particular in a human.

[0047] A further aspect of the present invention relates to a method for the treatment and / or prevention of oxygen deficiency and / or blood deficiency in an organ or patient, comprising the use of the artificial oxygen carrier according to the present invention as a perfusion or transfusion solution or as a synthetic blood substitute, particularly in humans. The composition of the oxygen carrier according to the invention (LENOX) preferably consists of albumin, perfluorodecalin (PFD), and lecithin in water, wherein the albumin concentration is between 2-20%, preferably 5-10%, more preferably about 5%, the PFD concentration is between 10-50% (preferably 17%), and the lecithin concentration is between 1-16% (preferably 2%). The mean particle diameter with BSA is 92.5 nm ± 8.5 nm, and the oxygen release is 3.99 pmol / mL ± 0.20 pmol / mL.

[0048] The addition of lecithin also surprisingly leads to significantly improved physicochemical properties of the resulting nanoparticles compared to previous albumin-based OLF particles. The oxygen carriers according to the invention (LENOX) are significantly smaller, less polydisperse, and less turbid than previous OLF oxygen carriers (Fig. 1). They can be synthesized particularly with HSA and BSA and are stable over a wider temperature range (-20 to +100 °C).

[0049] Furthermore, they exhibit a significantly lower viscosity than OLF particles (Fig. 2). In particular, the oxygen carriers according to the invention are long-term compatible with Ringer's solution, Sterofundin® ISO (Figure 3), Ringerfundin®, STEEN solution, OCS solution, and 6% HES (130 / 0.4) and can be synthesized directly and stably in these solutions. For example, it was previously not possible to synthesize stable oxygen carriers without additional excipients in approved volume replacement solutions such as Sterofundin® ISO.

[0050] The oxygen carriers according to the invention have very good properties with regard to stability, viscosity, particle size and oxygen release.

[0051] The present invention will now be further described in the following examples with reference to the accompanying figures, but is not limited thereto. For the purposes of the invention, all cited documents and publications are hereby incorporated by reference in their entirety. The figures show:

[0052] Figure 1: Viscosity curve of the OLF particles compared to the LENOX (Example Synthesis I, HSA). Figure 2: Diameter (A) and polydispersion index (B) of the OLF particles compared to the LENOX (Example Synthesis I, HSA).

[0053] Figure 3: Transmission of the OLF particles and the LENOX (Example Synthesis I, HSA).

[0054] Figure 4: Diameter and polydispersion index of the OLF particles and the LENOX synthesized in Sterofundin® ISO (Example Synthesis II).

[0055] Figure 5: Microscopy images of the OLF particles (state of the art) synthesized in Ringer's solution (A), HES 6% (B), and Sterofundin® ISO (C). The noisy background in images B and C indicates that these emulsions are disrupted. The emulsion in image A is still intact, but shows particles > 1 pm. The LENOX particles (example synthesis I, BSA) are not visible in the microscope because they are below the resolution limit (D). The artifacts in image D are caused by residues on the Neubauer chamber and do not originate from the emulsion droplets.

[0056] Figure 6: Viscosity of the OLF particles compared to the LENOX, synthesized in 6% HES (130 / 0.4) (Example Synthesis IV).

[0057] Figure 7: Viscosity of the OLF particles and the LENOX (Example Synthesis II) before and after storage for 4 h at 37 °C.

[0058] Examples

[0059] List of abbreviations

[0060] Lecithin-modified nano-oxygen carrier LENOX

[0061] (Lecithin-modified nano oxygen carrier)

[0062] Perfluorodecalin PFD

[0063] AOC in organ life fluid OLF

[0064] Hydroxyethyl starch HES

[0065] University of Duisburg-Essen UDE bovine serum albumin BSA human serum albumin HSA

[0066] Perfluorocarbon-based artificial oxygen carriers PFOCs

[0067] (Perfluorocarbon-based oxygen carrier) Albumin-based artificial oxygen carriers A-AOCs (Albumin-based artificial oxygen carrier) Artificial oxygen carriers AOC (Artificial oxygen carrier) 10th / 50th / 90th percentile dl0 / d50 / d90

[0068] Range of particle size distribution S

[0069] Materials and equipment:

[0070] For the syntheses of the artificial oxygen carriers, perfluorodecalin HP (F2 Chemicals, Lancashire, UK), Millipore water (Q-Pod, Merck, Darmstadt, DE) and either Albumin Fraction V (bovine serum albumin, Roth, Karlsruhe, DE) or Albunorm (human serum albumin, Octapharma, Langenfeld, DE) were homogenized with a Microfluidizer (LM20, Microfluidics, Waterloo, CAN) after a preemulsion had been prepared with an Ultraturrax (IKA).

