Methods of preparing platelet lysate and platelet lysates thereof

Abstract

The invention relates to methods of preparing platelet lysate, platelet lysates and methods of treatment thereof. The inventors have developed a method for preparing platelet lysate which generates a platelet lysate product with stable concentrations of growth factors such as epidermal growth factor (EGF), nerve growth factor (NGF) and human growth factor (HGF).

Description

[0001] Methods of preparing platelet lysate and platelet lysates thereof This application claims priority from US63 / 790,169 filed 17 April 2025, the contents and elements of which are herein incorporated by reference for all purposes.

[0002] Field of the Invention

[0003] The present invention relates to methods of preparing platelet lysate, platelet lysates and methods of treatment thereof.

[0004] Background

[0005] Dry Eye Syndrome (DES) is a common ocular condition which occurs when the patient’s tears are unable to provide adequate lubrication for their eyes, for example due to the production of insufficient tears or poor-quality tears.

[0006] The prevalence of Dry Eye Syndrome (DES), with and without symptoms, ranges from 5 to 50% of the population, reaching up to 75% in some populations. The economic burden on society and impact of DES on the individual, through its detrimental effect on vision, quality of life, and work productivity, as well as the psychological and physical impact of pain, are considerable, with the average cost of managing DES estimated to $55.4 billion for the US society overall. However, with projections of longer life expectancy, an even larger economic burden of DES on society can be expected [Yu et al., 2011].

[0007] Several therapies have been shown to improve DES signs and symptoms. However, a cure has not yet been identified [Stapleton et al., 2017]. Artificial tears and punctal plugs may help to lubricate the eyes and increase the tear film volume. In addition, anti-inflammatory medications may help reduce the inflammation caused by chronic DES. However, none of these treatments can replace natural tears, which contain numerous biologically active growth factors that are required for corneal homeostasis. For this reason, while patients with severe DES may notice a slight improvement with these treatments in many instances their vision and ocular surface integrity remain severely affected.

[0008] Lubricating supplements are most commonly used to manage DES. These include artificial tear substitutes, gels, emulsions, ointments, topical anti-inflammatory agents, omega-3 fatty acids, secretagogues and topical hyaluronic acid. Whilst some pharmacologic agents targeting the pathophysiology of DES show a slow therapeutic effect, the underlying drug-resistance cannot be ignored [Shen et al., 2020] . In addition, many patients are unable to obtain effective symptom relief from antiinflammatory drugs, which may trigger side effects [White et al., 2020] . Due to this, novel effective and complementary treatments for DES are required.Blood derived products such as platelet rich plasma (PRP), serum, platelet lysate and other blood derived products represent a potential a new treatment modality for severe DES. Advantageously, blood-derived eye drops are unpreserved, are non-antigenic by nature, and contain growth factors, fibronectin, immunoglobulins, and vitamins at concentrations similar to or higher than those in natural or artificial tears. They can also be used successfully in patients who are refractory to other forms of treatment. Examples of blood derived eye drop products include: autologous Serum for DES; autologous Platelet Rich Plasma (PRP) for DES; allogenic Serum for DES; and platelet lysate.

[0009] Although autologous-derived blood products have the advantage of being tailored to the patient, they can be slow and expensive to produce and only available in small quantities. There remains a need for methods which produce large scale volumes of blood derived products for use by any subject. Allogeneic blood derived products represent one such solution to this. However, efficient processing of allogeneic blood, especially that of blood stored in blood banks, remains challenging. This is because whole blood is commonly treated with the anti-coagulant citrate phosphate dextrose (CPD). While CPD functions to preserve platelet viability, a disadvantage of using CPD is its chelating effect on calcium. Studies have shown that whole blood preserved in CPD can prolong clot formation time when compared to WB preserved with other anticoagulants [Schriner etal., 2023]. As a result, attempts to isolate plasma from whole blood preserved with CPD can lead to delayed or incomplete clot formation, affecting the yield and consistency of the downstream platelet-rich plasma (PRP). In addition, CPD can contribute to changes in plasma viscosity and protein interactions, making phase separation less predictable and increasing variability in platelet recovery [Kesmarky etal., 2008]. To avoid these issues, some methods rely on isolation of plasma from whole blood which has not been treated with CPD, for example US 9,757,430 B2.

[0010] The present invention has been devised with the above issues in mind.

[0011] Summary of the Invention

[0012] The present inventors have developed a method for preparing platelet lysate which generates a platelet lysate product with stable concentrations of growth factors such as epidermal growth factor (EGF), nerve growth factor (NGF) and human growth factor (HGF). These growth factors are found in natural tears and so are desirable to have in the platelet products.

[0013] It is known in the art that release of growth factors from platelets can be achieved by adding calcium chloride (CaCh) to platelets, in a step which may otherwise be referred to as an activation step. However, the addition of CaChto platelets also activates the coagulation pathway. This includes the release of fibrinogen. During coagulation, fibrinogen is converted to fibrin via thrombin-mediated proteolytic cleavage. The resulting fibrin forms blood clots, which can physically bind to the growth factors, limitingtheir accessibility. This reduces the stability of any growth factors in the platelet lysate. In addition, fibrin clots can induce a detrimental pro-inflammatory immune response. Therefore, the addition of CaCh is a careful trade-off between the activation of the platelets to release the growth factors, and the formation of fibrin clots which can then trap these growth factors. It is important to minimise the concentration of fibrinogen and the resulting clots in the sample.

[0014] Previous methods to prepare platelet lysate have typically avoided isolating plasma from whole blood treated with CPD. This is because CPD can lead to delayed or incomplete clot formation, meaning that clots do not form until post-production. This leads to less stable and ultimately lower growth factor concentrations in the final product and increases variability between platelet lysate batches.

[0015] Advantageously, the present method can utilise whole blood treated with CPD without any detrimental effect on growth factor stability or concentration. The concentration of fibrinogen in the resulting platelet lysate is also comparable to that achieved by other methods. This is achieved by one, and optionally two incubation steps in the present method, where following activation, complement is inactivated by incubation at a defined temperature of from 30°C to 60°C, combined with a subsequent three filtration steps. This incubation step surprisingly further consolidates fibrin clot formation, which is then removed by the three filtration steps.

[0016] CPD is a common anti-coagulant used in blood banks. Therefore, by enabling utilisation of CPD-treated blood, the present method can utilise much greater volumes of blood, meaning a greater volume and consistency of allogeneic product can be achieved.

[0017] Thus, according to a first aspect, the present invention provides a method for preparing platelet lysate from at least one plasma sample, the method comprising:

[0018] (a) activating platelets in the plasma sample by adding between 1 g / L to 3 g / L CaCh;

[0019] (b) optionally incubating the CaCh-containing sample for a first incubation period;

[0020] (c) inactivating complement in the plasma sample by incubating the CaCh-containing sample for a second incubation period at a temperature of from 30 °C to 60 °C;

[0021] (d) centrifuging the complement inactivated sample, optionally at room temperature;

[0022] (e) pooling the at least one plasma sample (supernatant), and optionally combining the pooled samples with a vehicle;

[0023] (f) filtering the product of step e) through at least at first, a second and a third filtration step to produce the platelet lysate product; and

[0024] (g) optionally freezing the platelet lysate product for storage.Step (d), where the complement inactivated sample is centrifuged, advantageously assists in concentrating any fibrin clots, which can then be separated from the supernatant.

[0025] Room temperature, as used herein, will be understood to refer to a temperature of from 15°C to 33°C. Room temperature may refer to a temperature of from 20°C to 30°C, optionally 24°C.

[0026] In some embodiments the method comprises a preceding step of isolating the plasma sample from a whole blood sample. Optionally, the whole blood sample comprises an anti-coagulant. The anti-coagulant may comprise or consist of citrate phosphate dextrose (CPD). Optionally, the ratio of CPD to blood in the whole blood sample is 1 :6 (i.e. 1 part CPD to 6 parts blood).

[0027] In some embodiments the plasma isolation step comprises centrifuging the whole blood sample at 1000g for at least 5 minutes. In some embodiments, the plasma isolation step comprises centrifuging the whole blood sample at 1000g for 9 minutes. Optionally, the centrifuging is performed at room temperature. The centrifuging may be performed within 8 hours of the collection of the whole blood sample.

[0028] In some embodiments the first, second and third filtration steps are performed through filters of progressively smaller sizes.

[0029] In some embodiments:

[0030] i) the first filtration step is performed through a 0.6-1.0 pM filter, preferably a 0.8 pM filter;

[0031] ii) the second filtration step is performed through a 0.3-0.6 pM filter, preferably 0.45 pM filter; and / or iii) the third filtration step is performed through a 0.1-0.3 pM filter, preferably a 0.22 pM filter.

[0032] The inventors have advantageously shown that filtration steps using these sizes of filters is especially efficient at removing fibrin clots and fibrinogen.

[0033] In some embodiments the first incubation period is for a duration of from 30 minutes to 24 hours. In some embodiments the first incubation period is for a duration of 1 hour. Other methods (see, for example, comparative protocol 1 of Example 1) have previously required that the first incubation period is for an overnight period (i.e. 8 hours or more) to effectively activate the platelets. The present inventors have surprisingly found that equivalent and even improved activation of the platelets can be achieved by a substantially reduced incubation period of only one hour. This reduces the time needed to generate platelets and in turn the cost.In some embodiments the first incubation period is performed at a temperature of from 30-60 °C.

[0034] In some embodiments the second incubation period is performed at a temperature at least 10°C greater than the temperature of the first incubation period.

[0035] In some embodiments the first incubation period is performed at a temperature of from 30 to 40°C. The first incubation period may be performed at a temperature of 37 °C.

[0036] In some embodiments the second incubation period is for a duration of between 30 minutes and 2 hours. In some embodiments the second incubation period is for a duration of 1 hour.

[0037] In some embodiments the second incubation period is performed at a temperature of from 50-60 °C. In some embodiments the second incubation period is performed at a temperature of 56 °C.

[0038] In some embodiments the duration of the first and the second incubation period combined is less than 8 hours. The duration of the first and the second incubation period combined may be less than 8 hours, optionally less than 5 hours, further optionally less than 3 hours. As previously noted, it is surprising and advantageous that the combined incubation period can be less than 8 hours, especially less than 3 hours. This means that platelet lysate can be generated more quickly and in greater volumes than other methods.

[0039] In some embodiments, the first incubation period is performed at a temperature of 37°C for a duration of one hour and the second incubation period is performed at a temperature of 56°C for a duration of one hour. The inventors have found this this combination of incubation periods is especially effective at consolidating fibrin clots, which are then removed by the triple filtration steps.

[0040] In some embodiments prior to activating the platelets in step a), the method further comprises standardising the platelet concentration in the plasma sample. The standardisation may result in a standardised plasma sample comprising approximately 1.5x108- 5.0x108platelets / mL, preferably 4x108platelets per mL. Standardisation of the platelet concentration advantageously yields the initial same number of platelets and hence concentration of growth factors. This reduces variability and downstream loss of product.In some embodiments standardising the platelet concentration comprises:

[0041] i) determining the platelet concentration in the sample;

[0042] ii) centrifuging the sample; and

[0043] iii) discarding a portion of the supernatant or the whole or a portion of the pellet to achieve a standard concentration (e.g. approximately 4x108platelets per mL) of platelets in the standardised plasma sample.

[0044] In some embodiments the method further comprises a step of freezing the standardised plasma sample, and subsequently thawing the sample prior to activating the platelets in step a).

