Tolbutamide Eye Drops Increase Aqueous Humor Outflow and Lower Intraocular Pressure: A Proof of Concept for Glaucoma Treatment

Background: Glaucoma refers to a heterogeneous group of diseases characterized by retinal cell degeneration and optic nerve atrophy leading to blindness. Even though about 40% of patients have normal intraocular pressure (IOP), current treatment. focuses on lowering IOP. With time, current drugs become less effective, which has motivated the search for novel drugs.The objective was to establish whether modulators of ATPsensitive potassium channels influence IOP. J Ophthalmol Res 2021; 4 (2): 114-127 DOI: 10.26502/fjor.2644-00240031 Journal of Ophthalmology and Research 115 Methods: The double-blind, 5-day short duration Proof-Of-Concept study was carried out at the Ophthalmology Clinic, University of Cologne, Germany. The only inclusion criteria were a diagnosis of glaucoma, ability to understand that they would be treated with an experimental drug, and readiness to sign a consent form. Results: In rabbits, 1 h after topical application of 80 μL of 0.5% tolazamide, tolbutamide, glibenclamide, and chlorpropamide suspended in phosphate buffered saline IOP de-creased, whereas 0.5% diazoxide increased IOP. In Cynomolgus monkeys tolbutamide decreased IOP. In 9 glaucoma patients treated for 5 days with one drop of a 0.5% tolbutamide solution twice daily, IOP was an average of 17% lowered. In one patient with ocular hypertension, tolbutamide lowered IOP by a 5-day average of 29% and increased aqueous humor outflow by 185%. No local adverse effects were observed. Conclusions: The data presented show that blockers of the ATP-sensitive potassium channels lower IOP whereas diazoxide, an ATP-sensitive potassium channel opener, increases IOP suggesting that elevated IOP results from an ionic imbalance. The data suggest that sulfonylurea drugs are useful for the treatment of glaucoma.


Introduction
Glaucoma refers to a heterogeneous group of ocular diseases that are characterized by retinal ganglion cell (RGC) degeneration [1] and progressive optic nerve atrophy; left untreated glaucoma gradually leads to visual field loss and blindness. Although Elevated intraocular pressure (IOP) is considered the main risk for the onset and progression of POAG, even though about 40% of patients present IOP values within the normal range [3,4] suggesting that elevated IOP is neither essential nor sufficient [5,6] to cause glaucoma; in fact, the risk of unilateral blindness in POAG patients treated to lower IOP is estimated to be about 27% [7] indicating that lowering IOP retards but does not prevents RGC degeneration and blindness.
Since elevated IOP, which results from impaired drainage of aqueous humor through the trabecular meshwork-Schlemm's canal complex [8], is the only modifiable risk factor, current therapies seek to lower IOP even in patients with normal IOP. Even though effective surgical procedures have been developed to lower IOP, pharmaceutical therapy with drugs administered topically is the accepted management of glaucoma.

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Topical prostaglandin analogs are most frequently used to treat glaucoma. Used once daily, prostaglandin analogs lower IOP by 25-30% [9] and stabilize it at a lower level by increasing uveoscleral outflow [10] without significant systemic side effects; however, they have considerable local adverse reactions [11][12][13][14][15] and in rare cases cause cystoid macular edema [16]. Several drugs belonging to various chemical classes is also used to treat glaucoma; however, side effects have limited their use as first line treatment [17][18][19][20][21].

Cynomolgus Monkeys
The IOP in the two monkeys treated with tolbutamide in suspension decreased by an average of 7.5 mm Hg or 31% and in the two monkeys treated with 0.5% solution the IOP decreased by an average of 6 mm Hg or 26% from the pre-treatment values. In the monkeys treated with vehicle the IOP decreased by 4.5 mm Hg or 19%, which was likely the result of the anesthesia [39]. No evidence of irritation, or redness were noticed in the 4 monkeys.

Subjects with Elevated IOP
Both eyes of a patient with pseudoexfoliative glaucoma and of a patient with POAG were treated with one drop of 0.5% tolbutamide solution. IOP was measured at the times indicated in Table 1 by a nurse while the patients were hospitalized. Table 1 shows that 5 hours after administration of tolbutamide, IOP was substantially lower in both eyes of the patient with pseudoexfoliation glaucoma. In the POAG patient tolbutamide decreased IOP in both eyes as early as 1 hour and was meaningfully lower at 3-and 16-hours postadministration.

Subjects During a 5-Day Treatment
To deter-mine the longer-term effect of tolbutamide on IOP and possible side effects, 9 patients with diagnosed POAG were treated with one drop of 0.5% tolbutamide twice daily for 5 days. Table 2 shows that in seven of the nine POAG patients,  Table 3 shows that during the 5-day treatment, the IOP in the tolbutamide-treated eye ranged from a high of 22 mm Hg at 9:00 AM on day 2 to a low of 10 mm Hg on day 9. The average 5-day IOP in the tolbutamide-treated eye was -6.1 ± 3.8 mm Hg (33%) lower than the pre-treatment IOP and significantly different (p=0.0001) from the 5-day mean (+0.5 ± 2.1) of the vehicle-treated eye.