[0071] For the newly developed oxygen carrier (LENOX), additional lecithin (97%, Roth, Karlsruhe, DE) was used.

[0072] Sterofundin® ISO (B.Braun, Melsungen, DE), HES 6% (Fresenius Kabi, Bad Homburg, DE) and Ringer's solution (Fresenius Kabi, Bad Homburg, DE) were used as exemplary saline solutions.

[0073] Particle size and polydispersion index were determined using dynamic light scattering (Stabino Nano-flex, Particle Metrix, Inning am Ammersee, Germany). Oxygen capacity was measured using a respirometer (O2k, Oroboros Instruments, Innsbruck, Austria), and turbidity was measured using a photometer (Specord S600, Analytik ena, Jena, Germany). Viscosity was determined using a rheometer (MCR 92, Anton Paar, Ostfildern, Germany). Viscosity was determined at two different shear rates because some emulsions exhibited non-Newtonian behavior. A very low shear rate (50 / s) and a physiological shear rate (645 / s) were chosen.

[0074] A light microscope (AXIO Lab. Al, Zeiss, Oberkochen, Germany) was used to visualize the microparticles.

[0075] The synthesis of pure Organ Life Fluid (OLF) particles was carried out according to the procedure described in patent application DE102021211272.2. For this purpose, 20 mL of albumin (BSA or HSA) and 4 mL of PFD were pre-emulsified using an Ultra-Turrax (T25 Basic, IKA-Werke, Staufen, Germany) at 9,500 rpm. The pre-emulsion was then transferred to the microfluidizer, and the entire volume was processed once at a pressure of 20,000 PSI (approximately 1,379 bar).

[0076] During the process, the device's outlet coil, and thus also the product, must be cooled with ice. After homogenization, the product was stored in an ice-filled container for at least 30 minutes.

[0077] Synthesis I-IV is carried out at a pressure of 30,000 PSI and a cycle rate of eight times; from Synthesis V onwards the pressure changes to 20,000 PSI and the number of cycles changes to seven times.

[0078] Synthesis I

[0079] For the synthesis of LENOX, 20 mL of a 5% albumin solution (bovine serum albumin (BSA) or human serum albumin (HSA)) and 4 mL of PFD were added. The two-phase mixture was mixed with 0.4 g of lecithin and pre-emulsified using an Ultra-Turrax (9,500 rpm). The pre-emulsion was then transferred to the microfluidizer and processed eight times at a pressure of 30,000 PSI (approximately 2,068 bar). During this process, the device's outlet coil, and thus the product itself, must be cooled with ice. After homogenization, the product was stored in an ice-filled container for at least 30 minutes.

[0080] Synthesis II

[0081] For the synthesis of LENOX in Sterofundin® ISO, 1 g of bovine serum albumin was dissolved in 20 mL of Sterofundin® ISO. 4 mL of PFD and then 0.4 g of lecithin were added to this solution. The mixture was pre-emulsified using an Ultra-Turrax at 9,500 rpm. The pre-emulsion was then transferred to the microfluidizer, and the entire volume was processed eight times at a pressure of 30,000 PSI (approximately 2068 bar). During this process, the device's outlet coil, and thus the product itself, had to be cooled with ice. After homogenization, the product was stored in an ice-filled container for at least 30 minutes.

[0082] Synthesis III

[0083] For the synthesis of LENOX in Ringer's solution, 1 g of bovine serum albumin (BSA) was dissolved in 20 mL of Ringer's solution. 4 mL of PFD and then 0.4 g of lecithin were added to this solution. The mixture was pre-emulsified using an Ultra-Turrax at 9,500 rpm. The pre-emulsion was then transferred to the microfluidizer, and the entire volume was cycled through the microfluidizer eight times at a pressure of 30,000 PSI (approximately 2068 bar). During this process, the device's outlet coil, and thus also the product, must be cooled with ice. After homogenization, the product was stored in an ice-filled container for at least 30 minutes.

[0084] Synthesis IV

[0085] For the synthesis of LENOXs in hydroxyethyl starch (HES, 6%), 1 g of bovine serum albumin (BSA) was dissolved in 20 mL of HES (6%). First, 4 mL of PFD and then 0.4 g of lecithin were added to this solution. The mixture was pre-emulsified using an Ultra-Turrax at 9,500 rpm. The pre-emulsion was then transferred to the microfluidizer, and the entire volume was processed eight times at a pressure of 30,000 PSI (approximately 2068 bar). During the process, the device's outlet coil, and thus also the product, must be cooled with ice. After homogenization, the product was stored in an ice-filled container for at least 30 minutes.

[0086] Statistics:

[0087] The software OriginPro® (version 2020) was used for the statistical evaluation of the analysis data.

[0088] The experimental data presented below are the group means ± standard deviation. The standard deviation is shown as error bars. To determine the statistical significance between two data sets, the data sets were first tested for normal distribution using the Shapiro-Wilk test. Since all data sets were normally distributed, unpaired t-tests or one-way ANOVA tests followed by post hoc analysis were performed. Two data sets are considered significantly different if the population mean differs from the test difference at an error level of *p < 0.05. If a significant difference exists, the two data sets are marked as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****). If the error level is *p > 0.05, the two data sets are not significantly different (ns). All data sets consist of a sample size of n = 3 samples each.

[0089] Results

[0090] The LENOX particles have a mean particle diameter of 92.5 ± 8.5 nm (Synthesis I, BSA) or 123.5 ± 2.2 nm (Synthesis I, HSA) and release 3.99 ± 0.2 pmol oxygen per mL of sample (at 17 vol%). The emulsion exhibits the properties of a Newtonian fluid, with viscosity changing only minimally with increasing shear rate. Compared to the LENOX particles, the OLF particles form a non-Newtonian fluid, and their viscosity changes significantly with changing shear rate (see Figure 1).

[0091] The mean particle diameter and polydispersion index differ significantly between the OLF particles (HSA) and the LENOXs. The measured samples had an average particle diameter of 1020.8 ± 85.9 nm for the OLF particles and 123.5 ± 2.2 nm for the LENOX (Synthesis I, HSA) (*p = 0.003). With a polydispersion index of 20.99 ± 1.33, the OLF particle emulsion is significantly (*p = 0.031) more polydisperse than the LENOX (Synthesis I, HSA) emulsion, which has a polydispersion index of 8.58 ± 3.28.

[0092] A visually visible difference between the OLF emulsion and the LENOX emulsion is the turbidity. By measuring the transmittance of both emulsions, it was shown that the LENOX emulsion (Synthesis I, HSA) is significantly (*p < 0.001) more permeable to light with a wavelength of 860 nm than the OLF emulsion. Thus, the OLF emulsion appears very turbid, whereas the LENOX emulsion appears clear.

[0093] To investigate their applicability in a perfusion medium, both the OLF particles and the LENOX particles were synthesized in different perfusion media (Ringer's solution, Sterofundin® ISO, and HAES 6%). During synthesis in Sterofundin® ISO, significant differences in the mean particle diameter (*p = 0.019) and the polydispersion index (*p = 0.001) were observed. These differences are comparable to the differences in Figure 2. During synthesis with Ringer's solution, an intact emulsion was formed with both the OLF particles and the LENOX particles. However, the OLF particles were visible under a light microscope, whereas the LENOX particles were too small to be seen in the light microscope. The OLF particles synthesized in Ringer's solution are therefore significantly larger than the LENOX particles in the same medium. The light microscope images of the OLF particles in HES 6% and in Sterofundin® ISO show a strong background noise.This is a sign of a damaged emulsion. Therefore, stable OLF emulsions cannot form in HES 6% and Sterofundin® ISO.

[0094] This behavior is also reflected in the viscosity. For example, the viscosity in HES 6% is significantly higher for the OLF particles (*p = 0.031 at 50 / s and *p = 0.037 at 650 / s) than for the LENOX (Synthesis IV). Figure 6 shows the viscosities as a function of two different shear rates, as the OLF particles have a strong non-Newtonian character.