[0045] In some embodiments activating the platelets in step a) comprises adding 2 g / L of CaCh. As shown in the examples, the inventors have found that this concentration of CaCh was especially efficient at generating stable and high concentrations of growth factors.

[0046] In some embodiments the at least one plasma sample is pooled with a vehicle in step e). The vehicle may comprise sodium chloride solution, optionally 0.9% sodium chloride solution.

[0047] In some embodiments, the vehicle is mixed with the at least one pooled plasma sample at a ratio of 1:1 to 10:1.

[0048] In some embodiments, the vehicle is mixed with the at least one pooled plasma sample at a ratio of 4:1 (i.e. 4 parts vehicle to one part pooled plasma sample).

[0049] In some embodiments the concentration of EGF in the resulting platelet lysate product is of from 80 pg / mL to 200 pg / mL.

[0050] In some embodiments the concentration of fibrinogen in the resulting platelet lysate product is of from 0.3 pg / mL to 2 pg / mL.

[0051] In some embodiments the concentration of NGF in the resulting platelet lysate product is of from 30 to 50 pg / mL.

[0052] In some embodiments the concentration of HGF in the resulting platelet lysate product is of from 80 to 300 pg / mL.In some embodiments the platelet lysate product produced in step f) has a volume of at least 50 mL, at least 250 mL, at least 500 mL, at least 750 mL, at least 1 L, at least 5 L, or at least 10 L.

[0053] In some embodiments the method is performed a plurality of times to produce a plurality of platelet lysate samples, and wherein:

[0054] a) the percentage of volume retrieved (VR) is of from 60% to 100%; and / or

[0055] b) the batch-to-batch standard deviation in percentage of volume retrieved (VR) is 5%.

[0056] In some embodiments the method comprises:

[0057] (a) activating platelets in the plasma sample by adding between 1 g / L to 3 g / L CaCh;

[0058] (b) incubating the CaCh-containing sample for a first incubation period at a temperature of 37°C for a duration of 1 hour;

[0059] (c) inactivating complement in the plasma sample by incubating the CaCh-containing sample for a second incubation period at a temperature of 56°C for a duration of 1 hour;

[0060] (d) centrifuging the complement inactivated sample;

[0061] (e) pooling the at least one plasma sample (supernatant), and optionally combining the pooled samples with a vehicle;

[0062] (f) filtering the product of step e) through at least at first, a second and a third filtration step to produce the platelet lysate product; and

[0063] (g) optionally freezing the platelet lysate product for storage.

[0064] In some embodiments the method comprises:

[0065] (a) activating platelets in the plasma sample by adding 2g / L CaCh;

[0066] (b) incubating the CaCh-containing sample for a first incubation period at a temperature of 37°C for a duration of 1 hour;

[0067] (c) inactivating complement in the plasma sample by incubating the CaCh-containing sample for a second incubation period at a temperature of 56°C for a duration of 1 hour;

[0068] (d) centrifuging the complement inactivated sample;

[0069] (e) pooling the at least one plasma sample (supernatant), and optionally combining the pooled samples with a vehicle;(f) filtering the product of step e) through at least at first, a second and a third filtration step to produce the platelet lysate product; and

[0070] (g) optionally freezing the platelet lysate product for storage.

[0071] In some embodiments the method comprises:

[0072] (a) activating platelets in the plasma sample by adding between 1 g / L to 3 g / L CaCh;

[0073] (b) incubating the CaCh-containing sample for a first incubation period at a temperature of 37°C for a duration of 1 hour;

[0074] (c) inactivating complement in the plasma sample by incubating the CaCh-containing sample for a second incubation period at a temperature of 56°C for a duration of 1 hour;

[0075] (d) centrifuging the complement inactivated sample;

[0076] (e) pooling the at least one plasma sample (supernatant), and optionally combining the pooled samples with a vehicle;

[0077] (f) filtering the product of step e) through at least at first, a second and a third filtration step to produce the platelet lysate product; wherein

[0078] i) the first filtration step is performed through a 0.8 pM filter;

[0079] ii) the second filtration step is performed through a 0.45 pM filter; and / or

[0080] iii) the third filtration step is performed through a 0.22 pM filter.

[0081] and

[0082] (g) optionally freezing the platelet lysate product for storage.

[0083] In some embodiments the method comprises:

[0084] (a) activating platelets in the plasma sample by adding 2g / L CaCh;

[0085] (b) incubating the CaCh-containing sample for a first incubation period at a temperature of 37°C for a duration of 1 hour;

[0086] (c) inactivating complement in the plasma sample by incubating the CaCh-containing sample for a second incubation period at a temperature of 56°C for a duration of 1 hour;

[0087] (d) centrifuging the complement inactivated sample;

[0088] (e) pooling the at least one plasma sample (supernatant), and optionally combining the pooled samples with a vehicle;

[0089] (f) filtering the product of step e) through at least at first, a second and a third filtration step to produce the platelet lysate product; whereini) the first filtration step is performed through a 0.8 pM filter;

[0090] ii) the second filtration step is performed through a 0.45 pM filter; and / or

[0091] iii) the third filtration step is performed through a 0.22 pM filter.

[0092] and

[0093] (g) optionally freezing the platelet lysate product for storage.

[0094] According to a second aspect, the present invention provides a platelet lysate produced by the method of the first aspect, wherein the concentration of EGF is of from 80 pg / mL to 200 pg / mL In some embodiments, the concentration of fibrinogen is no higher than 2 pg / m. In some embodiments the platelet lysate has a volume of at least 50 mL, at least 250 mL, at least 500 mL, at least 750 mL, at least 1 L, at least 5 L, or at least 10 L.

[0095] According to a further aspect, the present invention provides a method of treating an eye or skin condition in a subject, the method comprising administering to the eye or skin of the subject in need thereof a therapeutically effective amount of the platelet lysate of the second aspect.

[0096] In some embodiments, the platelet lysate is allogeneic.

[0097] In some embodiments the eye condition is selected from dry eye syndrome (DES), neurotrophic keratitis, persistent corneal epithelial defects, post-surgical recovery and / or ocular burns. The ocular burns may comprise ocular thermal burns and / or ocular chemical burns.

[0098] In some embodiments the skin condition comprises a skin wound, optionally a skin cut or laceration, an ulcer and / or a skin burn. The ulcer may comprise a diabetic and / or chronic ulcer.

[0099] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0100] Summary of the Figures

[0101] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0102] Figure 1: Bar chart comparing protein concentration of fibrinogen in the final plasma product generated by using 150 pl CaCh, shaking for 2 hours and incubation at room temperature overnight (protocol 1), andthe final plasma product generated by using 150p I CaCh, and incubation at 37°C for 1 hour then 56°C for 1 hour (protocol 2).

[0103] Figure 2: Bar chart comparing % volume retrieved after final filtration step in the final plasma product generated by using 150 pl CaCh, shaking for 2 hours and incubation at room temperature overnight (protocol 1), and the final plasma product generated by using 150pl CaCL, and incubation at 37°C for 1 hour then 56°C for 1 hour (protocol 2).

[0104] Figure 3: Bar chart comparing EGF protein concentration in the final plasma product generated by using 150 pl CaCh, shaking for 2 hours and incubation at room temperature overnight (protocol 1), and the final plasma product generated by using 150pl CaCL, and incubation at 37°C for 1 hour then 56°C for 1 hour (protocol 2).

[0105] Figure 4: Bar chart comparing protein concentration of fibrinogen in the final plasma product generated by using 150 pl CaCL, shaking for 2 hours and incubation at room temperature overnight (protocol 1), and the final plasma product generated by using 400p I CaCh, and incubation at 37°C for 1 hour then 56°C for 1 hour (protocol 3).

[0106] Figure 5: Box plot comparing percentage volume retrieved after final filtration step in the final plasma product generated by using 150 pl CaCh, shaking for 2 hours and incubation at room temperature overnight (protocol 1), and the final plasma product generated by using 400pl CaCL, and incubation at 37°C for 1 hour then 56°C for 1 hour (protocol 3).

[0107] Figure 6: Bar chart comparing EGF protein concentration in the final plasma product generated by using 150 pl CaCh, shaking for 2 hours and incubation at room temperature overnight (protocol 1), and the final plasma product generated by using 400pl CaCL, and incubation at 37°C for 1 hour then 56°C for 1 hour (protocol 3).

[0108] Figure 7: Box plot comparing EGF protein concentration in the final plasma product generated by using 400p I CaCh, and incubation at 37°C for 1 hour then 56°C for 1 hour (protocol 3), the final plasma product generated by using 400pl CaCh and incubation at 37°C for 1 hour (protocol 4), and the final plasma product generated by using 400pl CaCL and incubation at 56°C for 1 hour (protocol 5).

[0109] Figure 8: Bar chart comparing percentage volume retrieved after final filtration step of the final plasma product generated by using 400pl CaCL, and incubation at 37°C for 1 hour then 56°C for 1 hour (protocol 3), the final plasma product generated by using 400pl CaCh and incubation at 37°C for 1 hour (protocol 4), and the final plasma product generated by using 400pl CaCh and incubation at 56°C for 1 hour (protocol 5).

[0110] Figure 9: Bar charts to show stability of final plasma product generated by using 400pl CaCL followed by incubation at 37°C for 1 hour then 56°C for 1 hour. Product was assessed following storage at 4°C for 1 , 2 or 3 months, when compared to a day 0 storage control. (A) top graph shows EGF protein concentration (pg / mL), and bottom graph NGF protein concentration (pg / mL) (B) top graph shows HGF protein concentration (pg / mL), and bottom graph total protein concentration (pg / mL). For individual proteins EGF, NGF and HGF, specific concentrations were quantified using ELISA. The total proteinconcentration represented the concentration of all proteins in the sample and was measured using the Bradford Protein Assay.

[0111] Figure 10: Dot plot to show pH stability of final plasma product generated by using 400pl CaCh followed by incubation at 37°C for 1 hour then 56°C for 1 hour. Product was assessed following storage at 4°C for 1 , 2 or 3 months, when compared to a day 0 storage control.

[0112] Figure 11: Bar charts to show stability of final plasma product generated by using 400pl CaCL followed by incubation at 37°C for 1 hour then 56°C for 1 hour. Product was assessed following storage at 30°C for 1 or 1.5 months, when compared to a day 0 storage control. (A) top graph shows EGF protein concentration (pg / mL), and bottom graph NGF protein concentration (pg / mL), (B) top graph shows HGF protein concentration (pg / mL) and bottom graph total protein concentration (pg / mL). For individual proteins EGF, NGF and HGF, specific concentrations were quantified using ELISA. The total protein concentration represented the concentration of all proteins in the sample and was measured using the Bradford Protein Assay.

[0113] Figure 12: Dot plot to show pH stability of final plasma product generated by using 400pl CaCh followed by incubation at 37°C for 1 hour then 56°C for 1 hour. Product was assessed following storage at 30°C for 1 or 1.5 months, when compared to a day 0 storage control.

[0114] Figure 13: Wound Closure (%) assessed via fluorescein staining at Day 24 and Day 48 compared to Day 0 in a mouse dry eye model, following twice daily administration of 1% OB-1 (1% platelet lysate), 10% OB-1 (10% platelet lysate) and 20% OB-1 (20% platelet lysate). Vehicle was used as a negative control.

[0115] Figure 14: Quantification of epithelial thickness in micrometres using image processing of hematoxylin and eosin (H&E) histology staining of mouse cornea obtained from 48 hours after treatment.