Effect Of 0.5% Tolbutamide On Aqueous
Humor Outflow Facility Table 4 shows that tolbutamide increases both aqueous humor production and outflow. Production in-creased by 158% whereas outflow increased by 342%, a net 185% increase in outflow. In the untreated eye both flow and outflow decreased from the pre-treatment values, which may be the result of diurnal fluctuations [42,43]. Eight hours after administration, IOP was 20% lower than 30 minutes after administration of a 0.5% suspension of tolbutamide and 24% lower after the administration of a 0.5% solution of sodium tolbutamide.  (a) Patient 1 was hospitalized for conditions unrelated to glaucoma; drops were administered by a nurse. The patient, the nurse, and the doctor measuring IOP were not aware of which eye received the drug and which the vehicle.
(b) Age not recorded.

3.Discussion
Glaucoma is a complex disease that presents significant obstacles to treatment because over time many patients require a combination of more than one drug to control IOP and even when IOP is controlled, optic nerve degeneration continues at a slower rate [41,42].
When IOP can no longer be controlled with medications surgical intervention is required, which often is not a permanent solution [43]. It is, therefore, essential to develop novel drugs that modulate the metabolic processes of trabecular meshwork, ciliary body, and retinal cells.
Here, we have shown that in rabbits blockers of KATP

Materials and Methods
The use of animals and all treatment protocols were approved by the responsible office for animal protection and adhered to the ARVO statement for the use of Animals in ophthalmic and vision research.

Effect of KATP Modulators on IOP of Rabbits
New Zealand White rabbits (2-3

Cynomolgus Monkeys
Tolbutamide, was converted into the water-soluble sodium salt by dissolving it in 0.25 M NaOH; the dissolved drug was added to 0.4% HPMC, the pH was adjusted to 6.7, tonicity to 300 mOsm with NaCl, and preserved with 0.001% thimerosal. The effect of 0.5%

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solubilized tolbutamide on IOP was compared to 0.5% tolbutamide suspended in PBS in Cynomolgus monkeys.

Human Subjects
Sodium tolbutamide was formulated as a 0.5% solution in 1.5% boric acid, 3% povidone, 0.002% thimerosal, pH 6.5, 281 mOsm. The drug was prepared under GMP guidelines by SK Pharmaceuticals (San Diego, CA, USA). Drug and vehicle control vials were color coded. Control vials were prepared in like manner without tolbutamide.

IOP of Cynomolgus Monkeys
Monkeys were anesthetized with ketamine hydrochloride (25 mg/kg) and baseline IOP determined. One drop of 0.5% tolbutamide suspension and one drop of 0.5% tolbutamide solution were each administered to the right eye of two monkeys; vehicle was administered to the left eyes. IOP was determined one hour after drug or vehicle administration. We are grateful to Dr. Craig Crosson for performing the study at Texas Tech University Health Sciences Center (Lubbock, Texas, USA).

Subjects
The human study was done with the approval of the Ethics Committee of the University of Cologne (Germany) and with the informed consent of each patient.

Subject
One drop of 0.5% tolbutamide suspended in PBS was instilled into the right eye and one drop of 0.5% tolbutamide solution was instilled into the left eye of a male, 62-year-old subject at 7:30 AM; IOP was measured by applanation tonometry at 8:00 AM and at 2-hour intervals until 4:00 PM.

Patients
Both eyes of a pseudoexfoliative glaucoma patient and of a POAG patient, hospitalized for conditions unrelated to glaucoma, were treated with one drop of 0.5% tolbutamide and IOP measured at "0" time and at the indicated times ( Fig. 1 and Table 1) by applanation tonometry.

Tolbutamide on the IOP of Glaucoma Patients
To

Statistical Analysis
Data was analyzed using the 2-sided paired t-test for 2 dependent means using GraphPad Prism (version 8.0.1, GraphPad software Inc., San Diego, CA, USA).
A p-value of ≤ 0.05 was considered significant.

Conclusion
We have shown that sulfonylureas administered topically to the eye lower IOP without any observable local side effects at a dose 500-fold lower than the lowest dose results from a greater increase in aqueous humor outflow via the trabecular meshwork than the increased formation; increased formation may represent a reversal of the decreased metabolic activity that occurs with age. The results presented here of the effect of sulfonylureas on IOP and aqueous dynamics are a proof-of-concept and validation on a larger number of glaucoma patients is necessary before conclusions can be reached as to the mechanism of the IOP lowering effect of sulfonylureas.

Funding
This research received no external funding.

Informed Consent Statement
Informed consent was obtained from all subjects involved in the study.

Data Availability Statement
The data presented in this study are openly available in Yareta at [doi]. 10.26037/ yareta:rfnal 7rkpzamzpcglubitcct6a.

Conflicts of interest
The authors are co-inventors of patent US No: