Use of valproic acid for reducing scarring after glaucoma surgery

Abstract

The present invention relates to the use of VPA to reduce scarring after glaucoma surgery. In one form, the glaucoma surgery is a glaucoma filtration surgery that involves creating a subconjunctival bleb. In another form, the glaucoma surgery is a minimally invasive glaucoma surgery that involves implanting a glaucoma tube shunt under the subconjunctival space.

Description

Detailed Description of the Invention

[0001] 〔Technical Field〕 The present invention relates to the use of valproic acid in the treatment of glaucoma surgery.

[0002] 〔Background〕 Eye surgeries, including those involving the conjunctiva, are frequently performed to slow the progression of eye diseases, particularly glaucoma. Glaucoma surgeries include those by conventional glaucoma filtration surgery (GFS) or less invasive glaucoma surgery, which is less invasive.

[0003] Complications of typical glaucoma surgeries are caused by postoperative fibrosis of the eye. In fact, the wound healing response to glaucoma surgery, regardless of whether it is conventional or minimally invasive, includes inflammation and scarring as the final outcome. The formation of scars mainly composed of disordered collagen not only destroys the normal blood / lymph vascular structure but also inevitably disrupts the conjunctival structure, which may in turn impair the biomechanical protective function of the postoperative conjunctiva.

[0004] Currently, additives such as mitomycin C (MMC) are routinely applied to improve the surgical outcome mainly by reducing the amount of collagen deposited in the scar. These drugs are effective in preventing ocular fibrosis and improving the outcome of glaucoma surgery, but are known to cause vision-threatening complications including extensive cell death, bleb leakage, hypotony, and / or endophthalmitis.

[0005] Furthermore, excessive or persistent inflammation after ophthalmic surgery has a high risk of scarring, so steroids are applied systemically, topically, or subconjunctivally as postoperative management to prevent failure, similar to other anti-inflammatory drugs. However, these dosing regimens generally involve taking immunosuppressive / anti-inflammatory drugs for a long time, and steroids in particular are associated with potentially serious side effects.

[0006] Therefore, an improved method for wound healing after glaucoma surgery is needed.

[0007] 〔Summary of the Invention〕 The present invention attempts to address these problems and / or provide a method for improving wound healing after glaucoma surgery.

[0008] According to a first aspect, the present invention provides the use of valproic acid (VPA) in the manufacture of a medicament for preventing tissue degeneration after glaucoma surgery.

[0009] According to a particular aspect, preventing the above tissue degeneration may include maintaining the conjunctival collagen structure.

[0010] The present invention also provides the use of VPA in the manufacture of a medicament for maintaining subconjunctival blisters formed by glaucoma surgery.

[0011] According to a particular aspect, maintaining the above subconjunctival blisters includes maintaining the conjunctival collagen structure.

[0012] The VPA according to any aspect may include VPA in any suitable form. According to a particular aspect, the above VPA may include derivatives, analogs, salts, their esters, or combinations thereof. For example, the above VPA may include sodium valproate, calcium valproate, semisodium valproate, divalproex, 2-n-propyl-3-aminopentanoic acid, 2-π-propyl-4-aminopentanoic acid, 2-n-propyl-4-hexynoic acid, or combinations thereof.

[0013] The glaucoma surgery may be any type of glaucoma surgery. According to a specific embodiment, the glaucoma surgery may include creating a subconjunctival bleb. For example, the glaucoma surgery may include glaucoma filtration surgery or minimally invasive glaucoma surgery (MIGS). According to a specific embodiment, the glaucoma surgery may be MIGS. In particular, the MIGS may include implanting a glaucoma tube shunt under the subconjunctival space. According to another specific embodiment, the glaucoma surgery may include ab externo glaucoma surgery or ab interno glaucoma surgery.

[0014] The glaucoma surgery may include the use of antimetabolites. The antimetabolites may be any suitable antimetabolites for the purposes of the present invention. For example, the antimetabolites may be mitomycin C (MMC), 5-fluorouracil (5FU), or a combination thereof.

[0015] The antimetabolites used in the glaucoma surgery may have an appropriate concentration. According to a specific embodiment, the concentration of the antimetabolites used in the glaucoma surgery may be 1.0 mg / mL or less.

[0016] The VPA may have any suitable concentration. For example, the VPA may have a concentration of 100 to 1000 μg / mL.

[0017] The drug may be suitable for administration to a subject by any suitable means. For example, the drug may be suitable for topical administration and / or subconjunctival administration.

[0018] The drug may be suitable for administration at any suitable time point. According to a specific embodiment, the drug may be suitable for administration immediately after glaucoma surgery.

[0019] According to another specific aspect, the above-mentioned drug may be suitable for administration from 6 to 120 months after the above-mentioned glaucoma surgery. In particular, the above-mentioned drug may be suitable for daily administration for at least 12 weeks after the above-mentioned glaucoma surgery.

[0020] The present invention also provides the use of valproic acid (VPA) in the manufacture of a drug for forming a weak subconjunctival scar after glaucoma surgery. The above-mentioned glaucoma surgery may be defined as described above. In particular, the above-mentioned glaucoma surgery may include implanting a glaucoma tube shunt under the subconjunctival cavity.

[0021] According to a specific aspect, forming the above-mentioned weak subconjunctival scar may include preventing encapsulation of the above-mentioned glaucoma tube shunt by collagen fibers. According to another specific aspect, forming the above-mentioned weak subconjunctival scar may enable the above-mentioned glaucoma tube shunt to maintain its aqueous humor outflow ability through its lumen.

