Patent ID: 12239644

BEST MODE FOR INVENTION

The present disclosure pertains to a pharmaceutical composition comprising fursultiamine or a salt thereof for prevention or treatment of macular degeneration.

MODE FOR CARRYING OUT THE INVENTION

A better understanding of the present disclosure will be obtained from the following examples which are set forth to illustrate, but should not be construed to limit, the present illustrative

Example 1: Inhibitory Effect of Fursultiamine on Neovascularization—HIF-1α Downregulation

Neovascular age-related macular degeneration is characterized by choroidal neovascularization. In the retina or choroid, hypoxia increases the expression of HIF-1α (hypoxia inducible factor1alpha) which, in turn, promotes VEGF to induce neovascularization in the choroid. This choroidal neovascularization results in a loss of vision.

In the present disclosure, the thiamine derivative fursultiamine was assayed for inhibitory activity against HIF-1α and preventive effect on choroidal neovascularization. To this end, in-vitro tests were conducted with ARPE-19 (Adult Retinal Pigment Epithelial cell line-19).

ARPE-19 cells are seeded into 60-mm dishes and incubated overnight to adhere to the dishes. On the next day, the cells were treated with fursultiamine-HCl (Toronto research chemical, F865230) or a vehicle (dimethyl sulfoxide (DMSO), Sigma-Aldrich) at the specific concentrations of 0 μM, 20 μM, 50 μM, and 100 μM. After one hour, the cells were exposed to 1% oxygen condition in a hypoxic chamber (INVIVO2400, Baker). After hypoxic exposure for 4-6 hours, the cells were lysed with a protein lysis buffer and protein isolation from the lysate was conducted.

Protein concentrations were measured using a BCA protein assay kit (Pierce BCA protein assay kit, Thermo Fisher Scientific). Samples with the same protein amounts were mixed with 4× loading buffer and boiled to denature the proteins into the primary structure before being loaded on SDS-PAGE gel. After electrophoresis, the proteins were transferred onto a PVDF membrane. The membrane was incubated overnight at 4° C. with a dilution of an anti-HIF-1α antibody (Novus, NB100-479) in 5% BSA (bovine serum albumin) solution.

On the next day, the membrane was incubated with a HRP (horseradish peroxidase)-conjugated secondary antibody, followed by reaction with ECL (enhanced chemiluminescence) reagent to induce chemiluminescence. The western blots were scanned with a chemiluminescence image analyzer (GE Healthcare, LAS-4000) to give an image exhibiting luminescence intensities depending on protein amounts. As a loading control, β-tubulin showed that the same protein amounts were loaded.

As a result, the expression of HIF-1α which was upregulated in the hypoxic condition (1% oxygen) was reduced by treatment with fursultiamine (FIG.1).

Example 2: Inhibitory Effect of Fursultiamine on Neovascularization—Suppression of Choroidal Vascular Endothelial Cell Growth

Effects of fursultiamine on the growth of choroidal vascular endothelial cells were evaluated by the ex vivo mouse choroid sprouting assay for age-related macular degeneration.

The eyeball was resected from C57BL/6J mice (Jackson Laboratory) at 3 or 4 weeks of age and the choroid/sclera was separated therefrom and sectioned into a size of 1 mm×1 mm. After being thawed on ice, Matrigel (Becton Dickinson, BD matrigel) in a liquid state was added in an amount of 300 μl to each well of 24-well plates and one of the cut choroid/sclera segments was seeded in the Matrigel in each well. Then, Matrigel was solidified for 10 min in a 37° C. incubator before adding 500 μl of an EGM medium (Lonza, Endothelial Growth Medium) to each well.

Subsequently, the growth of vascular endothelial cells was induced in a 37° C. incubator. The medium was exchanged every two days with a fresh one containing 20 μM or 50 μM fursultiamine-HCl. The growth of vascular endothelial cells from the choroid was monitored from the day of seeding the choroid/sclera and observed on day 3 to 5. The sprouting distances were measured at a total of 4 sites using the ImageJ software and averaged. Statistical analyses were performed with the Prism program, with statistical significance defined as p<0.05.

