Liposomal methotrexate formulations for intravitreal application

Abstract

The present invention relates to liposomal methotrexate formulations for antiproliferative therapy suitable for intravitreal application.

Description

[0001] Liposomal methotrexate formulations for intravitreal application The present invention relates to liposomal methotrexate formulations for antiproliferative therapy suitable for intravitreal application.

[0002] Background of the Disclosure

[0003] Proliferative and neoplastic diseases of the retina involve abnormal cell growth and proliferation within the eye, severely impacting the vision and the quality of life of the affected patients. Thus, the development of targeted pharmacological interventions has become of crucial importance for hampering disease progression and restoring normal ocular tissue conditions.

[0004] Currently, the treatment of proliferative retinal diseases such as intraocular lymphoma or proliferative vitreoretinopathy (PVR) involves intravitreal administration of therapeutic drugs.

[0005] Methotrexate (MTX), a small-molecule folic acid antimetabolite, has been proven effective in treating intraocular lymphoma and exhibited successful results in recent studies concerning the treatment of PVR. However, treatments involving intravitreal injection of free MTX require a weekly or bi-weekly injection frequency, as the short intravitreal half-life of MTX causes a rapid clearance from the patient’s eye, reaching concentrations below therapeutic MTX levels within 2-3 days after the intravitreal injection.

[0006] The high injection frequency places a significant burden on patients, which in turn leads to a low therapy compliance. The necessary frequent injections also place a greater burden on doctors and inflate treatment costs for the healthcare system. In addition, MTX levels peak after each injection, increasing the risk for cytotoxic side effects. With drug levels rapidly subsiding, the effective drug concentration heavily fluctuates within the patient’s eye, failing to achieve a reliable constant effective concentration across a prolonged period. Moreover, the frequency of the invasive procedure promotes the emergence of complications based on intravitreal infections such as endophthalmitis, uveitis and corneal decompensation.

[0007] To provide an alternative for repeated intravitreal injections, solid implants have been developed to sustain the release of MTX over an extended period. Solid implants such as poly lactic-co-glycolic acid (PLGA) -based implants have been investigated in vitro and demonstrated a promising potential of lowering the treatment burden and eliminating the high peak concentrations. However, the application of the implants requires highly invasive implantation techniques, increasing the risk of complications, and is not applicable in cases with intraocular lymphoma because of the high risk of tumor seeding. The manufacturing of the implants is expensive, and the implantation requires

[0008] January 23, 2026 1 / 39specific injectors which often lead to ocular hypo- or hypertension. Furthermore, due to the dimension of the implant, the procedure can lead to visual impairments due to the physical presence of the solid implant in the vitreous cavity.

[0009] The disadvantages of the current art summarized above underline the need for MTX formulations which retain or improve treatment efficacy while minimizing the invasive aspect of the interventions and reduce the risk of complications in the treatment of retinal diseases.

[0010] Brief Summary of the Disclosure

[0011] The present disclosure overcomes the drawbacks of the prior art.

[0012] An aspect of the present disclosure relates to a liposomal methotrexate formulation for use in antiproliferative therapy, wherein the therapy includes intravitreal application of the liposomal methotrexate formulation, the liposomes having a Z-average particle size of between about 70 nm and about 170 nm. The liposomal methotrexate formulation according to the first aspect is suitable for intravitreal application, and provides for minimized treatment burden on the patients and physicians by sustaining an effective MTX concentration within the patient’s eye for a prolonged period of time. This reduces the need for frequent intravitreal injections or implantations, instead the formulation is in a form that allows easy injection with a small needle gauge with long intervals between administrations. Experiments have shown that the formulations of this disclosure provide for long lasting MTX concentrations suitable for antiproliferative treatment. Because of the reduced treatment burden therapy adherence is improved.

[0013] The liposomal methotrexate formulations according to the present disclosure may effectively release methotrexate into the intravitreal space for an extended period with an essentially constant and / or controlled release rate, avoiding highly fluctuating therapeutic drug levels. Thus, exceedingly high and potentially toxic drug levels are avoided.

[0014] As antiproliferative treatment with the formulations of this disclosure require only few and less invasive administration compared to implants, the risk of intervention-based complications is reduced, reducing the costs for the overall healthcare system.

[0015] Results have shown that the formulations of this disclosure do not impair the visual system of the patient and demonstrate excellent biocompatibility.

[0016] In a second aspect, this disclosure relates to a liposomal methotrexate formulation for use in antiproliferative therapy, wherein the therapy includes intravitreal application of the liposomal methotrexate formulation, the liposomes having a polydispersity index of between about 0.1 and about

[0017] January 23, 2026 2 / 39In a third aspect, this disclosure relates to a liposomal methotrexate formulation for use in antiproliferative therapy, wherein the therapy includes intravitreal application of the liposomal methotrexate formulation, the liposomes having a negative zeta potential, such as between about - 15 mV and about - 5 mV.

[0018] In a fourth aspect, this disclosure relates to a liposomal methotrexate formulation for use in antiproliferative therapy, wherein the therapy includes intravitreal application of the liposomal methotrexate formulation, the formulation having a Therapeutical Serenity Score of at least 4.0.

[0019] In a fifth aspect, this disclosure relates to a liposomal methotrexate formulation for use in antiproliferative therapy, wherein the therapy includes intravitreal application of the liposomal methotrexate formulation, the liposomes comprising:

[0020] methotrexate,

[0021] one or more lipids, including at least one phospholipid, and

[0022] an aqueous salt solution (such as a balanced salt solution, BSS).

[0023] In a sixth aspect, this disclosure relates to a liposomal methotrexate formulation, having one or more or all of the following features:

[0024] the liposomes have a Z-average particle size of between about 70 nm and about 170 nm,

[0025] the liposomes have a polydispersity index of between about 0.1 and about 0.3, and / or

[0026] the liposomes have a negative zeta potential, such as between about - 15 mV and about - 5 mV.

[0027] In a seventh aspect, this disclosure relates to a liposomal methotrexate formulation, having

[0028] a Therapeutical Serenity Score of at least 4.0, and / or

[0029] the liposomes comprising:

[0030] o methotrexate,

[0031] o one or more lipids, including at least one phospholipid, and

[0032] an aqueous salt solution (such as a balanced salt solution, BSS).

[0033] January 23, 2026 3 / 39Preferably, the liposomal formulations of this disclosure are administered at an amount corresponding to 200 to 800 µg of MTX, preferably 300 to 500 µg of MTX, such as about 400 µg of MTX, per single administration.

[0034] The term "liposome" refers to artificially prepared vesicles composed of lipid bilayers. Liposomes can be used for delivery of APIs due to their unique property of encapsulating a portion of an aqueous solution inside a lipophilic bilayer membrane. Lipophilic compounds can be dissolved in the lipid bilayer, and in this way liposomes can carry both lipophilic and hydrophilic compounds. To deliver the molecules to sites of action, the lipid bilayer can fuse with other bilayers such as cell membranes, thus delivering the liposome contents.

[0035] The term “liposomal methotrexate formulation” may be referred to as “extended-release formulation”, or as “sustained release formulation”, or as “nanocarrier” or as “drug nanocarrier”, or as “liposome-based nanocarrier”, or as “phospholipid-based nanocarrier”, or as “liposome-based formulation”, or as “sustained-release liposome-based methotrexate formulation”, or as “formulation”, or as “liposomal formulation” or as any combination or variation derived from these terms.

[0036] “Intervention-based complications” are complications that arise as a consequence of an invasive intervention into the tissue of interest. In the context representative of the present disclosure, the tissue of interest is the eye.

[0037] As used herein, the term “about” is intended to be a modifier of ± 5% or ± 1% of the specified value.

[0038] The term “subject” may refer to a mammal, such as a landrace pig or a human.

[0039] The term “patient” may refer to a human.

[0040] The term “application” optionally in the context of “intravitreal application” may essentially refer to an injection, more precisely an intravitreal injection. In the context of solid implants, the term “application” may refer to implantation.