[0022] Also provided is the use of valproic acid (VPA) in the manufacture of a drug for preventing encapsulation of a glaucoma tube shunt implanted under the subconjunctival cavity.

[0023] Another aspect of the present invention is the use of valproic acid (VPA) in the manufacture of a drug for maintaining the aqueous humor outflow ability of a glaucoma tube through its lumen after the glaucoma tube has been implanted under the subconjunctival cavity.

[0024] The above-mentioned glaucoma surgery may include the use of antimetabolites. The above-mentioned antimetabolite may be any suitable antimetabolite. For example, the antimetabolite may be, but is not limited to, mitomycin C (MMC), 5-fluorouracil (5FU), or a combination thereof. The antimetabolite may have an appropriate concentration. According to a specific aspect, the above-mentioned antimetabolite may have a concentration of 1.0 mg / mL or less.

[0025] According to certain embodiments, the agent may further comprise an antimetabolite. The antimetabolite may be as described above.

[0026] [Brief Description of the Drawings] To fully understand the present invention and to put it into an easily practical and effective state, here, as a non-limiting example, only typical embodiments are described, and the description refers to the accompanying illustrative drawings. In the drawings: Figure 1 shows a mouse model of conjunctival scarring; Figure 2 shows the visualization of collagen structures treated with phosphate buffered saline (PBS) and VPA in a mouse model of surgical conjunctiva by hematoxylin and eosin (H&E) staining, picrosirius red staining, and second harmonic generation light (SHG) at the indicated time points after surgery; Figure 3 shows a decrease in the thickness of collagen fibers in the VPA-treated postoperative conjunctiva of a mouse model; Figure 4 shows a decrease in collagen fiber strength in the VPA-treated postoperative conjunctiva of a mouse model measured by collagen area ratio (CAR), collagen fiber density (CFD), and number of collagen fibers per mm 2 ; Figure 5 shows that no reticulation of collagen fibers was induced in the VPA-treated postoperative conjunctiva of a mouse model. The collagen structure is measured by collagen reticulation Index (CRI) and Collagen area Reticulation density (CARD); Figure 6A shows that VPA inhibits the steady-state type I collagen expression in primary rabbit conjunctival fibroblasts, and Figure 6B shows that VPA inhibits the steady-state type I collagen expression in human Tenon's conjunctival fibroblasts; Figure 7 shows the effectiveness of VPA in maintaining microshunt implants in a rabbit model for at least 28 days after surgery compared to PBS; Figure 8 shows the development of cysts and the maintenance of vascular structures in blebs imaged by confocal microscopy in rabbit models treated with PBS and VPA on the 28th day after surgery; Figure 9 shows the histological visualization of collagen characteristics in a rabbit model of microshunt implant surgery treated with VPA on the 28th day after surgery; Figure 10 shows the immunofluorescent visualization of the characteristics of collagen and fibronectin in a rabbit model of microshunt implant surgery treated with VPA on the 28th day after surgery; Figure 11 shows the expression of transcripts promoting fibrosis and angiogenesis in a rabbit model of microshunt implant surgery treated with VPA; Figure 12 shows the improvement of bleb morphology in a rabbit model of microshunt implant surgery when VPA is combined with a low dose of MMC; Figure 13 shows the maintained vascular structure in the blebs of a rabbit model when a low dose of MMC is used on the 28th day after surgery; Figure 12 shows the histological visualization of collagen characteristics in a rabbit model of microshunt implant surgery treated with high and low doses of MMC in combination with VPA on the 28th day after surgery; Figure 15 shows the expression of transcripts promoting fibrosis and angiogenesis in a rabbit model of microshunt implant surgery treated with high and low doses of MMC in combination with VPA on the 29th day after surgery; Figure 16 shows the protein expression of COL1A1 in a rabbit model of microshunt implant surgery treated with high and low doses of MMC in combination with VPA on the 29th day after surgery; Figure 17 shows the histochemical visualization of the implant tip leading to the subconjunctival space in a rabbit model of MIGS treated with MMC or a combination of MMC and VPA.

[0027] 〔Detailed Description〕 As described above, there is a need to improve the outcome of glaucoma surgery. In general terms, the present invention relates to an improved outcome of glaucoma surgery by restoring normal conjunctival tissue structure through the use of valproic acid. In particular, the present invention can protect the function of the ocular surface from adverse reactions to the above glaucoma surgery. The present invention also relates to the preservation of collagen structure, which can reduce the level of destruction in the scar collagen to be deposited next, and can preserve the normal vascular structure that can lead to improvement of surgical outcome. Furthermore, the present invention results in a reduction in the amount / concentration of antimetabolites used during glaucoma surgery.

[0028] According to a first aspect, the present invention provides the use of valproic acid (VPA) in the manufacture of a medicament for preventing tissue degeneration after glaucoma surgery.

[0029] The present invention also provides the use of VPA in the manufacture of a medicament for maintaining the subconjunctival bleb formed by glaucoma surgery. In particular, maintaining the subconjunctival bleb includes maintaining the conjunctival collagen structure. The maintenance of the conjunctival collagen structure may be as described below. For example, the bleb can be maintained by suppressing conjunctival scarring. Scarring leads to rupture of the bleb, thereby allowing the intraocular pressure (IOP) reduced by glaucoma surgery to persist.