[0095] To investigate the influence of normothermic perfusion temperature (37 °C) on the emulsions, samples of the OLF particles and LENOX were stored in Sterofundin® ISO (Synthesis II) for 4 h at 37 °C, and the viscosity was determined before and after storage. A significant difference (*p > 0.001, *p = 0.003, *p = 0.007) was observed between the two emulsions. The OLF particles had a significantly higher viscosity than the LENOX both before and after storage. Only after storage at a shear rate of 650 / s were the two emulsions not significantly different from each other (*p = 0.056).

[0096] Summary:

[0097] LENOX are significantly smaller, less polydisperse and less turbid than OLF particles.

[0098] LENOX can be synthesized with both HSA and BSA.

[0099] LENOX are more stable at different temperatures (-20 to +100 °C).

[0100] LENOX are compatible with Ringer's solution, Sterofundin® ISO, STEEN solution, OCS solution and HAES 6%. OLF particles show incompatible behavior (viscosity, stability, etc.) in many of these solutions.

[0101] Further experiments methods

[0102] Dynamic light scattering

[0103] The particle size distribution was determined by dynamic light scattering (DLS) using Nano-flex (Microtrac). The refractive indices were taken from previous work. The high- and low-temperature viscosity was determined individually for each batch and each test. After a blank measurement for 300 s with water, 1 ml of the sample was filled into a 2-mL reaction tube and measured three times for 300 s.

[0104] For data analysis, the percentiles d10, d50, and d90 were determined. Furthermore, the range (S) of the particle size distribution was calculated using Equation (1).

[0105] viscosity

[0106] A rheometer (MCR 92, Anton Paar) was used to measure the shear-rate-dependent viscosity. The software's internal calibration process was used to set up and calibrate the instrument. For the measurement, 1 mL of the sample was placed on the measuring stage and the system was placed in measurement mode. The system was heated to 20 °C, and the viscosity was measured at various shear rates. Immediately after the first measurement, the system was heated to 37 °C, and the measurement was repeated to determine the high-temperature viscosity.

[0107] Oxygen release

[0108] Oxygen release was measured using an SDR SensorDish Reader (PreSens) under hypoxic conditions. 0.5 ml of water or saline solution was prepared per well in a special 24-well plate equipped with oxygen sensors (OxoDish OD 24, PreSens). This plate was stored under a hypoxia workstation (Whitley H35) (1% O2, 5% CO2, 94% N2) until the oxygen level no longer changed. 2.5 mL of the sample was transferred to a gas-tight flask and oxygenated with 100% O2 for 15 minutes at a gas flow of 0.5 mL / min. 0.5 ml of the previously oxygenated sample was added to each well, and oxygen release was measured until the value returned to baseline.

[0109] Zeta potential: The zeta potential was determined by stream potential measurement (Stabino Zeta, Microtrac). For the zeta potential measurements, 1 ml of the sample was filled into the specific volumetric flask and the flask was connected to the measuring system (Stabino, Microtrac). The sample was measured ten times for 10 s each, and the results were averaged.

[0110] Further syntheses

[0111] Synthesis V

[0112] For the synthesis of LENOX, 20 mL of a 5% albumin solution (bovine serum albumin (BSA) or human serum albumin (HSA)) and 4 mL of PFD were added. The two-phase mixture was mixed with 0.4 g of lecithin and pre-emulsified using an Ultra-Turrax (9,500 rpm). The pre-emulsion was then transferred to the microfluidizer, and the entire volume was processed seven times at a pressure of 20,000 PSI (approximately 1,379 bar). During the process, the device's outlet coil, and thus also the product, must be cooled with ice. After homogenization, the product was stored in an ice-filled container for at least 30 minutes.

[0113] Synthesis VI

[0114] For the synthesis of LENOX in hydroxyethyl starch (HES, 6%), 1 g of bovine serum albumin (BSA) was dissolved in 20 mL of HES (6%). First, 4 mL of PFD and then 0.4 g of lecithin were added to this solution. The mixture was pre-emulsified using an Ultra-Turrax (9,500 rpm). The pre-emulsion was then transferred to the microfluidizer, and the entire volume was processed seven times at a pressure of 20,000 PSI (approximately 1,379 bar). During this process, the device's outlet coil, and thus also the product, must be cooled with ice. After homogenization, the product was stored in an ice-filled container for at least 30 minutes.

[0115] Synthesis VII

[0116] For the synthesis of LENOX in sodium chloride (NaCl, 0.9%), 1 g of bovine serum albumin (BSA) was dissolved in 20 mL of NaCl (0.9%). First, 4 mL of PFD and then 0.4 g of lecithin were added to this solution. The mixture was pre-emulsified using an Ultra-Turrax (9,500 rpm). The pre-emulsion was then transferred to the microfluidizer, and the entire volume was cycled through the microfluidizer seven times at a pressure of 20,000 PSI (approximately 1,379 bar). During this process, the device's outlet coil, and thus also the product, must be cooled with ice. After homogenization, the product was stored in an ice-filled container for at least 30 minutes.

[0117] Synthesis VIII

[0118] For the synthesis of LENOX in Ringer's solution, 1 g of bovine serum albumin (BSA) was dissolved in 20 mL of Ringer's solution. 4 mL of PFD and then 0.4 g of lecithin were added to this solution. The mixture was pre-emulsified using an Ultra-Turrax (9,500 rpm). The pre-emulsion was then transferred to the microfluidizer, and the entire volume was cycled through the microfluidizer seven times at a pressure of 20,000 PSI (approximately 1,379 bar). During this process, the device's outlet coil, and thus the product itself, must be cooled with ice. After homogenization, the product was stored in an ice-filled container for at least 30 minutes.

[0119] Synthesis IX

[0120] For the synthesis of LENOX in STEEN Solution, 4 mL of PFD and then 0.4 g of lecithin were added to 20 mL of STEEN Solution. The mixture was pre-emulsified using an Ultra-Turrax (9,500 rpm). The pre-emulsion was then transferred to the microfluidizer, and the entire volume was cycled through the microfluidizer seven times at a pressure of 20,000 PSI (approximately 1,379 bar). During this process, the device's outlet coil, and thus also the product, must be cooled with ice. After homogenization, the product was stored in an ice-filled container for at least 30 minutes.

[0121] Synthesis X

[0122] For the synthesis of LENOX in Sterofundin ISO, 1 g of bovine serum albumin (BSA) was dissolved in 20 mL of Sterofundin ISO. First, 4 mL of PFD and then 0.4 g of lecithin were added to this solution. The mixture was pre-emulsified using an Ultra-Turrax (9,500 rpm). The pre-emulsion was then transferred to the microfluidizer, and the entire volume was cycled through the microfluidizer seven times at a pressure of 20,000 PSI (approximately 1,379 bar). During this process, the device's outlet coil, and thus also the product, must be cooled with ice. After homogenization, the product was stored in an ice-filled container for at least 30 minutes.

[0123] Basic data of the syntheses The following data refer to an undiluted synthesis, as it results from the microfluidizer.

[0124] Synthesis V

[0125] Particle size distribution:

[0126] In the following table, d represents the hydrodynamic diameter of the particles and S represents the range of the particle size distribution. d10 means that 10% of the measured particles are exactly the same size or smaller than the specified particle diameter, d50 corresponds to 50%, and d90 corresponds to 90%. d10 75 nm therefore means that 10% of the measured particles were 75 nm or smaller.

[0127] Zeta potential: -72.9 ± 0.7 mV

[0128] Viscosity (at 200 s' 1 ):

[0129] 20 °C: 1.85 ± 0.10 mPa-s (n = 6)

[0130] 37 °C: 1.21 ± 0.07 mPa-s (n = 6)

[0131] Oxygen release: 309 ± 94 hPa (n = 6)

[0132] Long-term stability

[0133] LENOX was stored upright at 4 °C in fully filled 2 mL reaction tubes. The emulsion was analyzed every 7 days over a period of 42 days. Each batch of LENOX was divided into six reaction tubes, so that a closed tube was used each day, and the opened tubes were discarded.

[0134] Viscosity:

[0135] Particle size distribution:

[0136] Oxygen-free setting Day 0: 309 ± 94 hPa Day 42: 379 ± 75 hPa

[0137] LENOX in various perfusion solutions

[0138] The compatibility of LENOX with clinically relevant crystalloid and colloidal solutions is essential for potential application. Colloidal volume replacement solutions are only used in exceptional clinical situations, but are widely used in preclinical research. For this reason, LENOX was synthesized and analyzed in various solutions.

[0139] Synthesis VI, Synthesis VII, Synthesis VIII, Synthesis IX and Synthesis X

[0140] Particle size Viscosity (at 200 s' 1 )

[0141] Oxygen-free setting:

[0142] Literature:

[0143] Konakion Product Information, Status of Information, January 2015, 26.07.2004

[0144] Albunorm product information, status of information, July 2018, approval number PEI.H.04333.02.1

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[0146] Riess J. Oxygen Carriers (“Blood Substitutes”) Raison d’Etre, Chemistry, and Some Physiology. Chem. Rev. (2001): 101, 2797-2919

[0147] Riess J. Understanding the Fundamentals of Perfluorocarbons and Perfluorocarbon Emulsions Relevant to In Vivo Oxygen Delivery. Artificial Cells, Blood Substitutes, and Biotechnology (2005), 33 : 47-63, 2005

[0148] Jahr J. Blood Substitutes and Oxygen Therapeutics: A Review. Anesth Analg. (2021): 132(1): 119-129. doi: 10.1213 / ANE.0000000000003957.

[0149] Lambert E, Janjic JM. Quality by design approach identifies critical parameters driving oxygen delivery performance in vitro for perfluorocarbon based artificial oxygen carriers. Sei Rep (2021) 2021 Mar 10;l 1(1):5569. doi: 10.1038 / s41598-021-84076-l .

[0150] Jaegers J, Haferkamp S, Arnolds O, Moog D, Wrobeln A, Nocke F, Cantore M, Pütz S, Hartwig A, Franzkoch R, Psathaki OE, Jastrow H, Schauerte C, Stoll R, Kirsch M, Ferenz KB. Deciphering the Emulsification Process to Create an Albumin-Perfluorocarbon-(o / w) Nanoemulsion with High Shelf Life and Bioresistivity. Langmuir. 2022 Aug 30;38(34): 10351 - 10361. doi: 10.1021 / acs.langmuir. lc03388. Epub 2022 Aug 15. PMID: 35969658.

[0151] Jägers J, Kirsch M, Cantore M, Karaman O and Ferenz KB. Artificial oxygen carriers in organ preservation: Dose dependency in a rat model of ex-vivo normothermic kidney perfusion. Artif Organs (2022) Apr 18. doi: 10.1111 / aor. 14264. Artif Organs. 2022 Sep;46(9): 1783-1793. doi: 10.1111 / aor.14264. Epub 2022 Apr 23. PMID: 35435266

[0152] Jägers J, Wrobeln A and Ferenz KB. Perfluorocarbon based oxygen carriers: From Physics to Physiology. Pflügers Arch (2021) 473 (2): 139-150, doi: 10.1007 / s00424-020-02482-2

[0153] Wrobeln A, Jägers J, Quinting T, Schreiber T, Kirsch M, Fandrey J and Ferenz KB. Albuminderived Perfluorocarbon-based Artificial Oxygen Carriers Can Avoid Hypoxic Tissue Damage in Massive Hemodilution. Sei rep (2020) 10 (1):11950. doi: 10.1038 / s41598-020-68701-z.

[0154] Mayer, D, Guerrero F, Goanvec C, Hetzel L, Linders J, Ljubkovic M, Kreczy A, Mayer C, Kirsch M and Ferenz KB. Prevention of Decompression Sickness by Novel Artificial Oxygen Carriers. Med Sei Sports Exerc (2020) 52(10): 2127-2135, doi:

[0155] 10.1249 / MSS.0000000000002354.

[0156] Ferenz KB and Steinbicker A. Artificial oxygen carriers- past, present and future-a review of the most innovative and clinically relevant concepts. J Pharmacol Exp Ther (2019) 369: 300- 10, doi: 10.1124 / jpet.118.254664.

[0157] Wrobeln A, Schlüter KD, Linders J, Mayer C and Ferenz KB. Functionality of albumin-derived perfluorocarbon-based artificial oxygen carriers in the Langendorff-heart. Artif Cells Nanomed Biotechnol (2017) 45(4): 723-30, doi: 10.1080 / 21691401.2017.1284858.

[0158] Wrobeln A, Laudien J, Groß-Heitfeld C, Linders J, Mayer C, Wilde B, Knoll T, Naglav D, Kirsch M and Ferenz KB (2017). Albumin-derived perfluorocarbon-based artificial oxygen carriers: A physico-chemical characterization and first in vivo evaluation of biocompatibility. Eur J Pharm Biopharm (2017) 115: 52-64, doi: 10.1016 / j .ejpb.2017.02.015.

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Claims

Patent claims 1. A process for the preparation of artificial oxygen carriers based on perfluorocarbons (PFOCs), comprising a) providing a suitable aqueous albumin solution and mixing it with at least one suitable artificial oxygen carrier based on perfluorocarbons, b) suitably adding lecithin, c) pre-emulsification by rapid stirring under suitable cooling, d) emulsification of the pre-emulsion from step c) under pressure via a microfluidizer under suitable cooling and subsequent storage for at least 30 min, also under suitable cooling.

2. The method according to claim 1, wherein the albumin is suitably dissolved in an aqueous buffer, water, a balanced full electrolyte solution for infusion therapy, such as Sterofundin® ISO, Ringerfundin®, STEEN solution, OCS solution, Ringer's solution or hydroxyethyl starch.

3. The method according to claim 1 or 2, wherein the artificial oxygen carrier based on perfluorocarbons is selected from the group consisting of perfluorodecalin, perfluorodecyl bromide, perfluorotributylamine, perfluoromethylcyclohexylpiperidine, perfluorodichlorooctane, perfluorotertbutylcyclohexane, perfluoro-15-crown-5-ether, dodecafluoropentane, perfluorooctyl bromide and perfluorocyclohexane.

4. The method according to any one of claims 1 to 3, wherein the albumin is selected from the group consisting of mammalian albumin, human serum albumin (HSA) and bovine serum albumin (BSA), or derivatives thereof.

5. A process according to any one of claims 1 to 4, wherein the pre-emulsification is carried out by rapid stirring with a homogenizer at between 8000 and 12000 revolutions / min, such as an Ultra-Turrax® at 9500 revolutions / min.

6. A process according to any one of claims 1 to 5, wherein the emulsification of the pre-emulsion is carried out at a pressure between 10,000 PSI (approx. 689 bar) and 40,000 PSI (approx. 2758 bar), preferably at 30,000 PSI (2068 bar) and is optionally repeated several times, preferably between 1 and 12, more preferably between 5 and 10, even more preferably 8 times.

7. The method according to any one of claims 1 to 6, wherein the cooling is carried out at below 4°C, preferably on ice.

8. Method according to one of claims 1 to 7, wherein albumin in a concentration between 2-20%, preferably 5-10%, more preferably about 5%, perfluorodecalin (PFD) in a concentration between 10-50%, preferably 17%, lecithin in a concentration between 1-16%, preferably 2%, are emulsified in water.

9. The method of any one of claims 1 to 8, wherein the emulsion is prepared at 20,000 PSI with BSA in water and the particle diameter in the emulsion is between 50 nm and 400 nm, more preferably between 60 nm and 300 nm, optimally between 70 nm and 250 nm.

10. The method according to any one of claims 1 to 9, wherein the mean particle diameter with BSA is 92.5 nm ± 8.5 nm and the oxygen release is 3.99 pmol / mL ± 0.20 pmol / mL.

11. A method according to any one of claims 1 to 10, wherein the artificial oxygen carrier is produced exclusively from the specified materials.

12. An artificial oxygen carrier produced by a process according to any one of claims 1 to 11.

13. Artificial oxygen carrier according to claim 12, consisting of 5% albumin, perfluorodecalin (PFD) in a concentration of 17% and lecithin in a concentration of 2% in a medium selected from the following media: water, Sterofundin® ISO, Ringerfundin®, STEEN solution, OCS solution, Ringer solution or hydroxyethyl starch.

14. Artificial oxygen carrier according to claim 12 or 13, which has a higher stability than non-lecithin-containing artificial oxygen carriers based on perfluorocarbons (PFOCs), as determined by increased viscosity of the artificial oxygen carrier.

15. A synthetic blood substitute comprising the artificial oxygen carrier according to any one of claims 12 to 14.

16. Use of the artificial oxygen carrier according to any one of claims 12 to 14 as a perfusion or transfusion solution or as a synthetic blood substitute.

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