Technical Field

The present invention relates to the formation of therapeutic combinations of the natural hispidulin molecule for use in the treatment of proliferative vitreoretinopathy (PVR) by formulating it with single and/or 5 -fluorouracil (5-FU) or taxol group chemotherapeutic agents.

The invention is applicable in the pharmaceutical industry. Due to the potential of hispidulin to be used as a single or in combination with a low dose of 5-FU or docetaxel in the treatment of PVR, hispidulin has the property of being made into a single or combined preparation by pharmaceutical companies.

State of the Art (Background)

Proliferative vitreoretinopathy (PVR), a common cause of severe vision loss or blindness, is an important eye health problem that develops in the failure of a ruptured retinal detachment (RD) repair. Defects in the retina cause the migration of retinal pigment epithelium (RPE) and glial cells with proliferative potential to the vitreous cavity. It is a clinical syndrome associated with retinal traction and detachment in which cells proliferate and contract on the retinal surfaces and vitreous. Exposure of RPE and glial cells to growth factors and cytokines activates the pathogenic mechanisms of PVR. Cells migrate, multiply, and produce extracellular matrix proteins that suppress retinal traction. The critical step in the development of PVR is RPE metaplasia to fibroblast-like cells in the process known as mesenchymal transition from the epithelial. Severe PVR can complicate about 10% of cases of retinal detachment surgery, resulting in failure or requiring reoperation.

Although a number of molecular targets have been investigated for the prevention and treatment of PVR, none have yet shown evidence of systematic benefit in humans. PVR remains a challenging clinical condition that can limit the success of RD repair, especially in sedentary and severe cases. Although surgical innovations have greatly improved PVR outcomes in recent years, resistant cases remain. Therefore, there is a need for effective pharmacological agents that can be used in the treatment of PVR.

Currently, there is no proven pharmacological agent for the treatment or prevention of PVR. The ability to manage this disease has been significantly improved by modem surgical methods, especially vitrectomy. However, permanent limitations to surgical success lead to the investigation of molecular targets. The most commonly studied and clinically used molecules in the medical treatment of PVR are anti-proliferative agents that inhibit cell proliferation and reduce experimental tractional retinal detachment, especially compounds such as 5 -fluorouracil (5-fluorouracil, 5-FU), daunorubicin, taxol, vincristine, cisplatin, mitomycin, colchicine, retinoic acid, and daptomycin. However, the clinical value of these substances is limited due to toxic undesirable side effects. For example, the use of 5-FU in the posterior segment of the eye damages the photoreceptor cells. 5-FU transforms into 5- fluorouridine, which is its active metabolite in the cell, creating an antiproliferative and anticontractile effect by inhibiting timidylate synthetase. In human studies, it was reported that intraocular 5-FU combined with vitrectomy resulted in only 60% anatomical success in eyes with PVR.

The intraocular inflammatory environment developing in PVR is an important aspect of PVR pathophysiology, so steroids have been explored as a potential therapeutic option. Although corticosteroids have the potential to greatly reduce the production of growth factors that contribute to mechanisms of action and membrane formation, in a randomized, controlled clinical study of patients at high risk of developing PVR, treatment with systemic corticosteroids was shown not to improve visual acuity while causing a decrease in epiretinal membrane formation. Combination therapies of low molecular weight heparin (LMWH) or steroids, an anticoagulant that binds many growth factors with 5-FU, reduce fibrin after vitrectomy, inhibit RPE proliferation, bind fibrogenic growth factors, and reduce experimental tractional retinal detachment. However, further clinical research on these combinations has proven that they do not show a beneficial effect on established PVR or unselected primary retinal detachments, but rather worsen visual acuity. According to the results of a different clinical study, the combination of 5-FU and LMWH does not improve the primary or final anatomical success rate of primary vitrectomy surgery for retinal detachment, nor does it make a statistical difference in the incidence of PVR in the treatment and placebo groups.

Vascular endothelial growth factor (VEGF), anti-VEGF drugs involved in the pathogenesis of PVR are well tolerated and are widely used in the treatment of disorders such as age-related macular degeneration and diabetic macular edema, among others. However, according to the results of the study, there was no decrease in the rate of retinal detachment in the subjects who received 1.25 mg intravitreal bevacizumab at the end of vitrectomy surgery for RD with grade C PVR. In addition, according to the results of a prospective study conducted for treatment purposes, retinoic acid, a vitamin A derivative that regulates cellular differentiation, has been reported to increase the reconnection rate of the retina in the treatment of PVR-complicated RD.

According to several clinical trials, it wasobserved that the administration of vitamin A derivative oral 13-cis-retinoic acid or microtubule inhibitor colchicine after the operation reduces PVR and increases the retinal attachment rate after surgical repair. In recent years, substances obtained from foods with pharmaceutical potential have attracted increasing attention in various clinical disciplines and diseases. Flavonoid applications have been demonstrated to have different therapeutic potentials by reversing anti-inflammatory, antiproliferative, antioxidant effects and epithelial-mesenchymal transition. However, according to the results of all experimental and clinical research, PVR therapy does not yet have an effective treatment option. Therefore, there is a need to develop new approaches in the treatment of PVR.

Brief Description and Objects of the Invention

According to the experimental results for the treatment of proliferative vitreoretinopathy, hispidulin (5,7-Dihydroxy-2-(4-hydroxyphenyl)-6-methoxy-4H-l-benzopyran -4-one, 4',5,7- Trihydroxy-6-methoxyflavone, 6-Methoxyapigenin, Salvitin, 6-Methylscutellarein, Dinatin, Scutellarein 6-methyl ether) in natural flavone structure applied at low concentration can completely block the RPE cell proliferation and migration prominent in this disease. Demonstration of anti -PVR effect in commercial human RPE cell series (ARPE-19) and primary RPE cells obtained from bovine eyes in an in vitro experimental environment proved that hispidulin can be a potential candidate molecule. Transforming growth factor beta 2 (TGF-P2) is one of the peptides responsible for PVR development and is often used in modeling. According to the results obtained within the scope of the invention, proliferation increases by 30% in cells exposed to 10 ng/ml TGF-P2 for 48 hours, while the migration of cells is also encouraged. However, treatment of these TGF-P2 treated cells with 3 pM hispidulin inhibits cell proliferation by 100% while greatly suppressing cell migration. Moreover, the concentration of hispidulin used for control purposes does not cause toxicity to cells.

As a second alternative, it was also tested in the model created with 5-FU or taxol derivative docetaxel, which is most commonly used for treatment in PVR disease that develops in humans. Accordingly, 150 nM 5-FU or 3 nM docetaxel treatment to RPE cells exposed to 10 ng/ml TGF-P2 strongly suppresses cell proliferation and migration. However, the fact that these pharmacological agents are chemotherapy drugs causes the development of side effects. Therefore, 100% suppression of cell proliferation in PVR by reducing these chemotherapeutics by 2/3 at a dose of 50 nM 5-FU or 1 nM docetaxel and in combinations with 1 pM hispidulin indicates that hispidulin can be used as a single in the treatment of PVR or reduced by 2/3 with the specified chemotherapeutics. Reducing the use of 5-FU or docetaxel together with the non-toxic hispidulin molecule in its natural structure by 2/3 in treatment will also reduce or terminate its side effects.

The natural hispidulin molecule contained in the invention has not been previously used and reported in the treatment of PVR disease by being formulated with single and/or 5-FU or taxol group chemotherapeutic agents. There are also no patents covering the results obtained according to the scans. Therefore, the results obtained for PVR therapy within the scope of the invention are unique.

Definitions of Figures Describing the Invention

The figures and related descriptions required to better understand the subject of the invention are as follows. Figure 1: (A) % effect of hispidulin on concentration and time-dependent RPE cell survival. Molecular structure of hispidulin, (B) 24 hours, (C) 48 hours, and (D) 72 hours of treatment efficacy. *P<0.005 control group (0).

Figure 2: The efficacy of hispidulin in combination with single, 5-FU or docetaxel in the TGF-P2 induced PVR model; (A) hispidulin single or combined treatment with 5-FU in the RPE cell series, (B) apoptosis efficacy of treatment in the RPE cell series, (C) hispidulin single or combined treatment with 5-FU in bovine primary RPE cells, (D) hispidulin single or combined treatment with docetaxel in the RPE cell series. *p<0.005 compared to the control group (0), **p<0.001 TGF-P2 group. (His: hispidulin; 5-FU: 5-fluorouracil; Doc: docetaxel; RPE: retinal pigment epithelial cells; TGF-P2: transforming growth factor beta 2).

Figure 3: Single administration of hispidulin or its combination with 5-FU blocks RPE migration. Combined therapy of RPE cells with 3 pM hispidulin and 50 nM 5-FU in the TGF- P2-induced PVR model inhibits cell migration (A, B). Low-dose therapy significantly inhibited mRNA expression of the transcription factors Snail (C), Twist (D) and ZEB1 (E) involved in cell migration. *p<0.001 compared to the control (untreated), **p<0.001 compared to the TGF-P2 group. (His: hispidulin; 5-FU: 5-fluorouracil; Doc: docetaxel; RPE: retinal pigment epithelial cells; TGF-P2: transforming growth factor beta 2).

Figure 4: The combination modality regulates the expression of several genes involved in cell migration. The combination of low concentration hispidulin and 5-FU downregulates MMP1, MMP2, MMP7, MMP9 (Figure A-D) and fibronectin (F) mRNA expression induced by TGF-P2. The treatment modality upregulates mRNA expression of occludin (E) and N- cadherin (G)*p<0.001 compared to the control group (untreated), **p<0.05 compared to the TGF-P2 group, ^# p<0.01 compared to the TGF-P2 group. (His: hispidulin; 5-FU: 5- fluorouracil; Doc: docetaxel; RPE: retinal pigment epithelial cells; TGF-P2: transforming growth factor beta 2).

Detailed Description of the Invention

Demonstration of anti -PVR effect in commercial human RPE cell series (ARPE-19) and primary RPE cells obtained from bovine eyes in an in vitro experimental environment proved that hispidulin can be a potential candidate molecule. Transforming growth factor beta 2 (TGF-P2) is one of the peptides responsible for PVR development and is often used in modeling. According to the results obtained within the scope of the invention, proliferation increases by 30% in cells exposed to 10 ng/ml TGF-P2 for 48 hours, while the migration of cells is also encouraged. However, treatment of these TGF-P2 treated cells with 3 pM hispidulin inhibits cell proliferation by 100% while greatly suppressing cell migration. Moreover, the concentration of hispidulin used for control purposes does not cause toxicity to cells.

As a second alternative, it was also tested in the model created with 5-FU or taxol derivative docetaxel, which is most commonly used for treatment in PVR disease that develops in humans. Accordingly, 150 nM 5-FU or 3 nM docetaxel treatment to RPE cells exposed to 10 ng/ml TGF-P2 strongly suppresses cell proliferation and migration. However, the fact that these pharmacological agents are chemotherapy drugs causes the development of side effects. Therefore, 100% suppression of cell proliferation in PVR by reducing these chemotherapeutics by 2/3 at a dose of 50 nM 5-FU or 1 nM docetaxel and in combinations with 1 pM hispidulin indicates that hispidulin can be used as a single in the treatment of PVR or reduced by 2/3 with the specified chemotherapeutics. Studies have shown that 5-FU suppresses PVR pathogenesis. However, it shows certain toxic effects on photoreceptor cells in the retina at doses where it is effective. It is known that the expected visual levels may not be achieved in the postoperative period due to these toxic effects. For this reason, the doses at which the strongest effect could be achieved were tried by minimizing the toxic effect of 5- FU and docetaxel. In the dose studies, these chemotherapeutics were reduced in various doses and their combination with hispidulin was examined. In line with the data obtained, reducing the use of 5-FU and docetaxel together with the non-toxic hispidulin molecule in the natural structure by 2/3 in treatment may also reduce or terminate the toxic side effects on the retina.

In the experimental study, the commercially obtained hispidulin was dissolved in dimethyl sulfoxide (DMSO) to obtain the doses of 0.78 pM, 1.56 pM, 3.12 pM, 6.25 pM, 12.5 pM, 25 pM, 50 pM and 100 pM. By testing these concentrations, it was observed that the strongest PVR inhibitory effect was 3 pM with the least toxic effect. In combination treatments, 5-FU solution, which is sold as a ready-made preparation, is diluted with DMSO solution and doses of different concentrations were obtained. Among the doses obtained, the least toxic effect and the most PVR inhibitory effect were observed with 1 pM hispidulin + 50 nM 5-FU.

The natural hispidulin molecule contained in the invention has not been previously used and reported in the treatment of PVR disease by being formulated with single and/or 5-FU or taxol group chemotherapeutic agents.