As a result, it was observed that the growth of vascular endothelial cells was inhibited by fursultiamine (FIGS.2a-2band Table 1).

TABLE 1Fursultiamine (uM)02050Sprouting398.417 ± 40.827229.926 ± 28.348126.713 ± 22.810range(pixels)

Example 3: Inhibitory Effect of Fursultiamine on VEGF Secretion

ARPE-19 cells were seeded into 60-mm dishes and incubated overnight to adhere to the dishes. On the next day, the medium was replaced with serum-free medium. The cells were treated with fursultiamine-HCl (Toronto research chemical, F865230) or a vehicle (DMSO, Sigma-Aldrich) at the specific concentrations of 0 μM, 50 μM, and 100 μM and then exposed to 1% oxygen condition in a hypoxic chamber (INVIVO2400, Baker). After hypoxic exposure for 4-6 hours, the cells were lysed with a protein lysis buffer and protein isolation from the lysate was conducted.

After 12 hours later, the medium was recovered and secreted levels of VEGF in the medium were measured using human VEGF enzyme linked immunoassay (ELISA) (R&D systems). Quantitation methods and concentration calculations were conducted according to the manufacturer's manual. Statistical analyses were performed using the Prism program, with statistical significance defined as p<0.05.

As a result, it was observed that the VEGF secretion which was increased in the hypoxic condition (1% oxygen) was suppressed by treatment with fursultiamine (FIG.3and Table 2).

TABLE 2—Vehicle123Oxygen condition21% O21% O21% O21% O2Fursultiamine (μM)0050100VEGF (pg/mL)86.6 ± 3.2184.2 ± 12.7163.4 ± 7.447.4 ± 4.6

Example 4: Comparison of Grade of Vascular Leakage Through Fluorescein Angiography

For use in assessing the therapeutic potential of fursultiamine in AMD, macular degeneration animal models in which retinopathy was induced by laser was constructed from C57BL/6J mice at 7-8 weeks of age. Ten mice were used for each of fursultiamine-administered and control groups. Fursultiamine was orally administered at a dose of 50 mg/kg for 8 days from one day before laser irradiation to one week after laser irradiation. To the control, sterile distilled water used as a solvent was administered.

The mice were anesthetized with Avertin (Sigma-Aldrich) and administered a mydriatic to induce dilation of the pupil. An argon laser (OcuLight GL, IRIDEX) was projected to both eyes to form four laser spots in each eyeball.

After one week, the mice were anesthetized and intraperitoneally injected with fluorescein (AK-FLUOR 10%, Akorn). Using MICRON IV Basic System (Phoenix Research Labs), retina base and fluoresceine images were captured.

Images for vascular leakage were obtained within 3 minutes (early phase) and about 7 minutes (late phase) after intraperitoneal injection of fluorescein to monitor fluorescence size and intensity between the two time points.

Hyperfluorescence with increasing size and intensity was scored as 2B; hyperfluorescence with constant size but increasing intensity was scored as 2A; hyperfluorescence with differences in none of size and intensity was scored as 1; and faint fluorescence with no spots was scored as 0. More spots scored as 2B accounted for higher neovascular leakages. Statistical analyses were performed using the Prism program, with statistical significance defined as p<0.05.

As a result, the vascular leakage for the late phase was decreased in the fursultiamine-treated choroidal retinopathy model, demonstrating that fursultiamine decreases vascular leakage (FIGS.4a-4b).