[0030] VPA is known as a first-generation antiepileptic drug and has been clinically used for many years. VPA and its salts are widely prescribed for other neurological diseases such as bipolar mania and migraine. VPA not only has a relatively good safety profile but also has good efficacy and a pharmaco-economic profile for neurological diseases.

[0031] VPA according to any aspect of the present invention can include any suitable form of VPA. According to a particular aspect, the VPA may include, but is not limited to, VPA derivatives, VPA analogs, VPA salts, VPA esters, or combinations thereof. For example, the VPA derivatives may include, but are not limited to, divalproex, 2-n-propyl-3-aminopentanoic acid, 2-π-propyl-4-aminopentanoic acid, or combinations thereof. The VPA analogs may include, but are not limited to, 2-n-propyl-4-hexynoic acid. The VPA salts may include, but are not limited to, sodium valproate, calcium valproate, semisodium valproate, and other alkali salts and alkaline earth salts of valproic acid, or combinations thereof. In particular, the VPA may include sodium valproate.

[0032] VPA can have any suitable concentration. For example, the VPA can have a concentration of 100 to 1000 μg / mL. In particular, the VPA can have a concentration of 150 to 950 μg / mL, 200 to 900 μg / mL, 250 to 850 μg / mL, 300 to 800 μg / mL, 350 to 750 μg / mL, 400 to 700 μg / mL, 450 to 650 μg / mL, 500 to 600 μg / mL. More particularly, the VPA can have a concentration of 150 to 300 μg / mL.

[0033] The glaucoma surgery in any aspect of the present invention can be any suitable glaucoma surgery. For example, the glaucoma surgery may include glaucoma filtration surgery or minimally invasive glaucoma surgery (MIGS). The glaucoma surgery may include ab externo glaucoma surgery or ab interno glaucoma surgery. In particular, the glaucoma surgery may include creating a subconjunctival space or bleb. The subconjunctival space / bleb can serve as a reservoir for aqueous humour.

[0034] According to certain aspects, the glaucoma surgery may be MIGS and may include implanting a glaucoma tube shunt under the subconjunctival space. In particular, the glaucoma surgery may be ab externo glaucoma surgery, may include glaucoma filtration surgery, or may include implanting a glaucoma tube shunt under the subconjunctival space, but is not limited thereto. The glaucoma tube may be any suitable glaucoma tube known in the art. In particular, the glaucoma tube may be PRESERFLO® MicroShunt (previously known as “InnFocus MicroShunt”). PRESERFLO® MicroShunt is an implantable glaucoma drainage device made of a highly flexible SIBS [poly(styrene-block-isobutylene-block-styrene)] polymer having a tube with an outer diameter of 350 μm and an inner lumen of 70 μm. It has triangular fins that prevent the tube from moving into the anterior chamber. The device may be designed to be implanted under the subconjunctival / Tenon's space. PRESERFLO® MicroShunt is manufactured and provided by InnFocus, Inc.

[0035] Ab interno glaucoma surgery may include a surgery for implanting a glaucoma stent from the cornea under the subconjunctival space. The glaucoma stent may be any suitable glaucoma stent known in the art. In particular, the glaucoma stent may be Allergan’s Xen Gel Stent.

[0036] According to certain aspects, the glaucoma surgery may include the use of antimetabolites. The antimetabolite may be any suitable antimetabolite for use in the glaucoma surgery. In particular, the antimetabolic drug may be used during the glaucoma surgery. The antimetabolite may include, but is not limited to, mitomycin C (MMC), 5-fluorouracil (5FU), or a combination thereof. According to certain aspects, the antimetabolite may be MMC.

[0037] The metabolic antagonist used in the glaucoma surgery may have an appropriate concentration. For example, the concentration of the metabolic antagonist used in the glaucoma surgery may be less than 1.0 mg / mL. In particular, the concentration of the metabolic antagonist may be 0.9 mg / mL or less, 0.5 mg / mL or less, 0.4 mg / mL or less, 0.2 mg / L or less, 0.1 mg / L or less. More particularly, the concentration of the metabolic antagonist may be 0.1 mg / mL or less.

[0038] The above-mentioned drug and / or the above-mentioned VPA may be in any appropriate form. For example, the above-mentioned drug and / or the above-mentioned VPA may be suitable for ophthalmic administration. In particular, the above-mentioned drug and / or the above-mentioned VPA may be suitable for subconjunctival, intravitreal, or topical administration. The above-mentioned drug and / or the above-mentioned VPA may be set for various ophthalmic delivery routes such as subconjunctival injection or administration by other ophthalmic delivery routes and / or dosage forms known in the art. The above-mentioned drug or VPA may be prepared in a liquid form for administration by eye drops or may be in a dry powder form such as a lyophilized form.

[0039] The above-mentioned drug may be suitable for any appropriate dosing schedule. Therefore, the above-mentioned drug may be suitable for administration at any appropriate time. The dosing schedule can be based on various factors (such as age, condition, weight, gender, the subject's diet, the severity of the condition, and other clinical factors).

