Protein-enhanced compositions for topical delivery

Abstract

Serum-based topical compositions for use in companion animals and associated methods are described herein. The use of xenogeneic serum can decrease preparation time as well as decrease the risk of infections and incompatibilities between donor and recipient. Moreover, by lysing serum, additional growth factors and proteins are made bioavailable, enhancing antimicrobial and wound-healing properties. The use of bovine serum as a starting material further reduces albumin concentrations relative to autologous human or animal serum, minimizing antibiotic metabolism and enabling synergistic efficacy when co-administered with antimicrobial agents.

Description

PROTEIN-ENHANCED COMPOSITIONS FOR TOPICAL DELIVERYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application No.63 / 711,906, entitled “COMPOSITIONS WITH PROTEIN ADDITIVE FOR TOPICAL OPHTHALMIC DELIVERY IN COMPANION ANIMALS ’, filed on October 25, 2024, the contents of which are hereby incorporated herein by reference in their entirety.

[0002] This application is related to International Patent Application No. PCT / US2025 / 013143. entitled “Serum Compositions With Additive For Ocular Delivery in Animals,” filed on January 27, 2025, the contents of which are hereby incorporated herein by reference in their entirety.BACKGROUND

[0003] In the veterinary field, serum-based lubricants are known to effectively mitigate comeal degradation and ulcer development, while also tending to stabilize the cornea scaffolding. Additionally, serum-based lubricant products can be beneficial for certain other conditions, such as indolent ulcers, corneal trauma, allergic conjunctivitis, KCS (dry eye), and post-operative care.

[0004] Eye diseases and comeal disorders in veterinary medicine are often treated with autologous or allogeneic blood serum compositions. These compositions contain natural growth factors, proteins, and vitamins beneficial for ocular surface healing. However, existing serum-based therapies present limitations:• Conventional autologous serum requires time-consuming in-clinic preparation and quality inconsistencies.• Allogeneic serums present the risk of infections due to same-species pathogens as well as incompatibilities between donor and recipient• Albumin, a predominant serum protein, has been shown to metabolize antibiotics, thereby reducing the efficacy of adjunct antimicrobial therapies.• Serum growth factors are incompletely liberated in unmodified serum, limiting their therapeutic potential.

[0005] Accordingly, there is a need for improved serum-based topical compositions that increase therapeutic factor availability', reduce components that interfere with adjunct therapies, and offer synergistic antimicrobial and wound-healing benefits.TECHNICAL FIELD

[0006] The present disclosure relates generally to compositions for topical use (e.g. ocular delivery) in animals, including methods of manufacture and apparatuses for preparing the same.TERMS AND DEFINITIONS

[0007] The term “companion animal'’ as used herein refers to non-food chain animals that are not typically used for human consumption.

[0008] Each instance of the term “serum” as used herein refers to blood-derived serum except where expressly stated otherwise.

[0009] In some examples a "therapeutic effect” can be an ameliorating effect that includes any therapeutically beneficial result in the treatment of a condition. For instance, an ameliorating effect can include a lessening in the severity or progression of a condition, remission, or a cure thereof.

[0010] In certain examples, a “therapeutic effect” can be a preventative effect or a prophylactic effect. The term “preventing” is art-recognized to include administration of a pharmaceutical or other remedy which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition.DETAILED DESCRIPTION

[0011] Disclosed herein are topical compositions for veterinary use in animals, particularly companion animals. In various embodiments, the compositions include blood components, particularly plasma components and selected formed components. In some embodiments, the compositions can themselves be derived from blood, for example a blood-derived serum. A blood-derived serum can refer to the fluid and solvent component of blood that does not play a role in clotting. In other terms, blood-derived serum can be defined as blood plasma without the clotting factors (or plasma without fibrinogen). Serum can, in particular, include serum albumin, serum globulins and electrolytes. In certain examples, the blood-derived serum includes bovine serum (e.g., to impart a compatible cross-species advantage over typical autologous or allogeneic serums). By using bovine serum, for instance, transmission of species-specific diseases or conditions can be mitigated.

[0012] Topical compositions as described herein particularly include blood components that are useful in promoting ocular healing such as growth factors and other proteins. Said compositions can be configured to enhance the bioavailability of such beneficial agents and / or to deliver them over an extended period of time after each application.

[0013] In accordance with the present disclosure, a topical composition can comprise a blood-derived serum, serum-derived proteins, a plurality of platelets, and various platelet cytosolic components, particularly platelet-derived proteins. The serum-derived and platelet-derived proteins can collectively include a number of growth factors. The serum-derived growth factors can include one or more of: platelet-derived growth factor (PDGF); epidermal growth factor (EGF); transforming growth factor beta (TGF-0); platelet-derived endothelial cell growth factor (PDEGF); insulin-like growth factor (IGF), particularly IGF-1; fibroblast growth factor (FGF), particularly FGF2; vascular endothelial growth factor (VEGF); nerve growth factor (NGF); fibronectin; vitronectin; and thrombospondin.

[0014] In various embodiments, the composition can be a product of the serum component and the platelet component of a volume of whole blood, while the red blood cells and white blood cells that would normally be a part of that volume of whole blood are absent from the composition. In the composition a fraction of said platelet component is present as intact platelets, while cytosolic components of another fraction of the platelet component are present in the composition. Stated another way, where a volume of whole blood includes a platelet count, in a topical composition derived from that volume the platelet count can be divided into a first plurality that is present in the topical composition as intact platelets and a second plurality from which the cytosolic components, particularly platelet-derived growth factors, are extracted and reconstituted into the serum. In addition, proteins may be extracted or liberated from other serum components. In certain embodiments, these serum-derived growth factors are extracted by lysing the serum, particularly so as to lyse a fraction of the total platelets in the serum. Consequently, the dissolved concentration of growth factors and other bioactive components such as antimicrobial peptides and protease inhibitors in the resulting blood-derived serum, also referred to herein as “lysed serum”, can be higher than if the whole platelet component were left intact (i.e., if the serum remained an “unlysed serum”.

[0015] One effect of the increased level of serum-derived growth factors is that, when applied to an eye of an animal in need of treatment, such compositions can provide enhanced healing effects by delivering a greater amount of bioavailable growth factors to the eye. Furthermore, the intact platelets in the composition can release the growth factors sequestered therein over a period of time after application, thereby providing extended therapeutic effect. For example, the concentration of growth factors in the composition can be greater than that of unlysed serum by a factor of about 1.5X to about 100,000X, such as about 1.5X to about 50,000X. about 1.5X to about 10,000X, about 1.5X to about 5,000X, about 1.5X to about E000X, about 1.5X to about 500X, about 1.5X to about 100X, about 1.5X to about 10X, or about 1.5X to about 3X. However, it will be understood that these values are not intended tobe limiting, as the enhancement of available protein — which can be quite significant — at least partially depends on the growth factor densify inside the platelets, which can vary.

[0016] The immediate and sustained therapeutic effects of the topical composition can be configured by selecting the ratio of dissolved serum-derived growth factors and bound growth factors. For example, this can be accomplished by selecting the relative fractions of the total platelet count in each of the first plurality7(i.e. intact platelets) and the second plurality (i.e. lysed platelets). For example, in some embodiments, the first plurality constitutes a larger portion of the total platelet count than the second plurality. In certain embodiments the first plurality can constitute from about 55% to about 95% of the total platelet count, or more particularly about 60% to about 90%, or about 70% to about 80%. In particular embodiments, the first plurality constitutes about 75% percent of the total platelet count.

[0017] The topical compositions described herein can include a blood-derived serum that is suited for the intended use. In some embodiments, the blood-derived serum is a non-autologous serum, or more particularly a xenogeneic serum selected to provide one or more benefits relative to an autologous serum. For example, using a xenogeneic serum can decrease the time needed to prepare the topical composition for use. The use of xenogeneic serum can also decrease the risk of infections and incompatibilities between a donor and conspecific recipient. Sources for the blood-derived serum can include mammals such as, without limitation, bovine, equine, porcine, ovine, caprine, murine, canine, or feline.

[0018] In some embodiments, the topical composition includes serum albumin but in lower concentration than that in autologous serum. Many antibiotic drugs bind to albumin and / or are metabolized at higher rates in the presence of albumin, consequently reducing the fraction of drug that is free and active. In some embodiments, the topical composition includes albumin at a concentration of less than about 100 g / L, less than about 90 g / L, less than about 85 g / L, less than about 80 g / L, less than about 75 g / L. less than about 70 g / L, less than about 65 g / L, less than about 60 g / L, less than about 55 g / L, less than about 50 g / L, less than about 45 g / L. or less than about 40 g / L. In various embodiments, the topical composition includes an albumin concentration that is at least the same or lower than the subject animal's autologous serum. In some embodiments, the concentration of albumin in the topical composition is about 10 mg / ml to about 30 mg / ml lower than that of autologous serum. In some embodiments, the concentration of albumin in the topical composition is about 1.5X to about 3X lower than that of autologous serum. The low albumin concentration allows the topical composition to be used in conjunction with antibiotic drugs with reduced interference with the drug's efficacy. In some embodiments, the serum is a bovine serum. Bovine serumnormally contains low concentrations of albumin (about 30g / L to about 40g / L in healthy, adequately nourished animals).

[0019] In some cases, compositions of the present disclosure can be implemented as an adjunctive or add-on treatment. Such treatment can include any treatment given to bolster or enhance the effectiveness of a previous treatment, in particular in cases where the first treatment proved not to be fully effective. Likewise, an adjunctive or add-on treatment can include treatment given to bolster or enhance the effectiveness of a different treatment or drug (e.g., in a combined treatment). For example, the composition can be used in combination with an antibiotic. Some non-limiting examples of an antibiotic can include oxy tetracycline, clindamycin, polymyxin, neomycin, gramicidin, tobramycin, gentamicin sulfate, bacitracin, fluoroquinolones (e.g., ciprofloxacin, levofloxacin, moxifloxacin, ofloxacin, gemifloxacin, and delafloxacin), etc. As discussed above, the efficacy of the adjunctive antibiotic may be preserved to a large degree due to the low albumin content of the topical composition disclosed herein.

[0020] The topical composition can itself possess antimicrobial properties. Without being bound to a particular theory, these properties can arise from antimicrobial peptides (such as cathelicidins and defensins) naturally present in serum, as well as the enhanced bioavailable growth factor content of the composition. In some cases, compositions of the present disclosure can be implemented as a replacement for another treatment or drug, particularly an antibiotic.

[0021] Disclosed herein are methods of making a topical composition as described above. In various embodiments, a method of making a topical composition, comprising steps including: providing a blood-derived serum comprising a starting amount of platelets; and lysing the blood-derived serum so as to leave a first plurality of the platelets intact and to disrupt a second plurality of the platelets so that they release serum-derived growth factors. One or more clarifying techniques (e.g. centrifugation and / or filtration) may be used to remove resulting cellular debris from the blood-derived serum, thereby producing a lysed serum comprising the first plurality of platelets and the serum-derived growth factors.

[0022] In some embodiments, the methods can comprise a preliminary process of obtaining blood and subjecting a volume of the blood to centrifugation to separate its solid and liquid components. In some embodiments, the blood is centrifuged at a speed and for a time selected to produce a precipitate containing white blood cells and red blood cells, while retaining substantially all platelets in the plasma. In various embodiments, the centrifugation speed is selected to provide a relative centrifugal force (ref) of about 300 ref to about 2000 ref or anyspeed therebetween. In particular embodiments, the centrifugation speed is from about 500 ref to about 1000 ref.

[0023] The centrifugation time can be selected to accomplish the desired degree of separation in combination with the selected speed. In various embodiments, the centrifugation time can be from about 5 minutes to about 30 minutes. In particular embodiments, the centrifugation time is about 15 minutes to about 25 minutes.

[0024] The centrifugation step yields a supernatant that is largely free from whole cells. In various embodiments, one or more stages of filtration can be employed to remove any residual cells from the supernatant.

[0025] A number of known effective cell-lysis techniques may be used to lyse the blood-derived serum. Non-limiting examples include freeze-thaw cycling, ultrasonication, enzymatic digestion, and chemical disruption. In some embodiments, the lysing process can include a freeze-thaw process. In some examples, multiple freeze-thaw cycles can be implemented, for example from two to five freeze-thaw cycles. The number, durations and temperatures of the freeze-thaw cycles is not particularly limited: rather, any combination of these may be selected so as to obtain a desired degree of lysis in the serum. Furthermore, the duration of a freeze period and a thaw period can differ (or in some cases, be the same) from cycle to cycle — where one freeze period and one thaw period constitute a single freeze-thaw cycle. In various embodiments, at least one of the one or more freeze-thaw cycles can comprise freezing at atemperature that is effective to disrupt cellular structures. For example, the temperature can be about -100 °C to about 0 °C, or about -80 °C to about 0 °C, or about -80 °C to about -20 °C. In various embodiments, at least one of the one or more freeze-thaw7cycles comprises freezing for a time of at least about 1 hour, for example about 1 hour to about 48 hours. In some examples, the duration of at least one of the freeze period or the thaw period can be selected based upon the temperature of the blood-derived serum.

[0026] The freeze-thaw process can be configured to result in lysis of a target percentage of the starting amount of platelets. In some embodiments, this target percentage constitutes about 55% to about 95% of the starting amount of platelets. In a particular embodiment, this target percentage constitutes about 75% of the starting amount of platelets. Additionally or alternatively, the duration of at least one of the freeze period or the thaw period in one or more freeze-thaw cycles can be selected based upon a target percentage of the starting amount of platelets to be lysed. For example, a first freeze-thaw cycle may be implemented to lyse a first threshold amount of platelets; a second freeze-thaw7cycle may be implemented to lyse a second threshold amount of platelets; and so forth. In one or more examples, filteringprocesses, centrifuging processes, etc. can be implemented at the conclusion of one or more thaw periods in the freeze-thaw cycles.

[0027] In some embodiments, the method can further comprise formulating the lysed serum for ophthalmic administration. In certain embodiments, this can include adding one or more additives. Non-limiting examples of additives include antihistamines, antibiotics, eye lubricants, vitamins, homeopathic remedies, herbal remedies, corticosteroids, glucocorticoids, nonsteroidal anti-inflammatory drugs, anticholinergics, analgesics, pH balancers, ocular hypertension drugs, glaucoma drugs, carbonic anhydrase inhibitors, beta blockers, antioxidants, immunosuppressive drugs, and transporter-delivery aids.

[0028] Disclosed herein are methods of treating an ophthalmic disorder in a companion animal, the method comprising administering to the eye of the animal a therapeutically effective amount of a topical composition described herein. Ophthalmic disorders thus treatable can include, without limitation, indolent ulcers, corneal trauma, keratoconjunctivitis sicca (dry' eye), allergic conjunctivitis, and post-operative inflammation. In some cases, the method can comprise co-administering an antibiotic, wherein the lysed bovine serum reduces albumin-mediated degradation of the antibiotic. It will be understood that dosing regimes for the composition(s) may be selected by a skilled person based upon a number of factors such as administration technique, pharmacological response, subject tolerance, and observed symptoms.EXAMPLES

[0029] A representative example protocol for making a topical composition using a one-liter sample of whole blood is as follows:• 5-10% ACD-A or Sodium Citrate (TBD) is aspirated into a blood draw collection container.• Blood Draw 1000ml• Centrifugation to separate RBC and WBC; 500-1000rcf for 15-25 minutes (TBD) • This speed can retain all platelets in the plasma. White and red cells may be separated below the buffy coat line.• Draw off plasma down to top of buffy coat.• Filter plasma to remove remaining WBC.• 1stfreeze @ -30 °C for min 2 hours• Lyse 30% of platelets (70% remaining)• Thaw to 2 °C• 2nd freeze @ -30 °C for min 2 hours• Lyse 25% of platelets (45% remaining)• Thaw to 2 °C• 3rd freeze @ -30 °C for min 2 hours• Lyse 20% of platelets (25% remaining)• Thaw to 2 °C• Centrifuge @1000rcf for 15 minutes to push cell debris to bottom.• Draw off 990ml, leaving bottom 10ml for disposal• Optional: Add one or more additives to the solution; mix as needed• Freeze for shipment.

[0030] Any methods disclosed herein comprise one or more steps or actions for performing the described method. The method steps and / or actions may be interchanged with one another. In other words, unless a specific order of steps or actions is required for proper operation of the embodiment, the order and / or use of specific steps and / or actions may be modified.

[0031] References to approximations are made throughout this specification, such as by use of the terms “substantially’' and “about."’ For each such reference, it is to be understood that, in some embodiments, the value, feature, or characteristic may be specified without approximation. For example, where qualifiers such as “about” and “substantially” are used, these terms include within their scope the qualified words in the absence of their qualifiers. All ranges also include both endpoints.

[0032] Similarly, in the above description of embodiments, various features are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure. This method of disclosure, however, is not to be interpreted as reflecting an intention that any claim require more features than those expressly recited in that claim. Rather, as the following claims reflect, inventive aspects lie in a combination of fewer than all features of any single foregoing disclosed embodiment.

[0033] The claims following this written disclosure are hereby expressly incorporated into the present written disclosure, with each claim standing on its own as a separate embodiment. This disclosure includes all permutations of the independent claims with their dependent claims. Moreover, additional embodiments capable of derivation from the independent and dependent claims that follow are also expressly incorporated into the present written description.

[0034] Without further elaboration, it is believed that one skilled in the art can use the preceding description to utilize the invention to its fullest extent. The claims and embodiments disclosed herein are to be construed as merely illustrative and exemplary, andnot a limitation of the scope of the present disclosure in any way. It will be apparent to those having ordinary skill in the art, with the aid of the present disclosure, that changes may be made to the details of the above-described embodiments without departing from the underlying principles of the disclosure herein. In other words, various modifications and improvements of the embodiments specifically disclosed in the description above are within the scope of the appended claims. Moreover, the order of the steps or actions of the methods disclosed herein may be changed by those skilled in the art without departing from the scope of the present disclosure. In other words, unless a specific order of steps or actions is required for proper operation of the embodiment, the order or use of specific steps or actions may be modified. The scope of the invention is therefore defined by the following claims and their equivalents.

Claims

CLAIMSWhat is claimed is:

1. A topical composition for use in an animal, comprising:a bovine blood-derived serum;a plurality7of bovine platelets suspended in the blood-derived serum;serum-derived growth factors dissolved in the blood-derived serum, wherein said serum-derived growth factors are derived from lysis of the serum; andalbumin,wherein the albumin is present at a lower concentration than a serum that is autologous to the animal.

2. The topical composition of claim 1, wherein the serum-derived growth factors are present at an increased concentration as compared to unlysed bovine blood-derived serum.

3. The topical composition of claim 2, wherein the serum-derived growth factors are present in the blood-derived serum at a concentration that is about 1.5X to about 100,000X higher than in unlysed bovine blood-derived serum.

4. The topical composition of any one of claims 1 to 3, wherein the serum-derived growth factors comprise one or more of: platelet-derived growth factor (PDGF); epidermal growth factor (EGF); transforming growth factor beta (TGF-P); platelet-derived endothelial cell growth factor (PDEGF); insulin-like growth factor (IGF), particularly IGF-1; fibroblast growth factor (FGF), particularly FGF2; vascular endothelial growth factor (VEGF); nerve growth factor (NGF); fibronectin; vitronectin; and thrombospondin.

5. The topical composition of any one of claims 1 to 4, wherein the animal is a companion animal.

6. The topical composition of any one of claims 1 to 5, wherein the composition possesses intrinsic antimicrobial activity.

7. The topical composition of any one of claims 1 to 6, wherein the composition comprises one or more antimicrobial proteins.

8. The topical composition of claim 7, wherein the one or more antimicrobial proteins comprise cathelicidins, defensins, or both.

9. The topical composition of any one of claims 1 to 8, wherein the albumin is present at a concentration that is about 10 mg / ml to about 30 mg / ml less than that in a blood-derived serum that is autologous to the animal.

10. The topical composition of any one of claims 1 to 8. wherein the albumin is present at a concentration that is about equal to about 3X less as compared to a blood-derived serum that is autologous to the animal.

11. The topical composition of any one of claims 1 to 10. wherein the albumin is present at a concentration that is less than about 100 g / L.

12. The topical composition of claim 11, wherein the albumin is present at a concentration that is less than about 40 g / L.

13. The topical composition of any one of claims 1 to 12, further comprising one or more additives selected from antihistamines, antibiotics, eye lubricants, vitamins, homeopathic remedies, herbal remedies, corticosteroids, glucocorticoids, nonsteroidal anti-inflammatory drugs, anticholinergics, analgesics, pH balancers, ocular hypertension drugs, glaucoma drugs, carbonic anhydrase inhibitors, beta blockers, antioxidants, immunosuppressive drugs, and transporter-delivery' aids.

14. The topical composition of any one of claims 1 to 13, wherein the topical composition is formulated for ocular delivery.

15. A topical composition for use in an animal, comprising:a blood-derived serum that is not autologous to the animal;an amount of intact platelets suspended in the blood-derived serum;serum-derived growth factors dissolved in the blood-derived serum; and albumin,wherein the serum-derived growth factors are present at an increased concentration as compared to unlysed blood-derived serum.

16. The topical composition of claim 15, wherein the serum-derived growth factors are present at a concentration that is about 1.5X to about 100.000X higher than in unlysed bovine blood-derived serum.

17. The topical composition of claim 15, wherein the serum-derived growth factors comprise one or more of: platelet-derived growth factor (PDGF); epidermal growth factor (EGF);transforming growth factor beta (TGF-P); platelet-derived endothelial cell growth factor (PDEGF); insulin-like growth factor (IGF), particularly IGF-1; fibroblast growth factor (FGF), particularly FGF2; vascular endothelial growth factor (VEGF); nerve growth factor (NGF); fibronectin; vitronectin; and thrombospondin.

18. The topical composition of any one of claims 15 to 17, wherein the serum is xenogeneic to the animal.

19. The topical composition of any one of claims 15 to 18. wherein the blood-derived serum is bovine, equine, porcine, ovine, caprine, murine, canine, or feline.

20. The topical composition of claim 19, wherein the blood-derived serum is bovine, equine or porcine.

21. The topical composition of claim 19, wherein the blood-derived serum is bovine.

22. The topical composition of any one of claims 15 to 21. wherein the albumin is present at a concentration that is about 10 mg / ml to about 30 mg / ml less than that in a blood-derived serum that is autologous to the animal.

23. The topical composition of any one of claims 15 to 21. wherein the albumin is present at a concentration that is about equal to about 3X less as compared to a blood-derived serum that is autologous to the animal.

24. The topical composition of any one of claims 15 to 23. wherein the albumin is present at a concentration that is less than about 100 g / L.

25. The topical composition of claim 24, wherein the albumin is present at a concentration that is less than about 40 g / L.

26. The topical composition of any one of claims 15 to 25, wherein the topical composition is formulated for ocular delivery7.

27. A method of treating a disorder in a companion animal, the method comprising topically administering to the animal a therapeutically effective amount of the composition of any one of claims 1 to 26.

28. The method of claim 27, wherein the disorder is an ophthalmic disorder and comprising administering the composition to the eye of the animal.

29. The method of claim 28, wherein the ophthalmic disorder is selected from the group consisting of indolent ulcers, corneal trauma, keratoconjunctivitis sicca (dry eye), allergic conjunctivitis, and post-operative inflammation.

30. The method of any one of claims 27 to 29, further comprising co-administering an antibiotic, wherein the lysed bovine serum reduces albumin-mediated degradation of the antibiotic.

31. A method of making a topical composition, comprising:providing a blood-derived serum comprising a starting amount of platelets; lysing the blood-derived serum so as to leave a first plurality of the platelets intact and to disrupt a second plurality of the platelets so that serum-derived growth factors are released into the blood-derived serum;clarifying the blood-derived serum to produce a lysed serum comprising the first plurality of platelets and the serum-derived growth factors;formulating the lysed serum for topical administration.

32. The method of claim 31, wherein the first plurality of platelets constitutes about 55% to about 95% of the starting amount of platelets.

33. The method of claim 32, wherein the first plurality of platelets constitutes about 75% of the starting amount of platelets.

34. The method of any one of claims 31 to 33, wherein the blood-derived serum is bovine, equine, porcine, ovine, caprine, murine, canine, or feline.

35. The method of claim 34, wherein the blood-derived serum is bovine, equine or porcine.

36. The method of claim 34, wherein the blood-derived serum is bovine.

37. The method of any one of claims 31 to 36, wherein clarifying comprises centrifuging and / or filtering the blood-derived serum.

38. The method of any one of claims 31 to 37, wherein lysing the blood-derived serum comprises subjecting the blood-derived serum to one of freeze-thaw cycling, ultrasonication, enzymatic digestion, and chemical disruption.

39. The method of any one of claims 31 to 38, wherein lysing comprises subjecting the blood-derived serum to freeze-thaw cycling comprising one or more freeze-thaw cycles, whereineach of the one or more freeze-thaw cycles disrupts a fraction of the second plurality of platelets.

40. The method of any one of claims 31 to 39, wherein at least one of the one or more freezethaw cycles comprises freezing at a temperature of about -100 °C to about 0 °C.

41. The method of any one of claims 31 to 40, wherein at least one of the one or more freezethaw cycles comprises freezing for a time of at least about 1 hour to about 48 hours.

42. The method of any one of claims 31 to 41, wherein the freeze-thaw cycling comprises from two to five freeze-thaw cycles.

43. The method of any one of claims 31 to 42, wherein the clarifying step is performed after a plurality of the one or more freeze-thaw cycles.

44. The method of any one of claims 31 to 43, wherein formulating comprises adding one or more additives selected from antihistamines, antibiotics, eye lubricants, vitamins, homeopathic remedies, herbal remedies, corticosteroids, glucocorticoids, nonsteroidal antiinflammatory drugs, anticholinergics, analgesics, pH balancers, ocular hypertension drugs, glaucoma drugs, carbonic anhydrase inhibitors, beta blockers, antioxidants, immunosuppressive drugs, and transporter-delivery aids.

45. The method of any one of claims 31 to 44, comprising formulating the lysed serum for ophthalmic administration.

46. A topical composition made according to the method any one of claims 31 to 45.