Example 5: Size of Choroidal Neovascularization (CNV) Lesion in Laser-Induced CNV Model after Treatment with Fursultiamine

In order to construct laser-induced CNV models, C57BL/6J mice at 7-8 weeks of age were anesthetized with Avertin (Sigma-Aldrich) and administered a mydriatic to induce dilation of the pupil, followed by projecting an argon laser (OcuLight GL, IRIDEX) to both eyes to form four laser spots in each eyeball. Ten mice were used for each of fursultiamine-administered and control groups. Fursultiamine was orally administered at a dose of 50 mg/kg for 8 days from one day before laser irradiation to one week after laser irradiation. To the control, sterile distilled water used as a solvent was administered.

One week after laser irradiation, the mice were anesthetized and subjected to fluorescein angiography. For histochemical staining, the eyes were enucleated and fixed in 4% paraformaldehyde (4% PFA, EMS) for 30 min. After removal of the cornea and lens, retinas were separated from the underlying choroid. The separated choroid was incubated for 1 hour in a blocking buffer (0.2% bovine serum albumin, 5% normal goat serum, 0.5% Triton X-100) to which a dilution of an antibody (Isolectin IB4-Alexa Fluor 488, Invitrogen) against isolectin, which is one of vascular markers, in a blocking buffer was then added. After reaction, the choroid was spread flatly and loaded with a mount solution (Mountant, Thermo Scientific), followed by applying a cover glass thereto for stabilization.

On the next day, images were captured at ×100 magnification under a confocal microscope (LSM800, Zeiss). Sizes of laser spots were quantitated using ImageJ program. Statistical analyses were performed using the Prism program, with statistical significance defined as p<0.05.

As a result, fursultiamine reduced CNV lesion sizes (FIG.5and Table 3).

TABLE 3Fursultiamine—ControladministeredFursultiamine (mg/kg)050Lesion size (10−3mm2)23.9 ± 2.415.7 ± 1.0

Example 6: Fursultiamine-Mediated Reprogramming of Mitochondrial Metabolism Recovery in Retinal Pigment Epithelium

Inflammation is known to be a leading pathological mechanism in neovascular age-related macular degeneration. Thus, examination was made to see whether fursultiamine treatment reprograms mitochondrial metabolism and thus inhibits inflammation. Because a change in mitochondrial metabolism may cause or aggravate inflammation, fursultiamine was expected to find applications in preventing or treating inflammatory neovascular ocular diseases when reprogramming mitochondrial metabolism.

To be specific, ARPE-19 cells were seeded onto 96-well XF plates, with the exchange of a fresh medium every two days. On day 5 after cell seeding, the cells were treated with 10 μg/ml LPS (lipopolysaccharides, Sigma-Aldrich) and then with 50 μM fursultiamine on day 6. On day 7, the medium was exchanged with an XF medium (Seahorse XF DMEM medium, Agilent) and the plates were left for 30 min at 37° C. in a non-CO2incubator.

In order to assess mitochondrial spare capacity, the following mitochondrial electron transport inhibitors and respiratory uncouplers were used in corresponding steps: oligomycin (2 μM), FCCP (0.5 μM), rotenone (2 μM), and antimycin A (2 μM).

The oxygen consumption rate (OCR) was measured using an XFe96 analyzer (Agilent) according to the manufacturer's manual.

As a result, fursultiamine treatment enhanced the mitochondrial spare capacity decreased by LPS. These data imply that fursultiamine reprograms mitochondrial energy metabolism and suppresses inflammation (FIGS.6a-6band Table 4).

TABLE 4LPS + FurConLPS (48 h)50 uM (24 h)LPS (ug/ml)—1010fursultiamine——50(uM)OCR (%)153.614 ± 36.22196.3486 ± 46.440189.438 ± 21.392

Taken together, the data thus obtained indicate that fursultiamine has inhibitory activity against the pathological mechanism of neovascular age-related macular degeneration and a composition comprising fursultiamine is expected to exhibit preventive and therapeutic effects on macular degeneration.

INDUSTRIAL APPLICABILITY

The present disclosure pertains to a composition for prevention or treatment of macular degeneration. More specifically, the present disclosure pertains to a composition comprising fursultiamine or a salt thereof for prevention or treatment of macular degeneration.