[0040] According to certain aspects, the above-mentioned drug may be suitable for administration immediately after the above-mentioned glaucoma surgery. For example, immediately after a surgical event, a single administration of the drug may be provided. In addition to the single administration, further repeated administrations of the drug may be provided. In particular, in addition to the single administration, drugs for daily, weekly, bi-weekly, monthly, and bi-monthly administrations may be provided. The above-mentioned drug may be suitable for repeated administrations for several years after the above-mentioned glaucoma surgery. In particular, the above-mentioned drug may be suitable for repeated administrations for 1 to 120 months, 2 to 96 months, 3 to 72 months, 4 to 60 months, 5 to 48 months, 6 to 36 months, 8 to 24 months, 12 to 18 months after the above-mentioned glaucoma surgery. More particularly, the above-mentioned drug may be suitable for repeated administrations up to 4 months after the above-mentioned glaucoma surgery.

[0041] According to certain aspects, the above-mentioned drug may be suitable for daily administration up to 6 months, 4 months, 3 months, 2 months, 1 month, 3 weeks, 2 weeks, 1 week after the above-mentioned glaucoma surgery. In particular, the above-mentioned drug may be suitable for daily administration up to 12 weeks after the above-mentioned glaucoma surgery.

[0042] According to certain aspects, preventing tissue degeneration may include maintaining the conjunctival collagen structure. For the purposes of the present invention, maintaining the conjunctival collagen structure may be defined as reducing the thickness of collagen fibers by about 25% and / or reducing the reticulation of collagen by about 30%.

[0043] In particular, maintaining the above-mentioned conjunctival collagen structure includes suppressing changes in the collagen structure and maintaining the integrity of the conjunctival vasculature structure. More particularly, maintaining the above-mentioned conjunctival collagen structure includes reducing the average thickness of collagen fibers formed after the above-mentioned glaucoma surgery. The maintenance may further include inhibiting the reticulation of collagen. In particular, the maintenance may include inhibiting the reticulation of collagen by 30%. The maintenance may further include enhanced expression of Vegfa.

[0044] VPA can prevent the disruption of the collagen structure during wound healing after glaucoma surgery by reducing the thickness of collagen fibers and the fenestration of collagen, so that the conjunctival structure can be preserved, thereby maintaining the biomechanical properties of the conjunctiva and its role in supporting the vascular structure of blood and lymphatic vessels. From the perspective of preserving the above conjunctival structure, the conjunctiva may also act as a protective barrier against infection in glaucoma surgery.

[0045] In addition, by using the above VPA, the concentration of the antimetabolite used during the above glaucoma surgery can be reduced. Thereby, the toxic effect of the antimetabolite on the conjunctival tissue can be significantly reduced, and thereby, the health of the conjunctival tissue can be maintained.

[0046] According to a third aspect, the present invention provides the use of valproic acid (VPA) in the manufacture of a medicament for forming a weak subconjunctival scar after glaucoma surgery.

[0047] The above glaucoma surgery may be defined as above. In particular, the above glaucoma surgery may include implanting a glaucoma tube shunt under the subconjunctival space.

[0048] According to a specific aspect, forming the above weak subconjunctival scar may include preventing encapsulation of the glaucoma tube shunt by collagen fibers. According to another specific aspect, forming the above weak subconjunctival scar may enable the above glaucoma tube shunt to maintain its ability to drain water through its lumen. In particular, the medicament is suitable for administration to a subject, whereby the medicament results in the development of a weaker subconjunctival scar through the presence of smaller (reduced collagen content) and thinner collagen fibers, promotes water outflow, and results in a favorable blister morphology that maintains the function of the microshunt.

[0049] The glaucoma surgery may include the use of antimetabolites. The antimetabolite may be any suitable antimetabolite. For example, the antimetabolite may be mitomycin C (MMC), 5-fluorouracil (5FU), or a combination thereof, but is not limited thereto. The antimetabolite may have an appropriate concentration. According to a particular embodiment, the concentration of the antimetabolite may be 1.0 mg / mL or less. In particular, when used in combination with a low dose of antimetabolite, the combination of VPA and antimetabolite may further reduce the length of collagen fibers, thereby further weakening the subconjunctival scar formed.

[0050] The use of VPA is also provided in the manufacture of a medicament for preventing encapsulation of a glaucoma tube shunt implanted under the subconjunctival space.

[0051] Another aspect of the invention is the use of VPA in the manufacture of a medicament for maintaining the aqueous outflow ability of a glaucoma tube through its lumen after the glaucoma tube has been implanted under the subconjunctival space.

[0052] According to a further aspect, the invention provides a method for preventing tissue degeneration after glaucoma surgery, which includes administering an effective amount of VPA.

[0053] According to a further aspect, a method for maintaining a subconjunctival bleb formed in glaucoma surgery is provided, which includes administering an effective amount of VPA.

[0054] The invention also provides a method for forming a weak subconjunctival scar after glaucoma surgery, which includes administering an effective amount of VPA. The glaucoma surgery may be defined as above. In particular, the glaucoma surgery may include implanting a glaucoma tube shunt under the subconjunctival space.

[0055] In particular, forming the weak subconjunctival scar may include preventing encapsulation of the glaucoma tube shunt by collagen fibers. According to another specific aspect, forming the weak subconjunctival scar enables the glaucoma tube shunt to maintain its aqueous humor outflow ability through its lumen.

[0056] According to a specific aspect, the agent may further include an antimetabolite. In particular, the antimetabolite may be as described above.

[0057] The use of VPA in the manufacture of an adjuvant for glaucoma surgery is also provided. The adjuvant may be able to sustain the IOP reduced by glaucoma surgery. In particular, the VPA may be used as an adjuvant for glaucoma surgery.

[0058] Not only the antimetabolite, but also the VPA and glaucoma surgery may be as described above.

[0059] The present invention also provides a method for preventing tissue degeneration after glaucoma surgery, which includes administering VPA to a patient in need thereof.

[0060] A method for maintaining a subconjunctival bleb formed in glaucoma surgery, which includes administering VPA to a patient in need thereof, is also provided.

[0061] The present invention also provides a method for forming a weak subconjunctival scar after glaucoma surgery, which includes administering VPA to a patient in need thereof. A method for preventing encapsulation of a glaucoma tube shunt implanted under the subconjunctival space, which includes administering VPA to a patient in need thereof, is also provided.

[0062] The present invention also provides a method for maintaining the aqueous humor outflow ability of a glaucoma tube through its lumen after the glaucoma tube is implanted into the subconjunctival space, which includes administering VPA to a patient in need thereof.

[0063] The glaucoma surgery may be as described above. The VPA may be as described above.

[0064] VPA is also provided for use in preventing tissue degeneration after glaucoma surgery. Another aspect of the present invention is VPA for use in maintaining a subconjunctival bleb formed during glaucoma surgery. The present invention also provides VPA for use in forming a weak subconjunctival scar after glaucoma surgery.

[0065] Another aspect of the present invention is VPA for use in preventing encapsulation of a glaucoma tube shunt implanted under the subconjunctival space. Yet another aspect of the present invention is VPA for use in maintaining the ability of water to flow through the lumen of a glaucoma tube after the glaucoma tube has been implanted under the subconjunctival space.

[0066] The above-mentioned glaucoma surgery may be as described above. The above VPA may be as described above.

[0067] The present invention also provides a medicament for preventing tissue degeneration after glaucoma surgery, the medicament comprising VPA. Another aspect of the present invention is a medicament for maintaining a subconjunctival bleb formed during glaucoma surgery, the medicament comprising VPA.

[0068] A medicament for forming a weak subconjunctival scar after glaucoma surgery, the medicament comprising VPA, is also provided.

[0069] Another aspect of the present invention is a medicament for preventing encapsulation of a glaucoma tube shunt implanted under the subconjunctival space and / or for maintaining the ability of water to flow through the lumen of the glaucoma tube after the glaucoma tube has been implanted under the subconjunctival space, the medicament comprising VPA.

[0070] The above-mentioned glaucoma surgery may be as described above. The above VPA may be as described above.

[0071] Although the invention has been generally described herein, it is provided by way of illustration and is not intended to be limiting. The same will be more readily understood by reference to the following examples.

[0072] [Example] [Example 1 - Mouse Model of Conjunctival Scarring] As shown in Figure 1, a mouse model of conjunctival scarring was prepared.

[0073] The conjunctiva was incised to expose the sclera. In the sclera, the incision was made up to the anterior chamber. The resulting fistula allowed aqueous humor to flow into the middle and lower parts of the conjunctiva. The liquid accumulated under the sutured conjunctiva was observed as a conjunctival vesicle.

[0074] The mouse model of conjunctival scarring was demonstrated using MMC. This mouse showed the same reaction as a human who had undergone glaucoma surgery when MMC was applied in exactly the same way.

[0075] To determine whether VPA has the ability to protect the collagen structure, the collagen properties in the mouse model of conjunctival scarring treated with VPA were measured using the quantitative multiphoton imaging described by Xu S et al. (J. Hepatol., 2014, 61(2): 260 - 269). The onset of scarring indicated by the peak production of collagen mRNA was measured on the 7th day after surgery. Mature scars were measured on the 14th day after surgery when the production of collagen mRNA was suppressed.

[0076] To examine the effect of VPA on the collagen structure during the onset of scarring on the 7th day, mice were injected directly into the surgical area with 300 μg / ml of VPA immediately after surgery and on the 2nd day. To determine the effect of VPA on mature scars on the 14th day, mice were injected as above and given an additional injection on the 7th day. By this method, it was easily observable that the collagen fibers in the VPA - treated eyes were thinner than those in the PBS - treated control eyes, not only in multiphoton scans but also in histologically stained sections. An exemplary collagen fiber showing the thinning effect of VPA is indicated by the white arrowhead in Figure 2.

[0077] Quantitative multiphoton analysis of the operated conjunctival region demonstrated that the thickness of collagen was indeed reduced in VPA-treated tissues (Figure 3). The entire range of thin, median, and thick collagen fibers in VPA-treated conjunctiva was relatively thinner than that in PBS-treated controls for all.

[0078] Quantitative analysis of collagen intensity (Figure 4) also confirmed that VPA decreased collagen production in a mouse model of conjunctival scarring. The number of fibers decreased in VPA-treated tissues, but the packing density did not differ significantly from the PBS control. Collagen intensity measured as the collagen area ratio (CAR) decreased significantly in both 7-day and 14-day VPA-treated tissues. The lack of a significant difference in collagen fiber density (CFD) in the state of VPA treatment, unlike the PBS control, indicated that the collagen fibers were not packed. Consistent with the ability of VPA to reduce the amount of collagen induced after surgery, the number of collagen fibers per mm 2 was significantly reduced in the operated tissues at both time points.

[0079] Most importantly, multiphoton analysis measured an aspect of the collagen structure that was not easily visualized by the eye. PBS treatment increased collagen fibrillogenesis (or branching), while VPA treatment inhibited this phenomenon, as shown by a significant decrease in the collagen reticulation index (CRI) when compared to PBS treatment on day 7 (Figure 5). The activity of VPA to suppress the increase in CRI on day 7 is of great significance as this is the time point of the peak of collagen induction in the wound tissue. What this means is that in the event of excessive and potentially prolonged wound healing, both of which are associated with pathological scarring, VPA can act to prevent obvious tissue structure changes. The CRI in the mature scar on day 14 is similar to that of the unoperated tissue. Therefore, the lack of effect of VPA in the mature scar on day 14 is important as it suggests that VPA does not change the structural integrity of the tissue in normal tissue and that these only affect excessive scarring. In the surgically induced postoperative conjunctiva treated with VPA, collagen fibrillogenesis was not induced. The collagen structure was measured as CRI and Collagen area Reticulation density (CARD).

[0080] These data measured at the micron scale indicate that VPA treatment prevents the disruption of the collagen structure with respect to the thickness of the collagen fibers and collagen fibrillogenesis, in addition to its ability to reduce collagen fiber strength. Therefore, VPA treatment may be a way to maintain the conjunctival structure, which is important for maintaining the biomechanical properties of the conjunctival structure and for supporting the vascular structures of blood and lymph.

[0081] [Example 2 - Rabbit Model of Microshunt Implant Surgery and Human Microshunt Implant Surgery] To apply to a rabbit model of microshunt implantation surgery (PRESERFLO® MicroShunt, Santen), as shown in Figure 6, using primary rabbit conjunctival fibroblasts, the dose of VPA to be used was determined with respect to (1) the effect on the reduction of type I collagen and (2) the non-toxicity to cell proliferation. The data showed that 300 μg / ml of VPA is the minimum effective concentration to significantly reduce the expression of Col1a1 in rabbit conjunctival fibroblasts without interfering with cell proliferation.

[0082] As can be seen from Figure 6A, VPA inhibits the expression of steady-state type I collagen in primary rabbit conjunctival fibroblasts.

[0083] Similarly, for application to humans, the dose of VPA to be used was determined with respect to the effectiveness in reducing type I collagen in the same manner as in the above rabbit model. The effect of VPA on the expression of type I collagen in human Tenon's conjunctival fibroblasts derived from three independent donors was examined, and the results were as shown in Figure 6B. Similar to the above rabbit model, it can be seen that 300 μg / ml of VPA is the minimum effective concentration to significantly reduce the expression of Col1a1 without interfering with cell proliferation.

[0084] [Example 3 - Efficacy of VPA in a Rabbit Model of Microshunt Implantation Surgery] For a rabbit model of microshunt implantation surgery using PRESERFLO® MicroShunt (Santen), immediately after surgery, once a day for the first 7 days, and then on the 10th, 14th, and 21st days after surgery, a total of 11 injections of 300 μg / ml VPA were performed. The eyes of the rabbits were evaluated by slit lamp photography (Figure 7). As found, the PBS-treated bleb had already ruptured by the 14th day, while VPA was effective in maintaining the filtering bleb for at least 28 days.

[0085] The confocal microscopy method was used to correlate the appearance and function of the blisters. It is known that the features that are significantly positively correlated include the size of the cysts and the density / tortuosity of the vascular structures.

[0086] In this example, the PBS-treated blisters were characterized by smaller cysts that were in the background of densely packed collagen fibers and an amorphous-looking tissue (Figure 8). Clearly, the vascular structures appeared to be tortuous. In contrast, the VPA-treated blisters were characterized by larger cysts that were more regularly arranged and less amorphous, surrounded by loosely packed collagen fibers. Notably, the vascular structures were straight in the VPA-treated conjunctiva. These observations support the ability of VPA to maintain the conjunctival vascular structures and, in combination with the development of larger cysts, support the possibility of contributing to the improvement of blister function and survival.

[0087] The ability of VPA to maintain the collagen structure of the conjunctiva was demonstrated by histological analysis. When tested against normal, unoperated conjunctiva, the PBS-treated blisters were characterized by thick, disordered collagen fibers (Figure 9). In contrast, the VPA-treated blisters were characterized by a thinner, more uniformly organized arrangement of collagen fibers when compared to normal tissue (Figure 9). These data suggest that treatment with VPA can maintain the collagen structure of the conjunctiva when an eluting implant is worn. Thus, VPA can maintain the biomechanical / scaffolding properties and the role of the protective wall of the conjunctiva. By preventing structural degenerative reactions in the conjunctiva, VPA can therefore increase the success of the surgery.

[0088] Differences in collagen structure between VPA treatment and PBS control treatment were further visualized by immunofluorescent staining of frozen sections of the conjunctiva. Antibodies specific for type I collagen (COL1A1) and fibronectin (FN) confirmed the development of thicker collagen and fibronectin fibers in PBS-treated conjunctiva (Figure 10). In contrast, VPA-treated blisters were permeated by thinner and more diffusely distributed fibers of both proteins (Figure 10). These data suggest that VPA affects not only the distribution and structure of collagen but also that of other extracellular matrix proteins such as fibronectin. Fibronectin is involved in wound contraction during wound healing, among other functions. By preventing the formation of excessive or thick fibronectin fibers, VPA may prevent the occurrence of pathological collagen contractures in the wounded conjunctiva.

[0089] The decrease in the thickness of collagen and fibronectin fibers suggests that less collagen and fibronectin may be produced in the state of VPA treatment. This was demonstrated by analyzing the amounts of collagen and fibronectin transcripts in the treated rabbit conjunctiva. As shown in Figure 11, the expression of both of these genes in rabbit tissues on the 28th day was significantly decreased in the state of VPA treatment when compared with the PBS control. Also, it was demonstrated that Smad6 was significantly induced by VPA treatment, supporting the previous finding from a mouse model of conjunctival scarring that the change in Smad6 expression is the mechanism of downregulation of Col1a1 by VPA. Surprisingly, the expression of Vegfa, a typical growth factor for angiogenesis, was significantly increased in the tissue treated with VPA. This finding is consistent with the ability of VPA to maintain vascular structures in surgically treated tissues, which can be inhibited by the development of denser and more disordered collagen scaffolds in the PBS control visualized by confocal microscopy.

[0090] In summary, the rabbit model of microshunt surgery implants showed that VPA treatment preserves not only the vasculature of the surgically treated tissue but also the tissue / collagen structure. And therefore, VPA treatment can be used to maintain extremely important functions of the normal conjunctiva. In other words, by reducing the degenerative reaction to surgery and at the same time enabling the development of large cysts, as a result, VPA can reduce the postoperative adverse effects while improving the bleb function, and can play a beneficial role as an adjunct for use with PRESERFLO® MicroShunt.

[0091] [Example 4 - Efficacy of VPA in Reducing MMC Exposure in a Rabbit Model of Microshunt Implant Surgery] A rabbit model of microshunt implant surgery using PRESERFLO® MicroShunt (Santen) was created and treated under the following conditions: (a) 0.4 mg / ml of MMC for 1 minute with a sponge; (b) 0.1 mg / ml of MMC for 1 minute with a sponge; and, (c) A combination of 0.1 mg / ml of MMC for 1 minute with a sponge and a total of 11 injections of 300 μg / ml VPA, once a day for the first 7 days immediately after surgery and then on the 10th, 14th, and 21st days after surgery.

[0092] The eyes of the rabbits were evaluated by slit lamp imaging (Figure 12). As found, all the blebs appeared to be functional by day 28. However, the morphology of the above blebs varied greatly depending on the treatment conditions.

[0093] Standard MMC treatment at 0.4 mg / ml resulted in completely avascular and cystic blisters. The treated area was clearly demarcated from normal conjunctiva. Considering that the vasculature supplies oxygen and nutrients and also provides an immune response against potential infections, treating with MMC at 0.4 mg / ml exposes the treated area to a high risk of tissue degeneration and increases vulnerability to infection.

[0094] MMC treatment at 0.1 mg / ml resulted in small avascular areas (marked by the "Star operator mark", Figure 12) remaining apparent in the treated area up to 28 days compared to treatment with 0.4 mg / ml of MMC, but with less avascularity and milder cystic blisters. Thus, when the MMC concentration was decreased, the risk of tissue degeneration and infection in these blisters was much lower.

[0095] MMC at 0.1 mg / ml in combination with VPA resulted in diffused blisters and normal angiogenesis that penetrated the entire treated area. The treated area, similar to normal conjunctival tissue, was expected to have the lowest risk of tissue degeneration and infection.

[0096] The confocal microscope revealed that standard treatment with 0.4 mg / ml MMC resulted in large cysts (marked by "Star operator mark", Figure 13) in the background of sparse collagen fibers. No vascular structures were detected. In contrast, blisters treated with 0.1 mg / ml MMC alone or in combination with VPA were characterized by smaller cysts (marked by "Star operator mark", Figure 13). In the latter two conditions, no differences that may exist in the collagen matrix could be discerned, but vascular structures could be readily visualized (arrowheads, Figure 13). These observations confirm that high doses of MMC enable the formation of large cysts that contribute to blister function. Lower doses of MMC resulted in smaller cysts, with no perceptible increase in size even with treatment with VPA. However, maintaining the presence of vascular structures is essential for the health of the conjunctiva.

[0097] The more subtle ability of VPA on maintaining the collagen structure of the conjunctiva is most clearly shown by histological analysis. When treated with standard 0.4 mg / ml MMC, large gaping spaces formed in the vesicular matrix where collagen fibers disappeared (Figure 14). The remaining collagen fibers were mature fibers, probably existing from the pre-operative tissue. In contrast, with a lower 0.1 mg / ml MMC administration, numerous immature and disordered collagen fibers could be detected (arrowheads, Figure 14), suggesting continuous production of collagen up to and beyond day 28 in the operated area. When low-dose MMC was applied simultaneously with VPA, the collagen network was sparse and mainly composed of mature fibers (Figure 14). This remarkable histology suggests that VPA inhibited the production of new fibers at some point before day 28, such that there was collagen production during the post-operative period, but the tested matrix was mainly highly reduced and composed of thinner mature collagen fibers (arrowheads, Figure 14). Overall, these data indicate that co-treatment with VPA enables the use of a lower dose of MMC. That dose preserves the vasculature and achieves a collagen matrix closer to normal tissue while maintaining the effectiveness of the microshunt from the perspective of vesicle function and integrity.

[0098] The different histologies of the extracellular matrix can be reflected in the differences in gene expression caused by treatment conditions. This was demonstrated by analyzing the amount of transcripts in the treated rabbit conjunctiva. As shown in Figure 15, where each symbol represents the eye of one rabbit (n = 5 for all conditions), the expression of Col1a1 mRNA in rabbit tissues on day 28 treated with 0.1 mg / ml MMC was significantly higher than both 0.4 mg / ml MMC treatment or 0.1 mg / ml MMC + VPA treatment, which supports the histological observations. Importantly, the level of expression of Col1a1 transcripts was similar between 0.4 mg / ml MMC treatment and 0.1 mg / ml MMC + VPA treatment, indicating that the latter can replace the use of high MMC doses in causing a similar decrease in collagen production. The expression of other fibrosis-related genes, including fibronectin, SPARC, and periostin genes, was also significantly reduced by co-treatment with VPA. The expression of Smad6 did not change similarly to VPA alone and is likely due to a drug interaction with MMC.

[0099] The mRNA data was verified by immunoblotting for COL1A1 production in rabbit tissues. As seen in Figure 16 where each symbol represents the eye of one rabbit (n = 5 for all conditions), the level of COL1A1 protein in the surgically treated conjunctiva was lowest in the tissues treated with low-dose MMC plus VPA. This data may be consistent with histological evidence. The collagen content measured in tissues treated with 0.4 mg / mL MMC likely represented the remaining collagen that persisted without further change after treatment because the tissues were metabolically inactive. In the case of the lower dose of 0.1 mg / ml MMC, the treated tissues appeared to be relatively active by showing significantly higher levels of Col1a1 transcription (Figure 15), and were also characterized by the appearance of clearly newly formed immature collagen fibers (Figure 14), and the deposited collagen content was similar to that at the high MMC dose. Notably, when co-treated with VPA, the levels of COL1A1 decreased more consistently among individual rabbits, resulting in a significant 2.4-fold average decrease compared to treatment with 0.1 mg / ml MMC alone. This finding suggests that co-therapy with VPA not only maintains tissue morphology but also ensures greater consistency in maintaining reduced COL1A1 levels compared to treatment with low-dose MMC alone.

[0100] In summary, VPA maintained the conjunctival collagen structure in both the mouse model of conjunctival scarring and the rabbit model of PRESERFLO® microshunt implantation surgery. This strongly supports the ability of VPA to maintain the biomechanical integrity of the conjunctiva after surgical implantation of the microshunt. Furthermore, the maintenance of the conjunctival vasculature by VPA suggests that the drug may also maintain the healthy state of the tissue and support its role as a protective barrier against infection. Additionally, the ability of VPA to maintain the number of goblet cells in the surgically treated conjunctiva indicates that the drug can be used pre- and pro-operatively to prevent the development of dry eye and improve the outcome of glaucoma surgery.

[0101] [Example 5 - Combination Therapy of VPA and Low - Dose MMC for Reducing Post - operative Scarring] To investigate the effect of VPA on post - operative scarring, the rabbit model of micro - shunt implant surgery was repeated as performed and treated under conditions (a), (b) and (c) in Example 4. Then, the rabbit eyes were evaluated.

[0102] Figure 17 shows the histochemical visualization of the implant tip leading to the sub - conjunctival space in the rabbit model of MIGS, treated as shown in the figure. Picrosirius red (pRed) - stained sections observed under polarized light revealed the presence of thick collagen fibers encapsulating the implant in the tissues treated with MMC alone, regardless of the concentration used.

[0103] In contrast, treatment with VPA (300 μg / mL) reduced the presence of thick fibers encapsulating the implant, especially the tip. This indicates that VPA treatment may reduce the risk of implant encapsulation and the resulting device failure.

[0104] The foregoing description has illustrated exemplary embodiments, but it will be understood by those skilled in the relevant art that many variations can be made without departing from the present invention. [Brief Description of the Drawings]

[0105]

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Claims

1. A drug for maintaining a subconjunctival bleb formed by minimally invasive glaucoma surgery (MIGS) and containing valproic acid (VPA), wherein the MIGS includes implanting a glaucoma tube shunt under the subconjunctival cavity and treating with mitomycin C (MMC) at a concentration of 0.1 mg / mL or less.

2. The drug according to claim 1, wherein the VPA has a concentration of 100 to 1000 μg / mL.

3. The drug according to claim 1 or 2, which is suitable for topical administration or subconjunctival administration.

4. The drug according to any one of claims 1 to 3, which is suitable for administration immediately after MIGS.

5. The drug according to any one of claims 1 to 4, which is suitable for daily administration for at least 12 weeks after MIGS.

6. The drug according to any one of claims 1 to 5, which is suitable for repeated administration from 3 to 120 months after MIGS.

7. MIGS includes creating a subconjunctival bleb, and the drug according to any one of claims 1 to 6.

8. Maintaining the subconjunctival bleb includes maintaining the conjunctival collagen structure, and the drug according to any one of claims 1 to 7.

9. A drug for preventing encapsulation of a glaucoma tube shunt implanted under the subconjunctival cavity and containing valproic acid (VPA) and mitomycin C (MMC), wherein MMC has a concentration of 0.1 mg / mL or less.

10. A drug for maintaining the aqueous humor outflow ability of the glaucoma tube through its lumen after a glaucoma tube shunt containing valproic acid (VPA) and mitomycin C (MMC) is implanted under the subconjunctival cavity, wherein MMC has a concentration of 0.1 mg / mL or less.

11. The drug according to claim 9 or 10, wherein the VPA has a concentration of 100 to 1000 μg / mL.

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