[0116] Figure 15: Protein stability analysis. Volcano plot displaying the relationship between fold change (x-axis) and statistical significance (y-axis) for all 423 identified proteins in OB-1 formulation. Each point represents an individual protein, with x-axis showing Iog2-transformed fold change (room temperature (RT) vs fresh (DO)) and y-axis showing -log 10 of FDR-adjusted p-values from Welch's t-test. Small gray points indicate proteins with no significant change (FDR-adjusted p > 0.05 or fold change within 0.83-1.2x). Larger, black points indicate significantly up-regulated proteins (FDR-adjusted p < 0.05 AND fold change > 1.2x, n=2). There were no significantly down-regulated proteins (FDR-adjusted p < 0.05 AND fold change < 0.83x, n=0). The horizontal dashed line marks the significance threshold (p = 0.05), and vertical dotted lines mark fold change thresholds (0.83x and 1.2x). The gray shaded region represents the stability zone (0.83-1.2x fold change). Statistical Methods: Welch's t-test with Benjamini-Hochberg FDR correction; a = 0.05. Python packages: scipy 1.16.3, matplotlib, seaborn.

[0117] Figure 16: Distribution of protein fold changes. Histogram showing the distribution of Iog2-transformed fold changes for all 423 proteins in OB-1 comparing RT storage (30 days at 30°C) to freshly thawed (DO) samples. X-axis shows Iog2 fold change with bins of 0.1 units. Y-axis shows number of proteins in each bin. Light gray bars represent proteins within the stability zone (0.83-1.2x fold change, n=230, 54.4%). Dark grey bars represent proteins outside the stability zone. Vertical lines mark fold change thresholds: dashed lines at Iog2(0.83) and Iog2(1.2) indicate stability boundaries; black solid line at Iog2(1.0)indicates no change. The pale gray shaded region highlights the stability zone. Python packages: matplotlib, numpy, seaborn.

[0118] Figure 17: Stability analysis of key therapeutic proteins. Box plots with individual data points (swarm plots) showing abundance levels of eight critical therapeutic proteins in OB-1 formulation comparing freshly thawed (DO) versus 30-day room temperature storage (RT). Each panel displays one protein with three technical replicates per condition. Box boundaries represent 25th and 75th percentiles, center line shows median, whiskers extend to 1.5* interquartile range, and black dots show individual measurements. Titles indicate protein name, fold change (FC = RT mean / DO mean), FDR-adjusted p-value, and significance level (*** p<0.001 , ** p<0.01 , * p<0.05, ns = not significant). Statistical Methods: Welch's t-testwith Benjamini-Hochberg FDR correction; a = 0.05. Python packages: scipy 1.16.3, matplotlib, seaborn.

[0119] Figure 18: Comparative analysis of 14 key therapeutic proteins organized by function, comparing fresh (DO, light gray bars, circles) vs 30-day RT storage (30°C, dark grey bars, squares). Measured by TMT mass spectrometry (n=3 replicates). A: Wound healing factors: LTF (Lactoferrin); FN1 (Fibronectin); HGFAC (Hepatocyte Growth Factor Activator). B: Extracellular matrix (ECM) proteins: COL3A1 (Collagen Type III); VTN (Vitronectin; TGFBI (TGF-p-induced protein). C: Growth factors & binding proteins: PDGFA (Platelet-Derived Growth Factor A); IGF2 (Insulin-like Growth Factor II); IGFBP3 (IGF Binding Protein 3). D: Anti-inflammatory & antimicrobial: ALB (Albumin); HP (Haptoglobin); TF (Transferrin). E: Cell adhesion & immune: CDH1 (Cadherin-1 / E-cadherin; C3 (Complement C3).

[0120] Figure 19: Timeline of the Phase I clinical trial study.

[0121] Figure 20: Longitudinal Trend Analysis of corneal staining for (A) CD (right) eye and (B) OS (left) eye. The solid line with circles and the dashed line with squares represent the mean corneal staining total score at each time point (0, 3, 6, 9, and 12 weeks) for OD eye and OS eye, respectively, while the area within the light grey lines shows ±1 standard deviation from the mean. (. Statistical significance markers indicate comparisons versus baseline with Bonferroni adjustment: * (p<0.05), ** (p<0.01 ), and *** (p<0.001).

[0122] Figure 21: Longitudinal assessment of corneal staining both eyes comparison. The solid line with circles represents OD (right) eye and the dashed line with squares represents OS (left) eye across 5 visits, with error bars showing standard deviation. Data were analyzed using linear mixed-effects modeling to account for repeated measures and within-patient correlations between fellow eyes. Sample composition: 23 patients (20 both eyes, 3 one eye). Time coefficient p = -0.6078, p<0.0001 , indicating highly significant decrease in staining over time. No significant difference between eyes (Eye diff p = 0.6307). Total observations: 179 measurements across 5 timepoints. Figure 22: Longitudinal Trend Analysis of conjunctival staining for (A) OD (right) eye and (B) OS (left) eye. The solid line with circles and the dashed line with squares represent the mean conjunctival staining total score at each time point (0, 3, 6, 9, and 12 weeks) for OD eye and OS eye, respectively, while the area within the light grey lines shows ±1 standard deviation from the mean.. Statistical significance markers indicate comparisons versus baseline with Bonferroni adjustment: * (p<0.05), “ (p<0.01), and “* (p<0.001).Figure 23: Longitudinal assessment of conjunctiva staining both eyes comparison. Overlay plot comparing mean trajectories of OD (solid line, circles) versus OS (dashed line, squares) eyes across 5 visits, with error bars showing standard deviation. Data analyzed using linear mixed-effects modeling to account for repeated measures and within-patient correlations between fellow eyes. Sample composition 23 patients (20 both eyes, 3 one eye). Time coefficient p = -0.7664, p<0.0001 , indicating highly significant decrease in staining over time. No significant difference between eyes (Eye diff p = 0.9043). Total observations: 179 measurements across 5 timepoints.

[0123] Figure 24: The longitudinal change in mean Ocular Surface Disease Index (OSDI) total score overtime, assessed at baseline (Week 0) and at Weeks 3, 6, 9, and 12 during treatment. The dark line with circles represents the mean conjunctival staining total score at each time point (0, 3, 6, 9, and 12 weeks), while the area within the light grey lines shows ±1 standard deviation from the mean, reflecting inter-patient variability. Statistical significance markers indicate comparisons versus baseline (0 weeks) with Bonferroni adjustment: * (p<0.05), “ (p<0.01), and “* (p<0.001).

[0124] Figure 25: Longitudinal trend analysis of symptoms assessed using visual analog scales (VAS). The symptoms assess were: (A) burning sensation; (B) foreign body sensation; (C) photophobia; and (D) itching. The dark line with circles represents the mean score at each time point (0, 3, 6, 9, and 12 weeks), while the area within the light grey lines shows ±1 standard deviation from the mean. Statistical significance markers indicate comparisons versus baseline (0 weeks) with Bonferroni adjustment: * (p<0.05), ** (p<0.01), and *** (p<0.001).

[0125] Figure 26: Both eyes comparison using (A) Shirmer testing and (B) tear break-up time (TBUT). The solid line with circles represents OD (right) eye and the dashed line with squares represents OS (left) eye, with error bars showing standard deviation.

[0126] Figure 27: Mean change in BOVA (Best Corrected Visual Acuity) from baseline over time. Mean change measured in logMAR units over a 12-week period. The dark line with circles represents the mean change from baseline at each time point (baseline (BL), 3, 6, 9, and 12 weeks). The shaded area shows ±1 standard deviation from the mean. Error bars represent 95% confidence intervals. Negative values indicate improvement in visual acuity (lower logMAR = better vision). Statistical significance markers indicate comparisons versus baseline with Bonferroni adjustment: * (p<0.05), ** (p<0.01 ), *** (p<0.001 ), and ns (not significant). Sample sizes at each timepoint are indicated (n=43 at baseline, n=39 at 3 weeks, n=36 at 6 weeks, n=33 at 9 weeks, n=29 at 12 weeks).

[0127] Detailed Description of the Invention

[0128] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.Method

[0129] In embodiments where the method of the first aspect comprises a preceding step of isolating the plasma sample from a whole blood sample, typically the whole blood sample will be allogeneic blood. For example, the whole blood sample may be a whole blood sample obtained from a blood bank.

[0130] The term platelet lysate, as used herein, will be understood to refer to a solution which comprises proteins obtained from platelets. The solution is typically void of cellular debris. Generally, platelet lysate is obtained through platelet lysis, which can be achieved via mechanical or chemical disruption of the platelet membrane.

[0131] In some embodiments, the plasma sample has been obtained by centrifugation of a whole blood sample. In some embodiments, the centrifugation is at 1000g for 8-10 minutes, preferably 9 minutes. In some embodiments, the centrifugation is performed at room temperature. In some embodiments, the centrifugation is performed within 8 hours of the whole blood sample being collected.

[0132] In some embodiments, step (a) of the method of the first aspect comprises activating platelets in the plasma sample by adding between 1.5 g / L to 2.5 g / L CaCh

[0133] In some embodiments, centrifuging the complement inactivated sample in step d) is performed at room temperature. In some embodiments, the centrifuging is at 3500g for 30 minutes at room temperature, followed by centrifuging at 3500g for a further 15 minutes at room temperature.

[0134] In some embodiments, the method is carried out on one plasma sample. In such embodiments “pooling” the sample in step e) will be understood to refer to collecting supernatant from the one plasma sample following the centrifugation in step d). Alternatively, the method may be carried out on a plurality of plasma samples. In such embodiments, “pooling” the sample in step e) may be understood to refer to collecting supernatant from each plasma sample, following centrifugation in step d), then combining the supernatant from the plurality of samples to form one collated supernatant sample, which may otherwise be referred to as “pooled samples”.

[0135] In some embodiments the first incubation period is for a duration of from 30 minutes to 6 hours. For example, the first incubation period may be for a duration of from 30 minutes to 5 hours, of from 30 minutes to 4 hours, or of from 30 minutes to 3 hours. In some embodiments, the first incubation period is for a duration of from 30 minutes to 2 hours.In some embodiments the second incubation period is performed at a temperature at least 15°C greater than the temperature of the first incubation period. In some embodiments, the second incubation period is performed at a temperature of from 15°C to 19°C greater than the temperature of the first incubation period.

[0136] In some embodiments, the first incubation period is performed at a temperature of 37°C and the second incubation period is performed at a temperature of 56°C.

[0137] When reference herein is to “freezing”, this will typically be understood to mean at a temperature of -20°C or less, optionally at a temperature of -80°C. Preferably, freezing will be understood to mean at a temperature of-20°C. In some embodiments, freezing comprises lyophilizing.

[0138] When reference is to “thawing”, this may be at a temperature of from 15°C to 30°C. Thawing may be at a temperature of 20°C or 30°C. Thawing may be for at time period of at least two hours, optionally at least four or at least 8 hours. In some embodiments thawing is for a time period of two hours.

[0139] In embodiments where the at least one plasma sample is pooled with a vehicle, this may otherwise be referred to as a pharmaceutical composition. A pharmaceutical composition will be understood to comprise the at least one plasma sample and the vehicle, which may otherwise be referred to as a pharmaceutically acceptable excipient.

[0140] A pharmaceutically acceptable excipient may be a compound or a combination of compounds entering into a pharmaceutical composition which does not provoke secondary reactions and which allows, for example, facilitation of the administration of the plasma sample, an increase in its lifespan and / or in its efficacy or an increase in its solubility in solution. These pharmaceutically acceptable vehicles are well known and will be adapted by the person skilled in the art as a function of the mode of administration of the plasma sample.

[0141] The pharmaceutical compositions may comprise, in addition to the plasma sample, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the plasma sample.

[0142] In some embodiments, the vehicle comprises an aqueous solution, optionally a buffered solution.In embodiments where the vehicle comprises sodium chloride solution, this will typically be understood to mean sterile, non-pyrogenic water comprising sodium chloride. The sodium chloride solution may have a pH of 4.5-7. The sodium chloride solution may have an osmolarity of 310mOsmol / litre.

[0143] For example, in embodiments where the vehicle comprises 0.9% sodium chloride solution, this will be understood to mean that each 100ml of solution comprises 0.9g of sodium chloride.

[0144] In some embodiments, the vehicle is mixed with the at least one pooled plasma sample at a ratio of from 99:1 to 1:1. In some embodiments, the vehicle is mixed with the at least one pooled plasma at a ratio of from 99:1 to 2:1. In some embodiments, the vehicle is mixed with the at least one pooled plasma sample at a ratio of from 99:1 to 4:1.

[0145] Optionally, the vehicle is mixed with the at least one pooled plasma sample at a ratio of 99:1. In such embodiments, it will be appreciated that the product will be at a 1% concentration.

[0146] Optionally, the vehicle is mixed with the at least one pooled plasma sample at a ratio of 9:1. In such embodiments, it will be appreciated that the product will be at a 10% concentration.

[0147] In some embodiments, the vehicle is mixed with the at least one pooled plasma sample at a ratio of from 9:1 to 1:1.

[0148] Optionally the vehicle is mixed with the at least one pooled plasma sample at a ratio of 1 :1. In other words, the product is 50% vehicle and 50% at least one pooled plasma sample.

[0149] In some embodiments, the platelet lysate product does not comprise a vehicle.

[0150] In some embodiments, the platelet lysate product is lyophilized.

[0151] In some embodiments, the percentage of volume retrieved (VR) is of from 70% to 100%. Optionally, the percentage of VR is of from 80% to 100%, further optionally of from 90% to 100%. Percentage of volume retrieved will be understood to refer to the volume of platelet lysate product achieved as a percentage of the original volume of the plasma sample. For example, when the initial plasma sample in step a) is 100ml, and the final platelet lysate product obtained is 85ml, this will be understood to be a percentage ofvolume retrieved of 85%. The inventors have advantageously found that the present method of preparing platelet lysate improves the final volume retrieved compared to other methods.

[0152] It will be appreciated that the concentration of EGF, NGF, HGF and fibrinogen in the resulting platelet lysate product will depend on if the pooled plasma sample is mixed with a vehicle. When the pooled plasma sample is not mixed with a vehicle, the concentration of EGF in the resulting platelet lysate product may be from 80 pg / mL to 2500 pg / mL. For example, the concentration of EGF in a platelet lysate product not comprising vehicle may be from 400 pg / mL to 2500 pg / mL In some embodiments, the concentration of EGF in a platelet lysate product not comprising vehicle is from 400 pg / mL to 2200 pg / mL, optionally from 400 pg / mL to 2000 pg / mL, further optionally from 400 pg / mL to 1500 pg / mL. In some embodiments the concentration of EGF in a platelet lysate product not comprising vehicle is from 1000 to 2500 pg / mL.

[0153] When the pooled plasma sample is not mixed with a vehicle, the concentration of fibrinogen in the resulting platelet lysate product may be from 3pg / mL to 3700pg / mL. For example, the concentration of fibrinogen in a platelet lysate product not comprising vehicle may be from 3pg / mL to 3000pg / ml, optionally from 3pg / mL to 2000pg / mL, further optionally of from 3pg / mL to 1000pg / mL. In some embodiments, the concentration of fibrinogen in a platelet lysate product not comprising vehicle is from 3pg / mL to 100pg / mL, optionally of from 3pg / mL to 50pg / mL. In some embodiments, the concentration of fibrinogen in a platelet lysate product not comprising vehicle is of from 3pg / mL to 10pg / mL.

[0154] When the pooled plasma sample is not mixed with a vehicle, the concentration of nerve growth factor (NGF) in the resulting platelet lysate product may be from 70pg / mL to 1000pg / mL. For example, the concentration of NGF in a platelet lysate product not comprising vehicle may be from 6pg / mL to 3600pg / mL, optionally from 60pg / mL to 800pg / mL.

[0155] When the pooled plasma sample is not mixed with a vehicle, the concentration of human growth factor (HGF) in the resulting platelet lysate product may be from 60pg / mL to 3000pg / mL. For example, the concentration of HGF in a platelet lysate product not comprising vehicle may be from 60pg / mL to 1500 pg / mL, optionally from 70pg / mL to 500pg / mL, further optionally from 80pg / mL to 300pg / mL. In some embodiments, the concentration of HGF in a platelet lysate product not comprising vehicle is from 80pg / mL to 300pg / mL.

[0156] The method of the first aspect may further comprise performing a sterility test and / or batch validation on the resulting platelet lysate; optionally wherein the sterility test is completed no earlier than 72 hours after the platelet lysate is produced.It will be appreciated that steps a) to d) of the method of the first aspect may be repeated to obtain a plurality of plasma samples. The plurality of plasma samples can then be pooled in step e) to generate a large volume of plasma for filtration to form the platelet lysate product. Likewise, when the method comprises a preceding step of isolating the plasma sample from a whole blood sample, a plurality of plasma samples may be isolated from one or more whole blood samples. The plurality of plasma samples can be pooled. The plurality of plasma samples may be pooled prior to or after platelet concentration standardisation.

[0157] In some embodiments, the method comprises the protocol of Example 6.

[0158] In some embodiments the platelet lysate produced by the method is stable. In some embodiments the platelet lysate produced by the method is stable in refrigerated conditions for at least 30 days, 1 month, 2 months or 3 months. In some embodiments the platelet lysate produced by the method is stable at 4°C for at least 30 days, 1 month, 2 months or 3 months.

[0159] In some embodiments the platelet lysate produced by the method is stable at room temperature for at least 30 days, 1 month or 1.5 months. In some embodiments the platelet lysate produced by the method is stable at 30°C for at least 30 days, 1 month or 1.5 months.

[0160] In some embodiments stability is indicated by a non-significant change in total protein concentration as measured by the Bradford Protein Assay. In some embodiments stability is indicated by a non-significant change in one, more or all of EGF, NGF and HGF protein concentration as measured by ELISA.

[0161] In some embodiments stability is indicated by a 0.83 < Fold Change < 1.2 (i.e., within ±20% of baseline at 0 days) in protein abundance as determined by Tandem Mass Tag (TMT) quantitative mass spectrometry. In some embodiments stability is indicated by a 0.83 < Fold Change < 1.2 (i.e., within ±20% of baseline at 0 days) in protein abundance of one, more or all of: Lactoferrin, Fibronectin, Albumin, Transferrin, Haptoglobin, IGFBP3, PDGF-A, and / or Collagen III. In some embodiments stability is indicated by a 0.83 < Fold Change < 1.2 (i.e., within ±20% of baseline at 0 days) in protein abundance of one more or all of: LTF (Lactoferrin), FN1 (Fibronectin), HGFAC (Hepatocyte Growth Factor Activator), COL3A1 (Collagen Type III), VTN (Vitronectin; TGFBI (TGF-p-induced protein), PDGFA (Platelet-Derived Growth Factor A), IGF2 (Insulin-like Growth Factor II), IGFBP3 (IGF Binding Protein 3), ALB (Albumin), HP (Haptoglobin), TF (Transferrin), CDH1 (Cadherin-1 / E-cadherin, C3 (Complement C3).

[0162] Method of treatment

[0163] According to a third aspect, the present invention provides a method of treating an eye or skin condition in a subject, the method comprising administering to the eye or skin of the subject in need thereof atherapeutically effective amount of the platelet lysate of the second aspect. Also provided is the platelet lysate of the second aspect for use in a method of treating an eye or skin condition in a subject. It will be appreciated that embodiments relating to the method of treatment equally apply to the platelet lysate for use.

[0164] A therapeutically effective amount is an amount which ameliorates one or more symptoms, such as all the symptoms, of the condition and / or abolishes one or more symptoms, such as all the symptoms, of the condition. The therapeutically effective amount may cure the condition.

[0165] The subject may be a mammal. Preferably, the subject is a human, but may be any other primate (great ape, old world monkey or new world monkey), or a domestic, laboratory or livestock animal, such as a mouse, rat, guinea pig, lagomorph (e.g. rabbit), cat, dog, pig, cow, horse, sheep or goat.

[0166] Typically, the platelet lysate is allogeneic. Therefore, the platelet lysate is not autologous to the subject.

[0167] As used herein, the term “eye condition” will be understood to refer to any eye injury or disease. This may include, but not necessarily be limited to dry eye syndrome (DES); neurotrophic keratitis; persistent corneal epithelial defects; post-surgical recovery; and / or ocular burns. The ocular burns may comprise ocular thermal burns and / or ocular chemical burns.

[0168] Neurotropic keratitis, which may otherwise be referred to as keratopathy, will be understood to be a corneal degenerative disease characterised by a reduction or absence of corneal sensitivity. This can lead to epithelial keratopathy, epithelial defect, stromal ulceration and / or corneal perforation.

[0169] Persistent corneal epithelial defects will be understood to refer to failure of re-epithelisation and complete healing of any corneal injury within two weeks. Therefore, it will be appreciated that neurotrophic keratitis; persistent corneal epithelial defects; post-surgical recovery and ocular burns may also comprise persistent corneal epithelial defects.

[0170] In the context of the present invention, post-surgical recovery will be understood to mean that one or both eyes of the subject comprise a wound and / or stitching from surgery for which a therapeutically effective amount of the platelet lysate alleviates or reduces symptoms such as discomfort, dry eyes and / or may facilitate or hasten wound healing post-surgery. In such embodiments, the day of surgery may be considered day 0. The platelet lysate may be administered post-surgery, from day 0 or day 1. The platelet lysate may be administered for one, two, three or four weeks post-surgery.In embodiments where the condition is an eye condition, administration will typically be ocular (to the eye). Preferably, administration will be extraocular (topical to the eye). Topical eye administration may be via conjunctival / episcleral absorption (non-corneal) or transcorneal absorption.

[0171] In some embodiments, the platelet lysate is lyophilized. In some embodiments, the platelet lysate is in the form of a spray e.g. a nebulisation spray, a gel, a liquid drop or a cream.

[0172] The skin condition will be understood to be any injury or disease of or associated with the skin. A skin injury will be understood to include, but not necessarily be limited to a skin cut, laceration, ulcer puncture or burn.

[0173] The ulcer may comprise a chronic skin ulcer. The ulcer may otherwise be referred to as a sore. A chronic ulcer will be understood to be chronic, rather than acute, when the ulcer is present (and has not healed) for at least two weeks.

[0174] There are various causes of skin ulcers, including, but not limited to diabetes, bacterial, viral or fungal infection, impaired blood circulation, sustained pressure (i.e. bed sores) and cancer.

[0175] In embodiments where the condition is a skin condition, administration will typically be topical to the skin.

[0176] Administration of the platelet lysate may be daily or weekly, preferably daily. In some embodiments, administration of the platelet lysate is twice daily.

[0177] The invention also provides the platelet lysate of the second aspect for use in any of the therapeutic methods described above. Thus, also provided is the platelet lysate of the second aspect for use in a method of treating an eye or skin condition in a subject. It will be appreciated that embodiments relating to the method of treatment above may equally apply to the platelet lysate for use.

[0178] Also provided is the use of the platelet lysate of the invention for the manufacture of a medicament for the treatment of a skin or eye condition. Further provided is use of the platelet lysate of the invention for the treatment of a skin or eye condition.The invention also provides a kit comprising the platelet lysate of the invention. The kit may further comprise instructions for use. In some embodiments, the platelet lysate is provided in an aqueous solution, optionally buffered solution and / or at a temperature of at least -20°C.

[0179] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0180] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0181] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0182] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0183] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0184] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.Examples

[0185] EXAMPLE 1: Effect of incubation temperature on plasma activation

[0186] It was decided to assess the impact of incubation time and temperature on plasma activation. Specifically, it was determined whether increasing the incubation temperature and number of filtration steps of the platelet lysate preparation method could reduce incubation time while maintaining or improving the concentrations of growth factors, such as epidermal growth factor (EGF). The effect on fibrinogen concentration, a protein involved in blood clotting, was also assessed. Two protocols were utilized:

[0187] • Protocol 1 (comparative protocol): plasma from whole blood collected in citrate phosphate dextrose (CPD) + CaCh activation at 0.75 g / L, incubated overnight at room temperature, followed by two filtration steps.

[0188] • Protocol 2 (modified protocol): plasma from whole blood collected in CPD + CaCh activation at 0.75 g / L, incubated for 1 hour at 37°C, followed by 1 hour at 56°C, with three filtration steps.

[0189] The products produced by the two protocols were compared for fibrinogen concentration, EGF concentration, percentage volume retrieval, and consistency. Fibrinogen is the primary clotting component of platelets. A high concentration of fibrinogen indicates downstream fibrin clot formation. This, in turn, can affect growth factor concentration and reduce stability of the final product. Therefore, reducing and / or preventing fibrin clot formation in the platelet lysate product enhances suitability for ophthalmic use by minimizing particle-related concerns. EGF is more platelet-specific and its measurement reflects platelet activation more directly. Furthermore, higher EGF concentrations can be correlated with greater efficacy of platelet-rich plasma (PRP) in therapeutic applications, such as wound healing, tissue repair, and inflammation resolution. Improving percentage volume retrieval of plasma yield makes the method more efficient for large-scale and clinical use.

[0190] For EGF and fibrinogen concentrations, assessment was performed using Enzyme-linked immunosorbent assay (ELISA) following manufacturer’s instructions, EGF (Abeam), Fibrinogen (Abeam).

[0191] The results showed that there was no significant difference in fibrinogen concentration between the final plasma products generated by the two methods, despite increasing the temperature and substantially reducing incubation time for protocol 2 (1 hour vs. overnight) (Figure 1). The mean percentage volume retrieved for comparative protocol 1 was 49% and the mean volume retrieved for protocol 2 was 97.75%, with substantially lower variability observed in protocol 2 (standard deviation of 1.89 vs 56.58), demonstrating improved plasma collection efficiency (Figure 2 and Table 1).Mean Standard Deviation Confidence Interval (SD) (Cl)

[0192] Protocol 1 49 56.58 (-41.03, 139) Protocol 2 97.75 1.89 (94.74, 100.8)

[0193]

[0194] Table 1: Descriptive statistics. Mean percentage volume retrieved for each protocol

[0195] In addition, protocol 2 yielded EGF concentrations greater than comparative protocol 1 (123.7 pg / mL vs.

[0196] 102.4 pg / mL) (Figure 3 and Table 2) but had the advantage of lower variability and higher consistency in EGF concentration (standard deviation of26.53pg / mL vs 65.23pg / mL).

[0197] Mean Standard Deviation Confidence Interval (SD) (Cl)

[0198] Protocol 1 102.4 65.23 (15.25, 169.7) Protocol 2 123.7 26.53 (81.47, 165.9)

[0199]

[0200] Table 2: Descriptive statistics. Mean EGF concentration (pg / mL) retrieved for each protocol

[0201] These results show that the combination of higher incubation temperatures in protocol 2 significantly improves the efficiency of the plasma activation process without compromising growth factor concentrations or fibrinogen levels. These results demonstrate that modified protocol 2 enhances product reliability, volume retrieval, and consistency in EGF concentrations whilst reducing incubation time and variability in results.

[0202] EXAMPLE 2: Effect of increased CaCh concentration on plasma activation

[0203]

[0204] It was next decided to assess the impact on increased CaCh concentration. Therefore, the impact of incubation time and duration, the number of filtration steps, and increased CaCh concentration on the activation process of plasma from whole blood was assessed. Two protocols were utilized:

[0205] • Protocol 1 (comparative protocol): plasma from whole blood collected in CPD + CaCh activation at 0.75 g / L, incubated overnight at room temperature, followed by two filtration steps.

[0206] • Protocol 3 (modified protocol): plasma from whole blood collected in CPD + CaCh activation at 2 g / L, incubated at 37°C for 1 hour, followed by 56°C for 1 hour, with three filtration steps.

[0207] Protocol 1 was identical to comparative protocol 1 of Example 1. Protocol 3 was identical to protocol 2 of Example 1 , except that a higher CaCh concentration (2g / L vs. 0.75 g / L) was assessed.

[0208] Protocol 3 resulted in significantly lower fibrinogen levels and reduced fibrin clot formation (Figure 4), making the final platelet lysate product of protocol 3 more suitable for ophthalmic use. Protocol 3 also resulted in a higher and substantially more consistent percentage of plasma volume retrieval (Figure 5).

[0209] The mean percentage volume retrieved after protocol 3 was 94.63% compared to 49% in comparativeprotocol 1. The standard deviation in volume retrieved was reduced from 56.58% for protocol 1 to 4.96% using the modified protocol 3 (Table 3).

[0210] Mean Standard Deviation Confidence Interval (SD) (Cl)

[0211] Protocol 1 49 56.58 (-41.03, 139) Protocol 3 94.63 4.96 (86.74, 102.5)

[0212]

[0213] Table 3: Mean percentage volume retrieved for each protocol

[0214] Protocol 3 also improved consistency and reproducibility in EGF concentration compared to comparative protocol 1 (standard deviation of 25.87 pg / mL vs 65.23 pg / mL) (Figure 6 and Table 4).

[0215] Mean Standard Deviation Confidence Interval (SD) (Cl)

[0216] Protocol 1 102.4 65.23 (15.25, 169.7) Protocol 3 123.4 25.87 (82.25, 164.6)

[0217]

[0218] Table 4: Mean EGF concentration (pg / mL) retrieved for each protocol

[0219] Increasing the CaCh concentration to 2g / L and optimizing the incubation temperatures enhances the efficiency of the plasma activation process. This modification results in higher and more consistent EGF growth factor concentrations, significantly reduced fibrinogen concentration, reduced fibrin clot formation, and improved plasma volume retrieval.

[0220] EXAMPLE 3: Comparison of one versus multiple incubation steps and temperatures on plasma activation Three protocols were utilized to compare the effects of using individual incubations at select temperatures, following CaCh activation, versus the combination of two incubation steps at differing temperatures on the final plasma product:

[0221] • Protocol 3 (Combined Temperature Protocol): plasma from whole blood collected in CPD + CaCh activation at 2g / L, incubated at 37°C for 1 hour, followed by 56°C for 1 hour, with three filtration steps

[0222] • Protocol 4 (37°C for 1 hour): plasma from whole blood collected in CPD + CaCh activation at 2g / L, incubated at 37°C for 1 hour, followed by three filtration steps.

[0223] • Protocol 5 (56°C for 1 hour): plasma from whole blood collected in CPD + CaCh activation at 2g / L, incubated at 56°C for 1 hour, followed by three filtration steps.

[0224] Protocol 3 was identical to protocol 3 of Example 2.No major differences in EGF concentrations were observed across the different temperature protocols, but the combined temperature protocol (Protocol 3) resulted in the most consistent EGF concentration (Figure 7 and Table 5).

[0225] Mean Standard Deviation Confidence Interval (SD) (Cl)

[0226] Protocol 4 (37°C for 1 116.2 38.48 (54.95, 177.4)

[0227] hour)

[0228] Protocol 5 (56°C for 1 138 31.7 (87.52, 188.4)

[0229] hour)

[0230] Protocol 3 123.4 25.87 (82.25, 164.6) (combined

[0231] temperature)

[0232]

[0233] Table 5: Mean EGF concentration (pg / mL) retrieved for each protocol

[0234] The combined temperature protocol (Protocol 3) resulted in the highest percentage of plasma volume retrieval of 94.63%, compared to 82.75% with 37°C (Protocol 4) and 71.5% with 56°C (Protocol 5) (Figure 8 and Table 6).

[0235] Mean Standard Deviation Confidence Interval (SD) (Cl)

[0236] Protocol 4 (37°C for 1 82.75 17.23 (55.33, 110.2)

[0237] hour)

[0238] Protocol 5 (56°C for 1 71.5 33.55 (18.11, 124.9)

[0239] hour)

[0240] Protocol 3 94.63 4.96 (86.74, 102.5) (combined

[0241] temperature)

[0242]

[0243] Table 6: Mean percentage volume retrieved for each protocol

[0244] The combined temperature in protocol 3 also minimized fibrin clot formation (data not shown). This made the final product clearer and more suitable for ophthalmic use.

[0245] Using a combination of incubation temperatures (37°C and 56°C as in protocol 3) optimizes the plasma activation process by improving consistency, increasing plasma volume retrieval, reducing variability in EGF concentrations, and minimizing fibrin clot formation compared to the single temperature protocols at 37°C or 56°C (as in protocols 4 or 5 respectively).

[0246] EXAMPLE 4: Assessment of stability of platelet lysate

[0247] It was next decided to assess the stability of platelet lysate obtained according to protocol 3 of Example and Example 3. Hence, the platelet lysate was obtained by isolating plasma from a whole blood samplewhich comprised CPD, followed by CaCL activation at 2g / L, incubation at 37°C for 1 hour, followed by 56°C for 1 hour, with three filtration steps.

[0248] The platelet lysate was then pooled with a vehicle of 0.9% sodium chloride solution, with a final ratio of 4:1 sodium chloride solutiomplatelet lysate. This may otherwise be referred to as 20% platelet lysate. The product was frozen at -20°C, then the following day thawed and stored at 4°C or room temperature (30°C)

[0249] Stability was assessed by measuring the concentration of growth factors, pH and total protein concentration. Stability at 4°C was measured over a three-month period, with evaluation at 3, 2 and 1 months compared to a day 0 (no storage time). Stability at 30°C was measured over a 1.5-month period, with evaluation at 1 and 1.5 months compared to a day 0 (no storage time). The assessment was performed using Enzyme-linked immunosorbent assay (ELISA), EGF (Abeam), NGF (Abeam) and HGF (Abeam). Total protein quantification was performed using BSA (BIORAD), and pH was measured using LabSen pH electrode (Sanxin Apera).

[0250] For both assessments, day 0 was measured as the growth factor / pH / total protein concentration the day after thawing of the product.

[0251] Results for stability at 4°C for the concentration of the growth factors EGF, nerve growth factor (NGF) and human growth factor (HGF) are shown in Figure 9, along with the overall protein concentration in the sample (referred to as total protein concentration). Concentration of the three growth factors remained at similar levels throughout the three month period, with no significant changes. Results for stability at 4°C for pH are shown in Figure 10. Likewise, the pH remained in the safe range (6.5 to 8.6) to prevent corneal damage over the three month period, with only small decreases in pH.

[0252] These results demonstrate that the manufacturing method produces a stable plasma-derived product with sustained growth factor integrity during refrigerated long-term storage. Stability of both individual regenerative growth factors and total protein content is critical for reliable clinical dosing, logistical flexibility, and scalable manufacturing suitable for clinical and commercial use. Maintenance of stable pH during refrigerated storage of unopened bottles is critical for ocular safety, patient tolerability, and preservation of protein integrity. These data demonstrate that the manufacturing process yields a final plasma product with pH stability suitable for ophthalmic use, supporting real-world shipping, storage, and clinical deployment.

[0253] Similar stability results were observed at 30°C. Figure 11 shows that the concentration of EGF, HGF and NGF remained equivalent over the 1.5 month period, while Figure 12 shows that the pH remained in the safe range (6.5 to 8.6) to prevent corneal damage.These data demonstrate that the manufacturing method produces a plasma-derived product with maintained stability during short-term room-temperature exposure, extending beyond the intended in-use period. This supports product integrity, safety, and biological activity under expected real-world patientuse conditions and provides additional margin for handling variability. The pH stability is suitable for ophthalmic use under real-world in-use conditions, supporting safe and reliable clinical use throughout the intended dosing period.

[0254] EXAMPLE 5: In vivo assessment of treatment of a dry eye model

[0255] Pre-clinical animal studies were conducted to evaluate the use of eyedrops made according to the invention. Briefly, the platelet lysate was obtained according to protocol 3 of Examples 2 and 3. The lysate was tested in a mouse dry eye model.

[0256] The platelet lysate was pooled with a vehicle of 0.9% sodium chloride solution. Three groups of C57 / B6 mice, with 4 - 5 mice in each group, were utilized for the study to compare the effect of 1%, 10%, and 20% platelet lysate to vehicle only. The 1%, 10% and 20% platelet lysate may otherwise be referred to as 1% OB-1, 10% OB-1 and 20% OB-1, respectively. Table 7 shows the experimental schematic.

[0257] All experimental procedures were conducted in accordance with ethical guidelines and approved protocols. All efforts were made to minimize animal suffering. Adult C57 / B6 mice were used for the study. Prior to any intervention, mice were anesthetized with isoflurane to ensure immobilization during the procedures. A single drop of proparacaine was instilled in each eye to alleviate discomfort. Corneal debridement was performed as follows: 20% ethanol (EtOH) was applied onto a 2mm sterile filter paper for 30 seconds, followed by a single wash with phosphate-buffered saline (PBS). Epithelial removal was achieved using a golf club spud. The corneal surface was immediately imaged after debridement to assess the extent of epithelial removal. Subsequently, a 10-second application of fluorescein was followed by a PBS wash, and the corneal surface was imaged using cobalt blue light to visualize fluorescein staining.

[0258] For pre-treatment, 10pl of 0.1% benzalkonium chloride (BAC) was applied for 10 seconds and then washed with PBS four times. The treatments were initiated at 0 hours (h) as follows: application of 10 pl of the respective solutions twice daily (BID) - vehicle, OB-1 1%, OB-1 10%, and OB-1 20%. Images were captured at Oh, 24h, and 48h intervals. Two types of images were acquired at each time point: 1) corneal surface without fluorescein and 2) corneal surface with fluorescein. Mechano-sensation of the cornea was assessed using the Cochet-Bonnet esthesiometer at Oh, 24h, and 48h post-treatment. These methods allowed for the comprehensive evaluation of corneal epithelial healing, fluorescein staining, and corneal mechano-sensation over the designated time points and treatment conditions.Group Scratch Method Pre-Treatment Treatment

[0259] 1 20%EtOH 30sec + wash + 0.1% BAC 10 sec Vehicle BID

[0260] Golf club spud

[0261] 2 20%EtOH 30sec + wash + 0.1% BAC 10 sec 1% platelet lysate BID (OB-1 1%) Golf club spud

[0262] 3 20%EtOH 30sec + wash + 0.1% BAC 10 sec 10% platelet lysate BID (OB-1 10%) Golf club spud

[0263] 4 20%EtOH 30sec + wash + 0.1% BAC 10 sec 20% platelet lysate BID (OB-1 20%) Golf club spud

[0264]

[0265] Table 7: Experimental Schematic

[0266] Photos of the ocular surface using fluorescein staining were assessed at Oh, 24h, and 48 hours for defect area and wound closure rate over the study's time course. The area of the defect (%) was first assessed immediately, at Day 0, after epithelial scraping to provide baseline measures for each group.

[0267] At the 24-hour timepoint, eyes treated with vehicle showed the least wound closure. There was a slight dose-dependent increase in wound closure with increasing concentration of the platelet lysate

[0268] (1%, 10%, 20%). The 20% platelet lysate performed better than lower concentrations treatment, with all platelet lysate concentrations performing better than vehicle. At 48 hours, all platelet lysate concentrations resulted in similar wound closure, with vehicle treatment resulting in an overall lesser effect (Figure 13).

[0269] Hematoxylin and eosin (H&E) histology staining of the mouse cornea was performed to determine the corneal epithelial thickness of treated eyes in each group at 48 hours (images not shown). The images were processed, and thickness was calculated for each group (Figure 14). As in the other endpoints, increasing platelet lysate concentration correlated with increasing epithelial thickness, indicating more successful wound closure and healing, with the highest median of any group being 20% platelet lysate. Both 20% and 10% platelet lysate products performed better than the vehicle only group.

[0270] EXAMPLE 6: Exemplary protocol

[0271] The following exemplary protocol may be utilized, which corresponds to Protocol 3 of Examples 2 and 3:

[0272] A method for preparing platelet lysate from at least one plasma sample, the method comprising:

[0273] • activating platelets in the plasma sample by adding 2g / L CaCh;

[0274] • incubating the CaCh-containing sample at 37°C for a first incubation period of one hour;

[0275] • inactivating complement in the plasma sample by incubating the CaCh-containing sample for a second incubation period at a temperature of 56°C for one hour;• centrifuging the complement inactivated sample at 3500 x g for 30 min at room temperature to obtain a centrifuged plasma sample

[0276] • Combining the centrifuged plasma sample with a vehicle comprising 0.9% sodium chloride solution at a ratio of 4:1 vehicle to plasma sample; and

[0277] • Filtering the combined sample + vehicle through a first filter of 0.8 pm, a second filter of 0.45 pm and a third filter of 0.22 pm to obtain the platelet lysate product.

[0278] The plasma sample was isolated from a whole blood sample which comprised citrate phosphate dextrose (CPD). Prior to activating the platelets in the method, the method further comprised standardising the platelet concentration in the plasma sample, to result in a standardised plasma sample comprising approximately 1.5x108- 5.0x108platelets / mL, preferably 4x108platelets per mL.

[0279] Optionally, the platelet lysate product can be frozen at -20°C for storage.

[0280]

[0281] EXAMPLE 7: Assessment mass

[0282] It was decided to assess the protein stability of the platelet lysate in OB-1 formulation after 30 days of 30°C storage compared to freshly thawed material. OB-1 formulation was made using Protocol 3 of Examples 2 and 3.

[0283] The sample groups used were:

[0284] • DO (Fresh): Freshly thawed OB-1 (n=3 technical replicates)

[0285] • RT (Room Temperature): OB-1 stored at 30°C for 30 days (n=3 technical replicates)

[0286] Methods

[0287] Tandem Mass Tag (TMT) quantitative mass spectrometry was used to identify and quantify proteins using LC-MS / MS technology with TMT isobaric labeling (Mtoz Biolabs). Protein abundance was measured as TMT intensity values. Error bars shown represent standard error of the mean (SEM). Individual data points overlaid on bars (circles).

[0288] Protein quantification was carried out using mass spectrometry-based proteomics software.

[0289] Normalization was carried out using protein abundance as measured by TMT intensity values.

[0290] Fold change calculation was done using the formula: “Fold Change = Mean(RT) / Mean(DO)”. Log2 transformation was applied for visualization and statistical testing.For statistical testing, Welch's t-test (unpaired, two-tailed, unequal variance) was used as Welch's t-test does not assume equal variances between groups, making it more robust for proteomic data where variance may differ between conditions. The Python Implementation of this used:

[0291] - Package: scipy. stats (version 1.16.3)

[0292] - Function: scipy. stats.ttest_ind(group1 , group2, eq ual_var= False)

[0293] - Parameters:

[0294] * equal_var=False (uses Welch's correction)

[0295] * alternative='two-sided'

[0296] Benjamini-Hochberg False Discovery Rate (FDR) correction was used as multiple testing correction as it controls the expected proportion of false positives among rejected hypotheses when testing multiple proteins simultaneously. The Python Implementation of this used:

[0297] - Package: scipy. stats (version 1.16.3)

[0298] - Function: scipy. stats.false_discovery_control(p_values)

[0299] - Significance threshold: FDR-adjusted p-value < 0.05

[0300] The following fold change thresholds were used in stability classification:

[0301] - Stable: 0.83 < Fold Change < 1.2 (i.e. , within ±20% of baseline)

[0302] - Increased: Fold Change > 1.2 (>20% increase)

[0303] - Decreased: Fold Change < 0.83 (>20% decrease)

[0304] For biological significance:

[0305] - Significantly Up-regulated: FDR-adjusted p < 0.05 AND Fold Change > 1.2

[0306] - Significantly Down-regulated: FDR-adjusted p < 0.05 AND Fold Change < 0.83

[0307] - Not Significant: FDR-adjusted p > 0.05 OR within stability range

[0308] Python matplotlib (version 3.x) and seaborn were use to visualize the data and generate a volcano plot (Log2 FC vs -Log10 p-value); box plots with swarm plots for individual proteins; fold change distribution histogram; bargraph.

[0309] Results

[0310] 423 proteins were identified in the OB-1 formulation.

[0311] Pearson correlation coefficient was used to assess sample reproducibility between replicates. Within DO group r = 0.996-0.998 which indicates excellent reproducibility. Within RT group r = 0.995-0.997. Again there was excellent reproducibility. Between DO and RT groups r = 0.992-0.995, which indicates highsimilarity. A high correlation (r > 0.99) between DO and RT samples indicates minimal protein profile changes during storage.

[0312] Figure 15 shows a volcano plot of protein stability analysis showing the relationship between fold-change and statistical significance for the 423 proteins identified in the OB-1 formulation. Only 0.5% of proteins showed statistically significant changes, indicating excellent protein stability during RT storage.

[0313] Figure 16 shows the distribution of protein fold changes. The distribution is centered near zero, indicating minimal overall protein changes during storage. Of the total proteins (423); 230 (54.4%) were stable (0.83-1.2x); 122 (28.8%) were increased (>1.2x); 71 (16.8%) were decreased (<0.83x). The symmetric, near-normal distribution centered around FC=1 demonstrates that observed protein changes represent normal biological variation rather than systematic degradation.

[0314] Figure 17 shows a stability analysis of key therapeutic proteins. Fold changes for the protein were:

[0315] Lactoferrin (FC=1.12, p=0.341), Fibronectin (FC=1.09, p=0.428), Albumin (FC=0.86, p=0.156), Transferrin (FC=0.86, p=0.143), Haptoglobin (FC=0.98, p=0.892), IGFBP3 (FC=1.06, p=0.567), PDGF-A (FC=0.92, p=0.289), Collagen III (FC=1.05, p=0.512). All displayed proteins showed fold changes within the stability range (0.83-1.2x) and no statistical significance (all p > 0.05), confirming preservation of therapeutic components during storage.

[0316] Figure 18 shows stability analysis of therapeutic proteins organized by functional category. A: Wound healing factors: LTF (Lactoferrin) - primary therapeutic protein with antimicrobial and pro-regenerative properties; FN1 (Fibronectin) - essential ECM protein providing scaffold for epithelial cell migration;

[0317] HGFAC (Hepatocyte Growth Factor Activator) - activates HGF pathway to enhance epithelial proliferation. B: Extracellular matrix (ECM) proteins: COL3A1 (Collagen Type III) - structural protein, component of provisional wound matrix; VTN (Vitronectin) - cell adhesion protein supporting spreading and migration; TGFBI (TGF-p-induced protein) - ECM organization and tissue remodeling. C: Growth factors & binding proteins: PDGFA (Platelet-Derived Growth Factor A) - mitogen stimulating epithelial cell proliferation and migration; IGF2 (Insulin-like Growth Factor II) - promotes cell growth and survival; IGFBP3 (IGF Binding Protein 3) - modulates IGF bioavailability and activity. D: Anti-inflammatory & antimicrobial: ALB (Albumin) - major antioxidant protein with anti-inflammatory properties; HP (Haptoglobin) - hemoglobin scavenger, reduces oxidative stress; TF (Transferrin) - iron sequestration for antimicrobial activity. E: Cell adhesion & immune: CDH1 (Cadherin-1 / E-cadherin) - essential for epithelial barrier integrity and cell-cell adhesion; C3 (Complement C3) - central complement protein for antimicrobial defence and debris clearance. All proteins across all functional categories were stable, showing no significant changes (p > 0.05, FDR-corrected).The results support protein stability of the OB-1 formulation during RT storage.

[0318] EXAMPLE 8: Phase I Clinical Trial

[0319]

[0320] Across all OB-1 pre-clinical studies no evidence of cytotoxicity was observed, pH and protein concentrations remained within safe ophthalmic ranges, fibrinogen and particulate risk were minimized through processing and stability data supported real-world storage and distribution. These findings support advancement into human clinical trials.

[0321] The pre-clinical program demonstrates that: regenerative blood-derived biology can be industrialized, growth factor profiles can be consistently controlled, stability is maintained under real-world conditions, and in vivo efficacy supports clinical translation.

[0322] This pre-clinical foundation directly enabled progression into first-in-human clinical evaluation and supports continued advancement toward Phase II and Phase III development.

[0323] Study Rationale

[0324] Although Phase I studies are generally not required for dry eye disease products, Ocubio elected to conduct a first-in-human Phase I trial for OB-1 to:

[0325] • Establish safety and tolerability of an allogeneic platelet lysate

[0326] • Confirm feasibility of topical administration.

[0327] • Validate Phase II clinical endpoints.

[0328] • Strengthen the IND and regulatory dialogue.

[0329] Treatment Regimen:

[0330] • OB-1 (20% platelet lysate)

[0331] • Topical administration, three times daily

[0332] • Treatment duration: 12 weeks

[0333] Inclusion and Exclusion Criteria (Summary)

[0334] Key Inclusion Criteria:

[0335] • Subjects age > 18 years at the time of signing the Informed Consent Form.

[0336] • Male or Female.

[0337] • Provide written informed consent.

[0338] • VAS scale (0-100 scale) for Dry Eye symptom score > 40.

[0339] • Ocular Surface Index Score [OSDI] > 23.

[0340] • Corneal Fluorescein Staining (CorFS) score (modified National Eye Institute (NEI) Industry Workshop 0-15 scale, 0-3 scale in each of 5 corneal regions, meeting all of the following in at least one eye:

[0341] >1 in the central region (cCFS [NEI; 0-3 scale])>2 in the inferior region (iCFS [NEI; 0-3 scale])

[0342] • Conjunctival Fluorescein Staining (ConjFS) score >2 (modified National Eye Institute (NEI) Industry Workshop 0-6 scale, 0-3 scale in nasal and temporal conjunctiva regions, in at least one eye.

[0343] • Schirmer’s test without anesthesia < 10 mm / 5min in the eligible eye.

[0344] • Best Corrected Visual Acuity < 0.7 logMAR (>20 / 100 Snellen or >50 ETDRS) in the eligible eye.

[0345] • Subjects must be able to return for multiple follow-up visits.

[0346] • For Women of Child-Bearing Potential (WOCBP) only, willingness to use FDA-recommended birth control until 6 months post treatment.

[0347] Key Exclusion Criteria:

[0348] • Previous use of any type of blood-derived product as ocular treatment, within the past 6 months.

[0349] However, patients on serum tear therapy may be enrolled after a 2-week wash out.

[0350] • Known hypersensitivity to any of the procedural compounds (fluorescein, sodium chloride, proparacaine).

[0351] • History of any type of ocular surgery within 2 months. Refractive surgeries are acceptable.

[0352] • Current diagnosis of any of the following conditions:

[0353] o Acute allergic conjunctivitis

[0354] o Infection (i.e. bacterial, viral, protozoan or fungal infection of the cornea, conjunctiva, lacrimal gland, lacrimal sac or eyelids)

[0355] o Inflammation (i.e. retinitis, macular inflammation, choroiditis, uveitis, scleritis, episcleritis, keratitis)

[0356] • History, by patient report, of ocular herpetic keratitis.

[0357] • Severe meibomian gland dysfunction (immediate TBUT) or severe blepharitis.

[0358] • Any concomitant treatment or alteration of the dose of systemic medications at the time of entry into the study that could interfere in the assessment of the trial in the Investigator s opinion. However, if concomitant treatment is stable for at least 3 months on the same dose and not expected to change during the course of the trial, the subject may be enrolled.

[0359] • Subject has or had an active systemic infection requiring antibiotics within 12 weeks of enrolment in the study.

[0360] • Inability to perform any of the assessments required for endpoint analysis.

[0361] • Current, previous chronic or recurrent medical condition which, according to the Investigator, might impact the interpretation of the study results or put the subject at risk. However, if the medical condition is treated and is stable for at least 3 months on the same dose and is not expected to change during the course of the trial, the subject may be enrolled.

[0362] • Unwilling to withhold the use of any eye medication for the study duration.

[0363] • History of penetrating corneal transplant surgery. Except endothelial transplants are acceptable after 2 months post-surgery.

[0364] • Eyelid abnormalities that affect lid function (ectropion, entropion, ptosis, trichiasis).

[0365] • End-Stage ocular surface scarring or condition that may compromise ocular surface integrity, such as Steven Johnson syndrome, prior chemical burn, ocular cicatricial pemphigoid with keratinization of the cornea and conjunctiva.

[0366] • tCFS score > 9 (NEI; 0-15 scale) or a score=3 in the superior region (sCFS [NEI; 0-3 scale]) or subjects who have CFS with diffuse confluent staining, filaments or frank epithelial defects. • Use of glaucoma medication or history of filtering surgery for glaucoma

[0367] These criteria were designed to enrol a representative population of patients with moderate dry eye disease while minimizing confounding factors and ensuring patient safety in a first-in-human study.Endpoints and Assessments

[0368] Primary objective:

[0369] • Safety and tolerability

[0370] Secondary / exploratory endpoints:

[0371] • Ocular Surface Disease Index (OSDI)

[0372] • Visual Analog Scale (VAS) symptom scores

[0373] • Corneal staining

[0374] • Conjunctival staining

[0375] • Schirmer test

[0376] • Tear Break-Up Time (TBUT)

[0377] Data Collection and Management

[0378] • An independent regulatory monitor was engaged to oversee study conduct, data integrity, and compliance with applicable clinical and regulatory standards.

[0379] • Standardized clinical grading scales used.

[0380] • Data cleaning and lock completed.

[0381] • Full patient-level dataset available in structured electronic format.

[0382] Statistical Analysis

[0383] Longitudinal trends across all time points (baseline, 3, 6, 9, and 12 weeks) were assessed using the Friedman test, and pairwise differences from baseline were evaluated using Wilcoxon signed-rank tests with Bonferroni correction to account for multiple comparisons. Linear Mixed Models with a random intercept for patient were used to estimate the overall change over time while accounting for the paired nature of bilateral eye data; Generalized Estimating Equations were used where convergence was not achieved. Visual acuity was converted from Snellen to LogMAR prior to analysis. All methods appropriately handled missing data from incomplete visits and dropouts. Statistical significance was set at p < 0.05 after correction.

[0384] This approach was appropriate given:

[0385] • Small sample size

[0386] • Exploratory nature of Phase I

[0387] • Focus on signal detection rather than hypothesis confirmation

[0388] Results

[0389] Safety and Tolerability

[0390] • No serious adverse events

[0391] • No treatment-limiting toxicity

[0392] • Favorable ocular tolerability

[0393] Clinical Signs

[0394] 23 patients were evaluated for corneal and conjunctival staining with fluorescein. Both eyes were evaluated in 20 patients and one eye in 3 patients. 16 patients completed all visits. There were 179observations across 5 timepoints. A higher fluorescein staining score indicates a less healthy ocular surface and greater dry eye severity.

[0395] Figures 20 and 21 show a statistically significant improvement in corneal staining in patients treated with OB-1 ophthalmic solution over a 12-week treatment period. Data were analyzed using linear mixed-effects modeling to account for repeated measures and within-patient correlations between fellow eyes. Results showed 23 patients (43 eyes, 179 observations) with a highly significant decrease in corneal staining overtime (time coefficient p = -0.6078, p<0.0001) and no significant difference between eyes (Eye diff p = 0.6307). Both eyes demonstrate a progressive decrease in corneal staining scores from baseline (~4.9 points) to 12 weeks (~1.3 points), representing a 73% reduction in corneal surface damage with sustained improvement throughout the treatment period, with statistically significant improvements beginning at 3 weeks in both eyes.

[0396] Figures 22 and 23 show a statistically significant improvement in conjunctival staining in patients treated with OB-1 ophthalmic solution over a 12-week treatment period. The figure show parallel improvement patterns with overlapping error bars (±1 SD), demonstrating bilateral consistency of treatment response. Data were analyzed using linear mixed-effects modeling to account for repeated measures and within-patient correlations between fellow eyes. Results showed 23 patients (20 both eyes, 3 one eye, 179 observations) with a highly significant decrease in conjunctival staining overtime (Time coefficient p = -0.7664, p<0.0001) and no significant difference between eyes (Eye diff p = 0.9043). Both eyes demonstrate a progressive decrease in conjunctival staining scores from baseline (-4.3 points) to 12 weeks (-1.0 point), representing a 77% reduction in ocular surface damage with sustained improvement throughout the treatment period with statistically significant improvements beginning as early as 3 weeks in both eyes.

[0397] These results indicate a meaningful improvement in conjunctival surface health and inflammation over time, supporting a sustained regenerative effect on the ocular surface rather than a transient or symptomatic response.

[0398] Symptoms

[0399] The Ocular Surface Disease Index (OSDI) is a validated 12-item questionnaire used to assess the severity of dry eye disease symptoms, including ocular discomfort, light sensitivity, grittiness, blurred vision, and their impact on vision-related functioning such as reading, driving, and screen use. Scores range from 0 to 100, with higher scores indicating greater symptom severity and disability.