[0041] The term “administration” optionally in the context of “intravitreal administration” may essentially refer to an injection, more precisely an intravitreal injection.

[0042] The “Therapeutical Serenity Score” (TSS) is a scoring parameter which assesses the extent to which a liposomal methotrexate formulation suitable for intravitreal application, such as the liposomal methotrexate formulation of the present disclosure, reduces the treatment burden for the patients and physicians and is more recommendable for a minimally invasive therapy. The TSS takes into account the time period during which the intravitreal methotrexate concentration is considered,

[0043] January 23, 2026 4 / 39the average concentration of methotrexate measured in the time period, the time period during which a therapeutically effective concentration is upheld, and the number of separate intravitreal applications (i.e. invasive interventions) performed during the considered time period. The TSS is calculated as follows:

[0044] c ■ e j ), with e as Euler’s number and:

[0045]

[0046] t = period of time during which intravitreal methotrexate concentrations are considered therapeutically effective (in weeks);

[0047] c = average therapeutically effective intravitreal methotrexate concentration (in µg / mL) in the considered period of time, the lower limit of c will be considered at 1 µg / mL, the upper limit of c will be considered at the critical concentration for intravitreal toxicity;

[0048] k = period of time during which the therapeutically effective intravitreal methotrexate concentration is below 1 µg / mL (in weeks);

[0049] j = number of separate intravitreal applications performed during the considered period of time, j cannot be 0.

[0050] For determining t and k when time periods are only available in days, one would need to convert the days into weeks (1 day = 0.143 weeks).

[0051] A higher TSS score indicates a higher adequacy of the formulation for use in a minimally invasive therapy. The intraocular MTX-levels may be determined by using aqueous taps from the anterior chamber at desired measuring days.

[0052] The measurement of the intraocular MTX-levels may occur in a human or non-human test subject. The measurement of the intraocular MTX-levels for determining the TSS may occur in a human or non-human test subject. The measurement of the intraocular MTX-levels may occur in pigs. The measurement of the intraocular MTX-levels for determining the TSS may occur in pigs. The measurement of the intraocular MTX-levels may occur in landrace pigs. The measurement of the intraocular MTX-levels for determining the TSS may occur in landrace pigs. The average therapeutically effective intravitreal methotrexate concentration (in µg / mL) would be calculated as the arithmetical average of the individual measurements for the considered period of time. In some embodiments of the present disclosure, the intraocular MTX-levels may be determined by using aqueous taps as used in the examples. In some embodiments of the present disclosure, the intraocular MTX-levels may be determined by using aqueous taps, wherein the MTX-levels may be measured every day in the considered period of time. In some embodiments of the present disclosure, the January 23, 2026 5 / 39intraocular MTX-levels may be determined by using aqueous taps, wherein the MTX-levels may be measured only in certain days in the considered period of time. In some embodiments of the present disclosure, the intraocular MTX-levels may be determined by using aqueous taps, wherein the MTX-levels may be measured as shown in the examples. In some embodiments of the present disclosure, the intraocular MTX-levels may be determined by using aqueous taps, wherein the MTX-levels may be measured multiple times per day in the certain measuring days in the considered period of time and the concentration value for each day may be considered as the arithmetic average of the multiple measurements of that day. In some embodiments of the present disclosure, the intraocular MTX-levels may be determined by using aqueous taps, wherein the MTX-levels may be measured multiple times per day in the measuring days in the considered period of time and the concentration value for each day may be considered as the arithmetic average of the multiple measurements of that day.

[0053] In a general definition, “therapeutically effective concentration” regarding intravitreal methotrexate is a concentration that is above 0.1 µg / mL. In a stricter definition, therapeutically effective methotrexate concentrations are at least 1 µg / mL. In some cases, concentrations between 0.1 µg / mL and 2 µg / mL are still considered to be therapeutically effective.

[0054] The “drug loading efficiency”, also called drug loading content or capacity, or referred to as “drug loading”, represents the amount of drug contained within the carrier system relative to the total weight of the drug-loaded carrier. This parameter indicates the mass ratio of the drug to the drug-loaded nanoparticles or carrier system. It is calculated as:

[0055] DLE (%) = (Weight of drug incorporated / Weight of drug-loaded carrier) × 100%

[0056] Brief

[0057]

[0058] of the

[0059]

[0060] Figure 1: Cryo-TEM image of the liposomes according to this disclosure.

[0061] Figure 2: Diagram showing cell viability over a wide range of lipid concentrations of the liposomes of this disclosure.

[0062] Figure 3A: Intravitreal MTX concentrations after administration of liposomal formulations of this disclosure measured in pigs over 42 days.

[0063] Figure 3B: Pharmacokinetics of methotrexate release in porcine eyes following intravitreal administration.

[0064] Figure 4: Intravitreal MTX concentrations after administration of comparative PLGA-MTX implant measured in pigs over 42 days.

[0065] January 23, 2026 6 / 39Figure 5: Diagram comparing intravitreal MTX concentrations achieved using liposomal formulations of this disclosure and comparative PLGA-MTX implants.

[0066] Figure 6A: Diagram comparing intraocular pressure with and without application of the formulations of this disclosure.

[0067] Figure 6B: Diagram comparing intraocular pressure with and without application of the formulations of this disclosure with an increased number of animal test subjects.

[0068] Figure 7A: Results of latency measurements in an ERG experiment.

[0069] Figure 7B: Results of latency measurements in an ERG experiment with an increased number of animal test subjects.

[0070] Figure 8A: Results of amplitude measurements in an ERG experiment.

[0071] Figure 8B: Results of amplitude measurements in an ERG experiment with an increased number of animal test subjects.

[0072] Figure 9: Results of amplitude measurements in an ERG experiment, wherein the test subjects have been injected with BSS.

[0073] Figure 10: Results of fundoscopy of animal eyes.

[0074] Figure 11: Results of OCT inspection.

[0075] Figure 12: Histological analysis showing comparison of H& E staining of eyes injected with MTX-loaded and unloaded liposomes.

[0076] Figure 13: Comparison of immunohistochemical staining of CD45 for leukocyte infiltration in MTX-loaded liposome-treated eyes and control eyes of animal test subjects.

[0077] Figure 14: Comparison of immunohistochemical staining of GFAP for Müller cell activation in MTX-loaded liposome-treated eyes and control eyes of animal test subjects.

[0078] Figure 15: Comparison of immunohistochemical staining of Iba1 for microglia in MTX-loaded liposome-treated eyes and control eyes of animal test subjects.

[0079] Detailed Description of the Disclosure

[0080] The present disclosure provides liposomal MTX formulations for use in antiproliferative therapy. In an embodiment, the therapy is intravitreal antiproliferative therapy. In an embodiment, the January 23, 2026 7 / 39formulations of this disclosure are for use in therapy of proliferative diseases of the eye. Optionally, the therapy is treatment of intraocular lymphoma or PVR.

[0081] In an embodiment, the liposomal MTX formulations of the present disclosure are for use in treatment of intervention-based complications of the eye, including for alleviation of intervention-based complications of the eye. In one embodiment, intervention-based complications of the eye may be infections of the eye, endophthalmitis or irritations. Irritations may include irritations of the eye surface. In another embodiment, an intervention-based complication of the eye may be bleeding of the eye. In another embodiment, intervention-based complications of the eye may be hemorrhages, such as intravitreal hemorrhages.

[0082] In embodiments, intervention-based complications of the eye may be bleeding of the eye and / or hemorrhages and / or irritations of the eye and / or infections of the eye. In embodiments, intervention-based complications of the eye may be endophthalmitis and / or irritations of the eye surface and / or bleeding of the eye.

[0083] In one embodiment, an intervention-based complication of the eye may be corneal decompensation. In another embodiment, an intervention-based complication of the eye may be uveitis.

[0084] In embodiments, intervention-based complications may relate to uveitis and / or to corneal decompensation and / or to endophthalmitis and / or to irritations of the eye and / or to irritations of the eye surface and / or to bleeding of the eye and / or to hemorrhages and / or to intravitreal hemorrhages and / or to infections of the eye and / or to combinations thereof.

[0085] The liposomes of this disclosure comprise methotrexate, one or more lipids, including at least one phospholipid, and an aqueous salt solution (such as a balanced salt solution, BSS). In this disclosure, a BSS is an essentially isotonic solution at essentially physiological pH.

[0086] The liposomes comprise one or more phospholipids. In an embodiment the phospholipids may be synthetic, semi-synthetic or natural phospholipids, or combinations thereof. Exemplary phospholipids include phosphatidylcholines, phosphatidylethanolamines, phosphatidylinosites, phosphatidylserines, cephalines, phosphatidylglycerols, lysophospholipids, and combinations thereof. In a preferred embodiment, the phospholipid is selected from phosphatidylcholine, such as lecithin, in particular soy lecithin.

[0087] The lipids may further include cholesterol. In an embodiment, the lipids of the liposomes of this disclosure have the following composition relative to the total amount of lipids in the liposomes:

[0088] phospholipid, such as phosphatidylcholine 70 to 95 mol%, such as 80 to 92 mol%

[0089]

[0090] January 23, 2026 8 / 39cholesterol 5.0 to 30 mol%, such as 8.0 to 20 mol%

[0091]

[0092] In some useful embodiments, the liposomes comprise phosphatidylcholine and cholesterol as lipids. In embodiments, the liposomal bilayers of the liposomes disclosed herein essentially consist of phospholipids and cholesterol.

[0093] For instance, the stable bilayers formed by the phospholipids may be relatively impermeable to aqueous molecules, providing an effective basis for controlled release formulations.

[0094] The stability and rigidity of the lipid bilayers can be further increased by adding cholesterol to the lipid composition of the liposomal formulations. Cholesterol effectively reduces the permeability of the liposomal membrane, decelerating the drug release. Too high amounts of cholesterol, on the other hand, may reduce the membrane fluidity, which could entail unsuccessful fusion of the liposomes with the target cellular membranes or impaired drug release. Therefore, the amount of cholesterol is crucial to the stability of liposomal formulations and should be set cautiously.

[0095] In embodiments of the present disclosure, the liposomes of the liposomal formulations may be mul-tilamellar and / or unilamellar and / or a combination thereof. In one embodiment of the present disclosure, the liposomes of the disclosed liposomal formulations are multilamellar. In one embodiment of the present disclosure, the liposomes of the disclosed liposomal formulations are unilamellar. In embodiments of the present disclosure, the liposomes of the disclosed liposomal formulations are multilamellar, but not multivesicular.

[0096] Generally, unilamellar liposomes have a higher surface-to-volume ratio, resulting in a faster MTX release. Their small size is beneficial for target delivery, as it enables them to penetrate tissues more easily. Generally, multilamellar liposomes encase the drug in internal compartments, resulting in a slower drug release. Before it can be released, the drug has to diffuse through multiple lipid bilayers. Generally, multilamellar liposomes provide a more controlled and sustained MTX release, due to the additional lipid bilayers that the drug needs to traverse before being released. The liposomal formulation of the present disclosure may comprise a mixture of unilamellar and multilamellar liposomes. In an embodiment, the majority (more than 50%) of the liposomes in the formulations of this disclosure are unilamellar or bilamellar. A low lamellarity provides for sufficient free volume within the liposomes to carry MTX.

[0097] In representative embodiments of the invention disclosed herein, the liposomal formulations are loaded with methotrexate. In representative embodiments of the invention disclosed herein, the liposomal formulations are loaded with methotrexate as the only therapeutic drug.

[0098] January 23, 2026 9 / 39The drug to lipid ratio, i.e. the mass ratio of MTX to lipids in the liposomes of this disclosure may range from 0.03 to 0.20, from 0.05 to 0.12, or from 0.07 to 0.10. Optionally, the drug to lipid ratio may be at least 0.03, at least 0.05, or at least 0.07. In an embodiment, this ratio ranges up to 0.20, up to 0.12 or up to 0.10.

[0099] In embodiments, the encapsulation efficiency of the liposomal MTX formulations of the present disclosure is of at least 30%, or of at least 40%, preferably of at least 50%, most preferably of about 60%. In special embodiments, the encapsulation efficiency of the liposomal MTX formulations of the present disclosure is of at least 60%.

[0100] In embodiments, the drug loading efficiency of the liposomal MTX formulations of the present disclosure is of at least 5%, or of at least 7.5%, preferably of at least 10%, more preferably of at least 15%. In special embodiments, the drug loading efficiency of the liposomal MTX formulations of the present disclosure is of about 15%. Methods of determining the encapsulation efficiency are known in the art. In embodiments, the encapsulation efficiency of MTX was determined by reverse phase HPLC using a C18 column. MTX concentration was measured prior and after non-entrapped MTX was removed by size exclusion chromatography of the liposomes (example section).

[0101] In embodiments, the liposomal formulations of this disclosure comprise an aqueous solution such as an aqueous salt solution, which may be isotonic and / or sterile. For example, the solution is balanced salt solution (BSS).

[0102] In one embodiment of the disclosure, the liposomal formulations disclosed herein comprise liposomes with average particle sizes (meaning the Z average) between about 70 nm and about 170 nm, preferably between 80 nm and 160 nm, more preferably between 90 nm and 150 nm, most preferably between 100 nm and 140 nm. In special embodiments, the average particle size of the liposomes can be about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, or about 135 nm.

[0103] In one embodiment of the present disclosure, the liposomes of the disclosed liposomal formulations, when loaded with MTX, have a polydispersity index (PDI) of between about 0.1 and about 0.3, preferably between about 0.15 and about 0.25, more preferably between about 0.175 and about 0.225, most preferably of about 0.2.

[0104] Methods for measuring the particle size (Z-Average) and PDI of liposomal formulation are known in the art. The particle size and the PDI of the liposomal formulations of the present disclosure have been measured by Dynamic Light Scattering, as described in the example section. Other methods for characterizing liposomes include but are not limited to Cryo-TEM, as described in the example section.

[0105] January 23, 2026 10 / 39The size of the liposomes is may be a relevant factor influencing MTX release. For example, smaller liposomes provide a shorter diffusion path for the drug between the interior and the exterior of the liposome, which leads to a faster drug release. Smaller liposomes are cleared more quickly from the body.

[0106] The effects of the liposomal size on drug release can be increased or attenuated by increasing or decreasing the number of lipid bilayers of the liposomes, resulting in unilamellar or multilamellar liposomes.

[0107] In the context of the present disclosure, the size of the liposomes is reflected by the parameter Z-average. Z-average is a parameter known in the art, often used with Dynamic Light Scattering. The Z-average represents a weighted average of the particle sizes in a sample. It describes the effective diameter of the liposomal particles in a suspension.

[0108] The Z-average is a weighted average, with larger particles having a stronger impact on the determined value, which is consistent with the physical optical phenomena encountered in Dynamic Light Scattering.

[0109] The polydispersity index (PDI) is a measure of the distribution of particle sizes in a given sample. The PDI is a parameter known in the art. In the context of the present disclosure, the PDI is used in order to indicate the homogeneity or heterogeneity of the liposomes within the liposomal formulations of the present disclosure. Methods of calculating the PDI are known in the art. In embodiments of the present disclosure, the PDI is calculated from the measurements obtained by Dynamic Light Scattering.

[0110] The PDI may be calculated using the following formula:

[0111] Standard Deviation2

[0112] PDI = - -; - 5 - ’

[0113]

[0114] Z — Average²,

[0115] where the standard deviation is the spread of the particle sizes in the sample. Alternatively, the PDI is calculated using the following formula:

[0116] (d₉₀ − d₁₀

[0117] PDI = —————,

[0118]

[0119] d-so

[0120] where:

[0121] d₁₀ is the diameter at the 10thpercentile of the sample; d₅₀ is the diameter at the 50th percentile of the sample (median diameter); d₉₀ is the diameter at the 90th percentile of the sample.

[0122] January 23, 2026 11 / 39A low PDI (a narrow size distribution) may correlate with a higher consistency in MTX loading or drug encapsulation across the liposomes. Thus, a low PDI ensures a more predictable drug loading and thus a better predictability for MTX release behavior. The release profile of a liposomal formulation with a low PDI shows a greater consistency in the release behavior.

[0123] Conversely, a high PDI (a broad size distribution) can lead to varying amounts of MTX across the liposomes. The release profile of a liposomal formulation with a high PDI shows greater differences in the release behavior of the individual liposomes in the liposomal formulation, with smaller liposomes generally demonstrating a faster MTX release and larger liposomes generally demonstrating a slower release, diminishing the predictability of the drug release behavior. In addition, a high PDI may indicate liposomal aggregates or fusion of liposomes, compromising the formulation’s stability and integrity. In the context of this disclosure, a PDI greater than 0.4 is considered a high PDI.

[0124] In one embodiment of the present disclosure, the liposomes of the disclosed liposomal formulations, when not loaded with drug particles, have a negative zeta potential of between about -15 mV and about -5 mV, preferably between about -12.5 mV and about -7.5 mV, more preferably between about -9 mV and about -11 mV.

[0125] In one embodiment of the present disclosure, the liposomes of the disclosed liposomal formulations, when loaded with MTX, have a negative zeta potential of between about -15 mV and about -5 mV, preferably between about -12.5 mV and about -7.5 mV, more preferably between about -9 mV and about -11 mV.

[0126] The liposomal formulations of the present invention are characterized by the fact that the zeta potential does not change significantly upon loading of the liposomes with MTX.

[0127] In embodiments, the change in zeta potential upon loading the liposomes with MTX is less than 40%, or less than 30%, or less than 20%, or less than 15%, or less than 10%, or less than 5%, or about 0%.

[0128] The zeta potential can be determined by methods known in the art. The zeta potential may be determined by Dynamic Light Scattering, as described in the example section.

[0129] The surface charge of the liposomes may play a role for MTX release.

[0130] Without wishing to be bound by this theory, liposomes of this disclosure with a negative surface charge (anionic liposomes) are more likely to increase the stability of the liposomal formulation, leading to a slower MTX release compared to positively charged liposomes. The negative charge may additionally play a role in the interaction of the liposomes with cells and tissues.

[0131] January 23, 2026 12 / 39In the context of the present disclosure, the liposomal surface charge is reflected by the zeta potential. Similarly charged liposomal particles are more likely to repel each other based on their electrostatic interaction. This promotes the diffusion of the liposomes, stabilizing the suspension and avoiding aggregation of liposomes. Charged particles have a highly positive or highly negative zeta potential. Thus, highly positive or negative zeta potential values correlate with increased stability of the liposomal formulations. Thus, highly positive or negative zeta potential values correlate with a lower probability of liposomes aggregating or fusing with each other. Conversely, low zeta potential or near-zero zeta potential indicates a weakened electrostatic repulsion between liposomal particles, which correlates with a higher probability of liposomes aggregating or fusing with each other.

[0132] Aggregation or fusion of liposomes may negatively impact the release profile, reducing the effectiveness of the liposomal formulations. Generally, liposomes with a negative zeta potential are more likely to increase the stability of the liposomal formulation, leading to a slower drug release compared to cationic liposomes. The negative charge may additionally play a role in the interaction of the liposomes with cells and tissues.

[0133] In embodiments, the liposomal methotrexate formulations of the present disclosure have a TSS of at least 2.0, or of at least 3.0, or of at least 4.0, or of at least 5.0, or of at least 6.0, or of at least 7.0, or of at least 8.0, or of at least 9.0, or of at least 10, or of at least 11, or of at least 12, or of at least 13, or of at least 14. In embodiments, the liposomal methotrexate formulations of the present disclosure have a TSS of from 2.0 to 30.

[0134] In embodiments, the liposomal methotrexate formulations of the present disclosure have a TSS of from 1.0 to 30, or from 2.0 to 29, or from 3.0 to 28, or from 4.0 to 27, or from 5.0 to 26, or from 6.0 to 25, or from 7.0 to 24, or from 8.0 to 23, or from 9.0 to 22, or from 10 to 21, or from 11 to 20, or from 12 to 19, or from 13 to 18, or from 14 to 17, or from 15 to 16.

[0135] In embodiments, the liposomal methotrexate formulations of the present disclosure have a TSS of from 2.0 to 30, or from 2.0 to 29, or from 3.0 to 28, or from 4.0 to 27, or from 5.0 to 26, or from 6.0 to 25, or from 7.0 to 24, or from 8.0 to 23, or from 9.0 to 22, or from 10 to 21, or from 11 to 20, or from 12 to 19, or from 13 to 18, or from 14 to 17, or from 15 to 16.

[0136] In embodiments, the liposomal methotrexate formulations of the present disclosure have a TSS of from 2.0 to 30, or from 2.0 to 25, or from 4.0 to 25, or from 5.0 to 20, or from 10 to 20, or from 10 to 15.

[0137] In embodiments, the liposomal methotrexate formulations of the present disclosure have a TSS of from 4.0 to 12, or from 5.0 to 11, or from 6.0 to 10, or from 7.0 to 9.0.

[0138] January 23, 2026 13 / 39In embodiments, the liposomal methotrexate formulations have a TSS of at least 7.0, or of at least 7.5, or of at least 8.0, or of at least 8.5, or of at least 9.0. In some embodiments, the liposomal methotrexate formulations of the present disclosure have a TSS of at least 10. In preferred embodiments, the liposomal methotrexate formulations of the present disclosure have a TSS of at least 12. In most preferred embodiments, the liposomal methotrexate formulations of the present disclosure have a TSS of at least 14. In representative embodiments, the liposomal methotrexate formulations of the present invention have a TSS of at least 4.0. Generally, a higher TSS score indicates a higher adequacy of the formulation for use in a minimally invasive therapy.

[0139] In embodiments, the liposomal MTX formulations of the present disclosure provide for MTX in the intravitreal cavity in therapeutically effective concentrations after a single administration over a period of at least 1 week, or at least 2 weeks, or at least 3 weeks, preferably of at least 4 weeks, more preferably of at least 6 weeks, most preferably of at least 8 weeks after injection.

[0140] In embodiments, the MTX released by the liposomal MTX formulations of the present disclosure is present intravitreally in a therapeutically effective concentration after a single administration for a time period of at least 4 weeks after injection.

[0141] In embodiments, the MTX released by the liposomal MTX formulations of the present disclosure is present intravitreally in a therapeutically effective concentration after a single administration for a time period of 6 to 10 weeks after injection.

[0142] In embodiments, the MTX released by the liposomal MTX formulations of the present disclosure is present intravitreally in a therapeutically effective concentration after a single administration for a time period of at least 8 weeks after injection.

[0143] In embodiments, the therapeutically effective concentration of MTX in the vitreous cavity is at least 1.0 pg / mL. In an embodiment, upon intravitreal administration of the liposomal MTX formulations of the present disclosure in a subject’s eye, the MTX concentration remains above a therapeutically effective concentration of at least 1.0 pg / mL, or from 2.0 to 8.0 pg / ml, over a period of at least 2 weeks after injection.

[0144] In an embodiment, upon intravitreal administration of the liposomal MTX formulations of the present disclosure in a subject’s eye, the MTX concentration remains above a therapeutically effective concentration of at least 1.0 pg / mL, or from 2.0 to 8.0 pg / ml, over a period of at least 4 weeks after injection.

[0145] In a special embodiment, upon intravitreal administration of the liposomal MTX formulations of the present disclosure in a subject’s eye, the MTX concentration remains above a therapeutically

[0146] January 23, 2026 14 / 39effective concentration of at least 1.0 pg / mL, or from 2.0 to 8.0 pg / ml, over a period of at least 6 weeks after injection.

[0147] In a special embodiment, upon intravitreal administration of the liposomal MTX formulations of the present disclosure in a subject’s eye, the MTX concentration remains above a therapeutically effective concentration of at least 1.0 pg / mL, or from 2.0 to 8.0 pg / ml, over a period of 6 to 10 weeks after injection.

[0148] Preferably, the liposomal formulations of this disclosure are administered at an amount corresponding to 200 to 800 µg of MTX, preferably 300 to 500 µg of MTX, such as about 400 µg of MTX, per single administration.

[0149] In embodiments, the intravitreal therapeutically effective concentration is at all times below the critical concentration of intravitreal toxicity.

[0150] In embodiments, the administration of the liposomal MTX formulations of the present disclosure into the vitreous cavity of a patient may relate to an intravitreal injection. A particular advantage of the invention of the present disclosure is that the liposomal MTX formulations disclosed herein can be administered to the patient via the use of a 30-gauge needle. The use of the liposomal formulations according to the present disclosure in an antiproliferative therapy advantageously minimizes the invasive character. The use of the liposomal formulations according to the present disclosure in an antiproliferative therapy advantageously reduces the risk of intervention-based complications. In special embodiments, the administration of the liposomal MTX formulations of the present disclosure into the vitreous cavity of a patient may relate to an intravitreal injection performed via a 30-gauge needle.

[0151] In embodiments, the interval between two intravitreal applications of the formulations of this disclosure is at most 2 weeks, or at most 3 weeks, or at most 4 weeks, or at most 5 weeks, or at most 6 weeks, or at most 7 weeks, or at most 8 weeks, or at most 9 weeks, or at most 10 weeks, or at most 11 weeks, or at most 12 weeks, or at most 13 weeks, or at most 14 weeks, or at most 15 weeks, or at most 16 weeks.

[0152] In embodiments, the interval between two intravitreal applications is at most 4 weeks, or at most 6 weeks, or at most 8 weeks, or at most 12 weeks.

[0153] Optionally, the interval between two applications is from 2 to 16 weeks, from 3 to 12 weeks, or from 4 to 8 weeks.

[0154] In embodiments, a method for obtaining the liposomal formulations according to the present

[0155] January 23, 2026 15 / 39disclosure comprises the following steps:

[0156] i) Dissolving the lipids in one or more solvents, such as chloroform and methanol (9:1 v / v)

[0157] ii) Mixing the lipids in the mixture

[0158] iii) Evaporating the one or more solvents in order to obtain lipid films, optionally under a nitrogen stream

[0159] iv) Drying the lipid films, e.g. in a vacuum chamber, for a time period ( e.g. about 1h)

[0160] v) Optionally adding beads to the lipid films

[0161] vi) Performing centrifugation, e.g. dual centrifugation,

[0162] vii) While performing centrifugation, adding an aqueous solution (such as balanced salt solution, BSS) with or without MTX in order to obtain the liposomal formulations of the present disclosure.

[0163] The method is characterized by a high encapsulation efficiency and a high drug loading efficiency.

[0164] The mixture of solvents may comprise one or more alcohols, and / or one or more organohalide. Among the alcohols, methanol, ethanol and propanol are preferred, whereas among the organo-halides, chloroform is preferred. The proportion of alcohol may be from 10% to 100% v / v, such as from 80% to 99%. The proportion of organohalide may be from 0% to 90%, such as from 1% to 20%.

[0165] In preferred embodiments, the MTX is added during the centrifugation step.

[0166] In embodiments, the encapsulation efficiency of the liposomal MTX formulations of the present disclosure is of at least 30%, or of at least 40%, preferably of at least 50%, most preferably of about 60%. In special embodiments, the encapsulation efficiency of the liposomal MTX formulations of the present disclosure is of at least 60%.

[0167] In embodiments, the drug loading efficiency of the liposomal MTX formulations of the present disclosure is of at least 5%, or of at least 7.5%, preferably of at least 10%, more preferably of at least 15%. In special embodiments, the drug loading efficiency of the liposomal MTX formulations of the present disclosure is of about 15%.

[0168] In embodiments, the lipid films are dried in a vacuum chamber for a time period of at least 30 minutes, or at least 45 minutes, or at least 60 minutes, or at least 120 minutes, or at least 180

[0169] January 23, 2026 16 / 39minutes.

[0170] The drug to lipid ratio, i.e. the mass ratio of MTX to lipids in the liposomes of this disclosure may range from 0.03 to 0.20, from 0.05 to 0.12, or from 0.07 to 0.10. Optionally, the drug to lipid ratio may be at least 0.03, at least 0.05, or at least 0.07. In an embodiment, this ratio ranges up to 0.20, up to 0.12 or up to 0.10.

[0171] In embodiments, the liposomal MTX formulations of the present disclosure are obtainable by the method of the present disclosure.

[0172] As no in vivo studies providing information about the intravitreal biocompatibility of liposomal formulations are known from the prior art, the inventors conducted a study to evaluate the intravitreal biocompatibility of the liposomal formulations disclosed herein. The studies were conducted in landrace pigs, the most human-like large animal model.

[0173] Methods to assess the intravitreal biocompatibility of drug formulations are known in the art. Measurement of the intraocular pressure, electroretinography, optical coherence tomography and fun-doscopy are exemplary methods which can be employed to this extent.

[0174] In the example sections, the inventors conducted biocompatibility assays of the liposomal formulations. The liposomal formulations of the present disclosure are characterized by excellent biocompatibility.

[0175] In embodiments, the fundoscopic analysis of the subject’s eyes shows physiological retinal and vascular structures with no evidence of inflammation or anterior chamber cells.

[0176] In embodiments, the intraocular pressure of the subject’s eyes following the administration of the liposomal formulations of the present disclosure remains within normal limits at all time points, with a mean value of lower than 21 mmHG, preferably of lower than 19 mmHg, most preferably of lower than 16 mmHg.

[0177] In embodiments, the subject’s eyes demonstrate preserved retinal layers without evidence of oedema following the administration of the liposomal formulations of the present disclosure when inspected with the use of optical coherence tomography (OCT).

[0178] In embodiments, the subject’s eyes demonstrate preserved retinal layers without evidence of atrophy following the administration of the liposomal formulations of the present disclosure when inspected with the use of OCT.

[0179] In embodiments, the subject’s eyes demonstrate preserved retinal layers without evidence of

[0180] January 23, 2026 17 / 39detachment following the administration of the liposomal formulations of the present disclosure when inspected with the use of OCT.

[0181] In embodiments, the subject’s eyes demonstrate preserved retinal layers without evidence of structural damage following the administration of the liposomal formulations of the present disclosure when inspected with the use of OCT.

[0182] In embodiments, the subject’s eyes demonstrate preserved retinal layers without evidence of oedema, atrophy, detachment or other structural damage following the administration of the liposomal formulations of the present disclosure when inspected with the use of OCT.

[0183] In embodiments, the subject’s eyes do not demonstrate significant changes in a- and b-wave amplitude following the administration of the liposomal formulations of the present disclosure compared to untreated control eyes when inspected with the use of electroretinography (ERG).

[0184] In embodiments, the subject’s eyes do not demonstrate significant changes latency over 6 weeks following the administration of the liposomal formulations of the present disclosure compared to untreated control eyes when inspected with the use of ERG.

[0185] In embodiments, the subject’s eyes do not demonstrate inflammatory cell infiltration, cell loss or degeneration after the administration of the liposomal formulations of the present disclosure.

[0186] In embodiments, the intravitreal administration of the liposomal formulations not loaded with a drug does not impair the cell viability of the subject’s eye.

[0187] The liposomal formulations of this disclosure may be sterile and / or isotonic.

[0188]

[0189] Throughout the examples, the term “nanocarriers” refers to the liposomes of this disclosure.

[0190] Preparation of Phospolipid-based nanocarriers

[0191] Nanocarriers were prepared by the thin film method and dual centrifugation using a Zentrimix 380 R (Hettich GmbH & Co. KG, Tuttlingen, Germany). Lecithine and cholesterol were used for nanocarrier formulation. First, the lipids were dissolved in a mixture of chloroform and methanol (9:1; 100 mM) and mixed according to the respective compositions, as shown in Table S1. The organic solvents were evaporated under a nitrogen stream. Lipid films were dried in a vacuum chamber for 1 hour at RT. Prior to dual centrifugation, 152 mg of Yttria stabilized zirconia ceramic beads (Sigmund Lindner GmbH, Warmensteinach, Germany) were added, and dual centrifugation was

[0192] January 23, 2026 18 / 39performed in three runs by addition of the required volumes of BSS without (Table S2) or with MTX (Table S3).

[0193] Table SI. Composition of the phospholipid-based nanocarriers

[0194] Ratio Volume Concentration Molecular Mass [mg]

[0195] [mo1-%] [pL] [mM] weight [g / mol]

[0196] Lecithine 90 225 100 760 17,1

[0197] Cholesterol 10 25 100 386,7 0,97

[0198] Table 82. Characteristic settings of the dual centrifugation process (Zentrimix) for nanocarrier fonnulations with BSS.

[0199] Run Time [min] Volume [pL]

[0200]

[0201] 1 15 27

[0202] 2 5 90

[0203] 3 vortex 133

[0204] Run 1 and 2 was performed with the Zentrimix (DC) at room temperature (20 °C) at 2500 rpm.

[0205] Table S3. Characteristic settings of the dual centrifugation process for nanocarrier formulations with BSS and Methotrexate.

[0206] Volume pure Volume Methotrexate in

[0207] Run Time [min] BSS [pL] BSS [pL]

[0208]

[0209] 1 15 0 27

[0210] 2 5 71.4 18.6

[0211] 3 vortex 133 0

[0212] Run 1 and 2 was performed with the Zentrimix (DC) at room temperature (20 °C) at 2.500 rpm.

[0213] Characterization of phospholipid-based nanocarriers by Dynamic Light Scattering

[0214] The average particle size, polydispersity index (PDI) and zeta potential of the liposomes were determined as the mean of three values by dynamic light scattering at room temperature using the automatic mode of a Zetasizer Nano ZS from Malvern™. To determine liposomal size (z-average) and PDI, liposomes were diluted with PBS to obtain a lipid concentration of 0.01 % (v / v). For zeta potential measurements, liposomes were mixed with 10 % PBS to a final concentration of 0.025 % (v / v) of the respective liposomal formulation.

[0215] The settings of the automatic mode of the zetasizer Nano ZS from Malvern™ (Malvern Instruments

[0216] January 23, 2026 19 / 39Ltd., Worcestershire, United Kingdom) were the following: number of measurements = 3; run duration = 10 s; number of runs = 10; equilibration time = 60 s; refractive index solvent 1.330; refractive index polystyrene cuvette 1.590; viscosity = 0.8872 mPa s; temperature = 25 °C; dielectric constant = 78.5 F / m; backscattering mode (173°); automatic voltage selection; Smoluchowski equation.

[0217] Nanocarrier formulations with BSS only or with MTX dissolved in BSS were characterized by dynamic light scattering in terms of size, polydispersity index (PDI) and zeta potential. The average size of the nanocarriers containing BSS only was 115.6 nm (SD = ± 0.43) with a PDI of 0.242 (SD ± 0.034) and zeta potential of -10.75 mV. The average size of nanocarriers obtained with MTX solution was 119.5 nm (SD = ± 0.45) with a PDI of 0.197 (SD = ± 0.009) and zeta potential of -10.66 mV. A high encapsulation efficiency of 57.13% with a drug loading of approximately 14.44% could be achieved as determined by reverse-phase HPLC.

[0218] Characterization of phospholipid-based nanocarriers by Cryo-TEM

[0219] Nanocarriers loaded with MTX were applied to holey carbon coated grids (Lacey, Tedpella, USA) initially glow-discharged during 45 sec at 20 mA in GloCube Plus (Quorum, UK). After 10 sec incubation at 10°C and 90% humidity, the grids are blotted from the back side using Whatman grade 1 filter paper and vitrified in liquid ethane at -180 °C with a Leica GP2 plunger (Leica Microsystems, Vienna, Austria). Subsequently, the vitrified grids were transferred to a Talos 200 electron microscope (FEI, Hillsboro, OR, USA) using a Gatan 626 cryo-holder (Gatan, Pleasanton, USA). Electron micrographs were acquired at an accelerating voltage of 200 kV using a low-dose system (40 e⁻ / Ų), while maintaining the sample at -175 °C. Defocus values ranged from -2 to -3 pm, and 25 images were captured on a 4 K x 4 K Ceta CMOS camera (Figure 1).

[0220] Encapsulation efficiency of MTX

[0221] The encapsulation efficiency of MTX was determined by reversed phase HPLC (Agilent 1100 Series) using a C18 column (Chromolith® Performance RP-18e, 100-3 mm) applying a linear gradient from 0-100% of 0.1% TFA in water (eluent A) to 0.1% TFA in acetonitrile (eluent B) within 5 minutes (flow rate 2 ml / min; UV absorbance λ = 214 nm). In short, MTX concentration was measured prior and after non-entrapped MTX was removed by size exclusion chromatography of the liposomes.

[0222] After the speed mixing process, the liposomes were divided into two parts with 100 pl each. Part 1 was used to calculate the 100% value obtained by destroying the liposomes by the addition of 50 pl 1% Triton™ X-100 and determining the area under the curve (AUC) of the model substance by HPLC. Part 2 was purified by Sephadex G-25 gel filtration chromatography (NAP™-5 columns) January 23, 2026 20 / 39UH0011P-WO

[0223] and quantified in the same way as part 1. The encapsulation efficiency E(%) was calculated using the following equation:

[0224] E(%)=([AUC] MTX part2 / [AUC] MTX parti) ×100%

[0225] whereby [AUC] MTX part 2 is the concentration of model substance in the purified liposomal fraction and [AUC] MTX part 1 is the concentration of the model substance in the liposomal suspension.

[0226] In-vitro cytotoxicity in ARPE-19 cell culture

[0227] The viability of human RPE cells (ARPE-19) was not impaired by the addition of unloaded nanocarrier formulation in a concentration range from 0.005 to 1.25 mg / ml as depicted in Figure 2.

[0228] The human retinal pigment epithelial cell line (ARPE-19) was obtained from American Type Culture Collection (Manassas, VA, USA). Cells were grown in Dulbecco’s modified Eagle’s medium containing nutrient mixture F12 (1:1) (Gibco / BRL Life Technologies, Grand Island, NY), and supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / mL streptomycin.

[0229] Cells were seeded into a 96-well plate, at a density of 1 x 104and 3 x 104cells per well, with 100 pL medium per well. The cells were allowed to grow for 24 hours at 37 °C in an incubator with 5% CO2. Liposomes were used for a 1:1 dilution series with medium starting from 2.5 mg / mL to 0.005 mg / mL. At 100% confluence, a volume of 100 pL of the liposome solution was added into each well. As negative control, cells were treated with a 1% Triton-X100 solution. As a positive control, cells were incubated with fresh medium. The cells were incubated at 37 °C with 5% CO2 for 24 hours. Afterwards, medium from each well was discarded and rinsed once with pre-warmed 1% PBS. 100 pL of a 1:10 dilution of PrestoBlue HS cell viability reagent (Invitrogen Eugene, USA) with medium was added to each well and incubated darkened for another 2 hours. Cell viability measurements were conducted with the automatic plate reader Infinite F200 pro from (Tecan, Männedorf, Switzerland).

[0230] HPLC and HPLC-MS methodology of aqueous humor samples

[0231] The analysis of methotrexate was carried out with HPLC using an Agilent 1100 system with a Chromolith® Performance RP-18e 100x3 mm column (Merck KGaA, Darmstadt, Germany) as stationary phase. The measurement was performed with a 5 min linear gradient starting at 100% water (+0.1% trifluoroacetic acid) to 100% acetonitrile (+0.1% trifluoroacetic acid) and the UV absorption at 258 nm was measured. As standard condition 20 pL of each sample were injected and analyzed.

[0232] January 23, 2026 21 / 39For the determination of the concentration of methotrexate, a calibration curve between 0.06 pg / mL and 62.5 pg / mL was created. The samples were measured in triplicates (y=11,138x-0,5303, R2=1).

[0233] Biocompatibility and Toxicity Evaluation of intravitreally administered MTX-loaded and unloaded liposomal formulations

[0234] The biocompatibility of empty and MTX-loaded liposomes was evaluated in four porcine eyes or in six porcine eyes over 6 weeks. Fundoscopy showed physiological retinal and vascular structures with no evidence of intraocular inflammation or anterior chamber cells (Figure 10). Figure 10 presents representative fundus photographs from one eye from 1, 2, 4 and 6 weeks after treatment with the MTX-liposomes showing no significant retinal changes over time.

[0235] Intraocular pressure (IOP) remained within normal limits at all time points (Figure 6A, each line representing one animal, and Figure 6B). Figure 6B also confirms that intraocular pressure was within normal limits throughout the experiment in all study and control eyes with no significant difference between the two groups, wherein multiple paired t-tests (fellow eyes paired) have been conducted for n=6 animal test subjects in total and n=3 test subjects for each of unloaded and MTX-loaded liposomes, respectively.

[0236] Optical Coherence Tomography (OCT) demonstrated preserved retinal layers without evidence of retinal oedema, atrophy, detachment or other structural damage or abnormalities (Figure 11). Figure 11 presents the corresponding OCTs from the same timepoints (1, 2, 4 and 6 weeks after treatment with the MTX-liposomes) showing no significant change in retinal structure over time. In particular, no signs of cystoid edema or retinal atrophy were observed. The mean central retinal thickness remained comparable before and after treatment (mean 278 ± 13 pm and 289 pm ± 18 pm (SEM) at baseline and 6 weeks respectively, p=0.14, paired t-test).

[0237] Electroretinography (ERG) showed no significant intra-individual changes in a- and b-wave amplitude (Figures 8A and 8B) or latency (Figures 7A and 7B) over six weeks compared to baseline or the (contralateral) control eyes. For an increased number of animal test subjects (n=3 unloaded liposomes and n=3 MTX-loaded liposomes), the comparison of animals treated with Balanced Salt Solution (BSS) (N=3, multiple paired t-tests, fellow eyes paired) presented in Figure 9 showed similar non-clinically relevant differences between injected and control eyes at 6 weeks similar to no clinically relevant changes in latency (Figure 7B) and amplitudes (Figure 8B) in the eyes of animals injected with liposomal formulations (n=6, n=3 unloaded liposomes, n=3 MTX-loaded liposomes) (multiple paired t-tests, fellow eyes paired).

[0238] Animals that underwent SHAM-injections with balanced salt solution showed similar biocompatibility January 23, 2026 22 / 39UH0011P-WO

[0239] results with ERG results presented in Figure 9.

[0240] Figures 12-15 show histological and immunohistochemical analysis of the retina following intravitreal injection of unloaded or methotrexate (MTX)-loaded liposomes and control eyes. Histological analysis showed no inflammatory cell infiltration, cell loss or degeneration.

[0241] Figure 12 shows the Hematoxylin and eosin (H& E) staining of retinal sections of the retina from an eye injected with unloaded liposomes (on the left) and the retina of an eye injected with MTX-loaded liposomes (on the right). No loss of cell density is visible even at higher magnification. In Figure 12, the abbreviations used are to be interpreted as follows: NFL - nerve fibre layer, GCL - Ganglion Cell Layer, I PL - Inner Plexiform Layer, INL - Inner Nuclear Layer, OPL - Outer Plexiform Layer, ONL -Outer Nuclear Layer, P = Photoreceptor Layers. Histological analysis showed no disruption of retinal architecture in H& E staining of eyes injected with unloaded liposomal formulations (Figure 12, on the left) or MTX-loaded liposomes (Figure 12, on the right).

[0242] Figures 13-15 show immunohistochemical staining of inflammatory and glial markers. In each one of Figures 13, 14 and 15, the control eyes are shown on the left and represent the left eyes of the animal test subjects, where no injection has been performed, and the MTX-loaded liposome-treated eyes are shown on the right and represent the right eyes of the animal test subjects. Figure 13 represents the CD45 staining for leukocyte infiltration with the control eyes shown on the left and the MTX-loaded liposome-treated eyes shown on the right. Figure 14 represents the GFAP staining for Muller cell activation with the control eyes shown on the left and the MTX-loaded liposome-treated eyes shown on the right. Figure 15 represents the Iba1 staining for microglia with the control eyes shown on the left and the MTX-loaded liposome-treated eyes shown on the right. For each one of Figures 13, 14 and 15, Phalloidin was used to visualize the actin cytoskeleton, and DAPI was used for nuclear staining in all panels. CD45 and Iba1 staining revealed no appreciable immune cell infiltration or microglial activation in either group. GFAP showed typical distribution in the inner retina. Immunostainings of eyes injected with MTX-liposomes showed no increased leukocyte infiltration, no increase in glial proliferation and no increased microglia activation compared to untreated left control eyes (Figures 13, 14 and 15).

[0243] In-vivo methotrexate release of liposomal MTX-formulation and MTX-PLGA-rods

[0244] Solid implants made from PLGA have never been evaluated in vivo. Therefore, the inventors conducted a study comparing the performance of the liposome-based nanocarriers of the present disclosure and PLGA-based implants. The liposomal MTX formulations of the present disclosure demonstrate a comparably good performance when compared to experimental PLGA-based implants of the prior art. Thus, the liposomal MTX formulations of the present disclosure demonstrate

[0245] January 23, 2026 23 / 39equally efficient pharmacokinetics, while not requiring surgical implantation or specialized injectors. The liposomal MTX formulations of the present disclosure successfully minimize the cost and the invasive nature of the intravitreal antiproliferative therapy.

[0246] Following intravitreal injection of 400 pg of liposomal methotrexate in 0.075 ml, drug release was monitored over a period of 6 weeks in four pigs. Results are presented in Figure 3A and Figure 3B.

[0247] In Figure 3A, the line with full black circle markers, the line with full black square markers, the line with full black diamond markers and the line with empty diamond markers each represents an individual eye of one animal, the dotted line with down-triangle markers represents the literature data of free MTX and the line with empty circle markers represents the mean ± SEM.

[0248] Figure 3B similarly shows the pharmacokinetics of methotrexate release in porcine eyes following intravitreal administration, in particular methotrexate concentrations in the aqueous humor after intravitreal injection of 400 pg free MTX (downward triangle, n = 1) or 400 pg liposomal MTX (n = 4; individual eyes: circle, square, up-triangle, pentagon). The black line with diamond markers depicts the group mean ± SEM. One animal injected with 400 pg of free MTX showed the expected exponential decay falling below therapeutic concentrations within 5 days (Figure 3B).

[0249] The release profile shows that the methotrexate concentration in the vitreous humor increased, reaching a Cmax of 8.2 ± 1.1 pg / ml at 14 days post injection (Figures 3A and 3B). Subsequently, the concentration gradually decreased but remained above the therapeutically effective threshold of 1 pg / mL throughout the 6-week observation period. At the end of the study (day 42), the mean concentration was 6.6 ± 0.9 pg / mL. The TSS of the tested formulation ranged between approximately 7.74 and approximately 8.01. These results confirm that a single intravitreal injection of liposomal methotrexate provides a sustained drug release, maintaining therapeutically relevant MTX concentrations in the aqueous humor of eyes similar in size to humans over an extended period of time and demonstrating the potential for long-term treatment strategies.

[0250] In vivo release of methotrexate (MTX) from PLGA-based rods in porcine eyes demonstrated sustained drug delivery over 42 days. Initial MTX concentrations on day 1 were approximately 4-5 pg / mL in both pigs. A stable release phase was observed between days 2 and 14, with concentrations ranging from approximately 4.5 to 6 pg / mL in both animals (Figure 4).

[0251] Animals

[0252] Ten landrace pigs from a local farmer with an initial weight of 30 to 35 kg were used for this study. The experiment adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the local ethics committee (ethical approval number: 35-9185.81 / G-

[0253] January 23, 2026 24 / 3911 / 24). Pigs were housed in the Interfaculty Biomedical Research Facility, Heidelberg University, with water ad libitum and restricted food access. All procedures were performed on the right eye under sterile conditions using an operating microscope. Pigs were either used for pharmacokinetics or biocompatibility studies.

[0254] Pharmacokinetics

[0255] The first group focused on analyzing drug release kinetics of either a dose of 400 pg MTX encapsulated liposomes (n=4) or PLGA implants loaded with 800 pg MTX (n=2). Aqueous taps were performed at 24 hours, 48 hours, 72 hours, 1, 2, 4 and 6 weeks after the IVI to determine drug concentration using high performance liquid chromatography (HPLC). Animals were euthanized with an overdose of potassium chloride solution (7.45%) six weeks after injection. Eyes were enucleated and MTX levels were determined in the vitreous, ciliary body and retina.

[0256] Biocompatibility

[0257] In some embodiments, four animals received intravitreal injections (I Vis) containing either unloaded (n=3) or MTX-loaded liposomes (n=1). In other embodiments, six animals received I Vis containing either unloaded (n=3) or MTX-loaded liposomes (n=3). Biocompatibility was assessed 1-, 2-, 4- and 6-weeks post-IVI. After euthanasia, the eyes were enucleated for histological processing.

[0258] Intravitreal injection, Implantation of PLGA-rods and aqueous tap protocols

[0259] For all procedures, animals were sedated with an intramuscular injection of Sedanol (6 mg / kg) and then anaesthetized with ketamine (11 mg / kg) and midazolam (2 mg / kg).

[0260] The right eye of eight pigs received an intravitreal injection of either 400 pg MTX-encapsulated liposomes or unloaded liposomes. The injection volume was 0.075 ml with slight variation depending on the drug loading of different MTX-liposome batches. The ocular surface was anesthetized with Conjucain (Bausch+Lomb). After iodine disinfection, injections were placed at the pars plana with a 30G microsyringe. Topical antibiotics (Floxal, Bausch+Lomb) and corticosteroids (Isopto-Max suspension, Novartis) were applied immediately after injection.

[0261] For the implantation of the PLGA implants, the eye of two pigs received a solid 4 mg-PLGA implant. Anesthesia was maintained throughout the procedure using intravenous propofol administration. The ocular surface was anesthetized with Conjucain. The sclera was thinned, and hemostasis was achieved using diathermy. A 1.8 mm incision was made through the pars plana, and the rod was inserted into the vitreous cavity. The incision site was closed with 7-0 Vicryl sutures and topical antibiotics and corticosteroids were applied after procedure.

[0262] January 23, 2026 25 / 39Aqueous taps

[0263] At 24, 28, 72 hours, 1-, 2-, 4- and 6-weeks post-implantation, the eyes of the abovementioned pigs received anterior chamber aqueous taps. Aqueous taps were performed using a 30G needle under direct visualization at the limbus. A volume of 50–100 µL of aqueous humor was aspirated.

[0264] In-vivo biocompatibility examination

[0265] In one experiment, four pigs underwent biocompatibility testing after the injection of either unloaded liposomes (n=3) or MTX-loaded liposomes (n=1) after 1-, 2-, 4-, and 6-weeks postopera-tively. In another experiment, six pigs underwent biocompatibility testing after the injection of either unloaded liposomes (n=3) or MTX-loaded liposomes (n=3) after 1-, 2-, 4-, and 6-weeks postopera-tively. First, intraocular pressure measurements were recorded using the iCare Tonovet Plus. After inducing mydriasis (cyclopentolate (0,5%), epinephrine (5%) and tropicamide (1%)), fundus photographs of the posterior pole including the optical nerve head were acquired using the ClearView2 veterinary fundus camera. Subsequently, optical coherence tomography of the central retina was performed using the Spectralis OCT (Heidelberg Engineering, Heidelberg, Germany). A 20° dense scan with a 30° infrared reflectance image was performed. The follow-up function was used to examine the exact same location at every subsequent biocompatibility visit. Lastly, electroretinogra-phy was performed using the RETevet™ system and the Dog, Cat, Nonhuman Primate ISCEV 6 Step Light First test protocol. This test includes light and dark adapted tests (15 min dark adaptation) to better assess cone and rod function. The ground electrode was placed centrally on the forehead. The reference electrode was placed 2.5 cm lateral the outer canthus. The active electrode was placed directly on the cornea.

[0266] Histopathologic Assay of the Retina

[0267] For the 4 or the 6 eyes, respectively, undergoing the biocompatibility testing, eyes were enucleated post-mortem, fixed in 4% paraformaldehyde for 4 h, embedded in Optimal Cutting Temperature Compound (O. C. T) and cut into 10 pm thick sections using a cryostat. Hematoxylin and eosin staining was performed, and the sections were evaluated under a light microscope.

[0268] Statistical analysis

[0269] Statistical analyses were performed using Prism 10 (GraphPhad Inc, USA). Normality was assessed using Kolmogorov-Smirnov-tests. Paired or unpaired t-tests were applied as appropriate.

[0270] January 23, 2026 26 / 39

Claims

Claims1. A liposomal methotrexate formulation for use in antiproliferative therapy, wherein the therapy includes intravitreal application of the liposomal methotrexate formulation, the liposomes having a Z-average particle size of between about 70 nm and about 170 nm.

2. The liposomal methotrexate formulation of claim 1, wherein the liposomes are multilamellar liposomes and / or unilamellar liposomes and / or a combination thereof.

3. The liposomal methotrexate formulation according to claim 1 or 2, wherein the liposomes have a polydispersity index of between about 0.1 and about 0.3.

4. The liposomal methotrexate formulation according to any of the preceding claims, wherein the liposomes have a zeta potential of between about - 15 mV and about - 5 mV.

5. The liposomal methotrexate formulation according to any of the preceding claims, wherein the formulation has a Therapeutical Serenity Score is at least 4.

6. The liposomal methotrexate formulation according to any one of the preceding claims, wherein the majority (more than 50%) of the liposomes in the formulation are unilamellar or bilamellar.

7. The liposomal methotrexate formulation according to any of the preceding claims, wherein the liposomes comprise one or more lipids, such as one or more phospholipids.

8. The liposomal methotrexate formulation according to claim 7, wherein the phospholipid is selected from phosphatidylcholine, such as lecithin, in particular soy lecithin.

9. The liposomal methotrexate formulation according to any of the previous claims, wherein the mass ratio of methotrexate to lipids in the liposomes is from 0.03 to 0.20, or from 0.05 to 0.12..

10. The liposomal methotrexate formulation according to any of the preceding claims, wherein an interval between two intravitreal applications is at least 2 weeks at least 4 weeks, or at least 6 weeks.

11. The liposomal methotrexate formulation according to any of the preceding claims, wherein the antiproliferative therapy is therapy of intraocular lymphoma, of proliferative vitreoretinopathy, or a combination thereof.

12. The liposomal methotrexate formulation according to any of the preceding claims, wherein the intravitreal application is performed using a needle with a size of at most 25 gauge.January 23, 2026 27 / 3913. The liposomal methotrexate formulation according to any of the preceding claims, wherein the liposomes comprise:methotrexate,an aqueous salt solution, andone or more lipids.

14. The liposomal methotrexate formulation according to any of the preceding claims, wherein the lipids of the liposomes have the following composition relative to the total amount of lipids in the liposomes:phospholipid, such as phosphatidylcholine 70 to 95 mol%,such as 80 to 92 mol%cholesterol 5.0 to 30 mol%,such as 8.0 to 20 mol%15. The liposomal methotrexate formulation according to any of the preceding claims, wherein the liposomal formulation is administered at an amount corresponding to 200 to 800 pg of methotrexate, preferably 300 to 500 pg of methotrexate, such as about 400 pg of methotrexate, per single administration.January 23, 2026 28 / 39

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