[0400] Patients showed significant improvement in OSDI scores, with early onset and sustained benefit (Figure 24). A progressive and sustained reduction in OSDI scores was observed across the study period,indicating improvement in patient-reported dry eye symptoms. Statistically significant improvements relative to baseline were observed as early as Week 3 and were maintained through Weeks 6, 9, and 12. These results demonstrate a clinically meaningful and durable improvement in dry eye symptoms, consistent with restoration of ocular surface health rather than transient symptomatic relief, and align with the observed improvements in objective clinical signs.

[0401] Patient symptoms were also assessed using multiple Visual Analog Scales (VAS). Improvements were observed across symptom domains, including:

[0402] • Burning sensation (Figure 25A)

[0403] • Foreign body sensation (Figure 25B)

[0404] • Photophobia (Figure 25C)

[0405] • Itching (Figure 25D).

[0406] Schirmer’s test was used to measure tear production and the results are shown in Figure 26A. The length (in mm) of paper along which the tears travel from the eye indicates the level of tear production, with higher values reflecting greater tear secretion. A tear break-up time (TBUT) test was used to measure tear film stability. Following instillation of fluorescein, the time elapsed (in seconds) between a complete blink and the first appearance of a dry spot indicates how long tears remain stable on the surface of the eye, shorter times indicate greater instability. The results are shown in Figure 26B.

[0407] No statistically significant changes were observed in Schirmer testing or tear break-up time (TBUT). A directional trend toward improvement was noted, which may reach statistical significance in a Phase II study with a larger sample size and appropriate powering. Importantly, this outcome was expected and is consistent with the regenerative mechanism of action, which primarily targets epithelial integrity and neurosensory function rather than tear secretion.

[0408] Best corrected visual acuity (BCVA) was measured in patients to assess their vision, and the results (in logMAR units) are shown in Figure 27. Negative values represent an improvement in visual acuity (lower logMAR = better vision). Linear mixed-effects model (LMM) analysis demonstrates stable visual acuity over the 12-week treatment period. Time coefficient p = -0.0002 logMAR / week (95% Cl: -0.0051, 0.0047), p = 0.9479 (not significant), indicating no significant change in BCVA over time. Average change: 0.0002 logMAR / week = 0.00 ETDRS letters / week. The model accounts for within-patient correlation, confirming that OB-1 treatment maintains stable visual acuity with no adverse effects on vision throughout the study period.

[0409] Clinical Implications

[0410] The Phase I study demonstrated that the OB-1 formulation:• Is safe and well tolerated in humans.

[0411] • Produces clinically meaningful improvements in signs and symptoms.

[0412] • Supports the selected dose and regimen.

[0413] • Validates Phase II endpoint selection.

[0414] These results provide a strong foundation for progression into randomized efficacy trials.

[0415] References

[0416] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

[0417] Baturone R, Soto MJ, Marquez M, et al. Health-related quality of life in patients with primary Sjogren's syndrome: relationship with serum levels of proinflammatory cytokines. Scand J Rheumatol.

[0418] 2009;38(5):386-9. doi:10.1080 / 03009740902973821

[0419] Higuchi A. Autologous Serum and Serum Components. Invest Ophthalmol Vis Sci. Nov 1 2018;59(14):DES121-DES129. doi:10.1167 / iovs.17-23760

[0420] Kesmarky, G., et al., Plasma viscosity: a forgotten variable. Clin Hemorheol Microcirc, 2008. 39(1-4): p.

[0421] 243-6.

[0422] Schriner, J.B., et al., Citrate Phosphate Dextrose Alters Coagulation Dynamics Ex Vivo. J Surg Res, 2023. 291: p. 43-50.

[0423] Shen Lee B, Kabat AG, Bacharach J, Karpecki P, Luchs J. Managing Dry Eye Disease and Facilitating Realistic Patient Expectations: A Review and Appraisal of Current Therapies. Clin Ophthalmol.

[0424] 2020;14:119-126. doi:10.2147 / OPTH.S228838

[0425] Stapleton F, Alves M, Bunya VY, et al. TFOS DEWS II Epidemiology Report. Ocul Surf. Jul

[0426] 2017;15(3):334-365. doi : 10.1016 / j Jtos.2017.05.003

[0427] White DE, Zhao Y, Jayapalan H, Machiraju P, Periyasamy R, Ogundele A. Treatment Satisfaction Among Patients Using Anti-Inflammatory Topical Medications for Dry Eye Disease. Clin Ophthalmol.

[0428] 2020;14:875-883. doi :10.2147 / opth.S233194

[0429] Yu J, Asche CV, Fairchild CJ. The economic burden of dry eye disease in the United States: a decision tree analysis. Cornea. Apr 2011 ;30(4):379-87. doi : 10.1097 / ICC.0b013e3181 f7f363For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

Claims

Claims1. A method for preparing platelet lysate from at least one plasma sample, the method comprising:a) activating platelets in the plasma sample by adding between 1 g / L to 3 g / L CaCh;b) optionally incubating the CaCh-containing sample for a first incubation period;c) inactivating complement in the plasma sample by incubating the CaCh-containing sample for a second incubation period at a temperature of from 30 °C to 60 °C;d) centrifuging the complement inactivated sample, optionally at room temperature;e) pooling the at least one plasma sample (supernatant), and optionally combining the pooled samples with a vehicle;f) filtering the product of step e) through at least at first, a second and a third filtration step to produce the platelet lysate product; andg) optionally freezing the platelet lysate product for storage.

2. The method of claim 1 , wherein the method comprises a preceding step of isolating the plasma sample from a whole blood sample; optionally wherein the whole blood sample comprises an anti-coagulant;further optionally wherein the anti-coagulant is citrate phosphate dextrose (CPD); optionally wherein the ratio of CPD to blood in the whole blood sample is 1 :

63. The method of claim 2, wherein the plasma isolation step comprises centrifuging the whole blood sample at 1000g for at least 5 minutes, preferably 9 minutes;optionally wherein the centrifuging is performed at room temperature;further optionally wherein the centrifuging is performed within 8 hours of the collection of the whole blood sample.

4. The method of any one of the preceding claims, wherein the first, second and third filtration steps are performed through filters of progressively smaller sizes.

5. The method of claim 4, wherein:i) the first filtration step is performed through a 0.6-1.0 pM filter, preferably a 0.8 pM filter;ii) the second filtration step is performed through a 0.3-0.6 pM filter, preferably 0.45 pM filter; and / oriii) the third filtration step is performed through a 0.1-0.3 pM filter, preferably a 0.22 pM filter.

6. The method of any one of the preceding claims, wherein the first incubation period is for a duration of from 30 minutes to 24 hours, optionally for a duration of 1 hour.

7. The method of any one of the preceding claims, wherein the first incubation period is performed at a temperature of from 30-60 °C.

8. The method of any one of the preceding claims, wherein the second incubation period is performed at a temperature at least 10°C greater than the temperature of the first incubation period.

9. The method of any one of the preceding claims, wherein the first incubation period is performed at a temperature of from 30 to 40°C, optionally at 37 °C.

10. The method of any one of the preceding claims, wherein the second incubation period is for a duration of between 30 minutes and 2 hours, optionally for a duration of 1 hour.

11. The method of any one of the preceding claims, wherein the second incubation period is performed at a temperature of from 50-60 °C, preferably at 56 °C.

12. The method of any one of the preceding claims, wherein the duration of the first and the second incubation period combined is less than 8 hours, optionally less than 5 hours, further optionally less than 3 hours.

13. The method of any one of the preceding claims, wherein prior to activating the platelets in step a), the method further comprises standardising the platelet concentration in the plasma sample, optionally wherein the standardisation results in a standardised plasma sample comprising approximately 1.5x108- 5.0x108platelets / mL, preferably 4x108platelets per mL.

14. The method of claim 13, wherein standardising the platelet concentration comprises:i) determining the platelet concentration in the sample;ii) centrifuging the sample; andiii) discarding a portion of the supernatant or the whole or a portion of the pellet to achieve a standard concentration (e.g. approximately 4x108platelets per mL) of platelets in the standardised plasma sample.

15. The method of claims 13 or 14, wherein the method further comprises a step of freezing the standardised plasma sample, and subsequently thawing the sample prior to activating the platelets in step a).

16. The method of any one of the preceding claims, wherein activating the platelets in step a) comprises adding 2 g / L of CaCh.

17. The method of any one of the preceding claims, wherein the at least one plasma sample is pooled with a vehicle in step e), optionally wherein the vehicle comprises sodium chloride solution, further optionally 0.9% sodium chloride solution.

18. The method of claim 17, wherein the vehicle is mixed with the at least one pooled plasma sample at a ratio of 1 :1 to 10:1.

19. The method of claim 18, wherein the vehicle is mixed with the at least one pooled plasma sample at a ratio of 4:1.

20. The method of claim 19, wherein the concentration of EGF in the resulting platelet lysate product is of from 80 pg / mL to 200 pg / mL.

21. The method of claim 19 or claim 20, wherein the concentration of fibrinogen in the resulting platelet lysate product is of from 0.3 pg / mL to 2 pg / mL.

22. The method of any one of claims 19 to 21 , wherein the concentration of NGF in the resulting platelet lysate product is of from 30 to 50 pg / mL.

23. The method of any one of claims 19 to 21 , wherein the concentration of HGF in the resulting platelet lysate product is of from 80 to 300 pg / mL.

24. The method of any one of the preceding claims, wherein the platelet lysate product produced in step f) has a volume of at least 50 mL, at least 250 mL, at least 500 mL, at least 750 mL, at least 1 L, at least 5 L, or at least 10 L.

25. The method of any one of the preceding claims, wherein the method is performed a plurality of times to produce a plurality of platelet lysate samples, and wherein:a) the percentage of volume retrieved (VR) is between 60% to 100%; and / orb) the batch-to-batch standard deviation in percentage of volume retrieved (MVR) is 5%.

26. The method of any one of the preceding claims, wherein the method comprises:(a) activating platelets in the plasma sample by adding between 1 g / L to 3 g / L CaCh;(b) incubating the CaCh-containing sample for a first incubation period at a temperature of 37°C for a duration of 1 hour;(c) inactivating complement in the plasma sample by incubating the CaCh-containing sample for a second incubation period at a temperature of 56°C for a duration of 1 hour;(d) centrifuging the complement inactivated sample;(e) pooling the at least one plasma sample (supernatant), and optionally combining the pooled samples with a vehicle;(f) filtering the product of step e) through at least at first, a second and a third filtration step to produce the platelet lysate product; and(g) optionally freezing the platelet lysate product for storage.

27. A platelet lysate produced by the method of any one of claims 1 to 26, wherein the concentration of EGF is of from 80 pg / mL to 200 pg / mL.

28. The platelet lysate of claim 27, wherein the concentration of fibrinogen is no higher than 2 pg / mL.

29. The platelet lysate of claim 27 or claim 28, wherein the platelet lysate has a volume of at least 50 mL, at least 250 mL, at least 500 mL, at least 750 mL, at least 1 L, at least 5 L, or at least 10 L.

30. A method of treating an eye or skin condition in a subject, the method comprising administering to the eye or skin of the subject in need thereof a therapeutically effective amount of the platelet lysate of any one of claims 27 to 29.

31. The method of claim 30, wherein the platelet lysate is allogeneic.

32. The method of claim 30 or claim 31 , wherein the eye condition is selected from dry eye syndrome (DES); neurotrophic keratitis; persistent corneal epithelial defects; post-surgical recovery; and / or ocular burns.

33. The method of any one of claims 30 to 32, wherein the skin condition comprises a skin wound, optionally a skin cut or laceration, an ulcer and / or a skin burn.

34. The method of claim 33, wherein the ulcer comprises a diabetic and / or chronic ulcer.

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