2024 Volume 72 Issue 4 Pages 381-384
Bietti’s crystalline dystrophy (BCD) is an autosomal recessive chorioretinal degeneration caused by mutations in the CYP4V2 gene. It is characterized by cholesterol accumulation and crystal-like deposits in the retinas. Hydroxypropyl-β-cyclodextrin (HP-β-CyD) exerts therapeutic effects against BCD by reducing lysosomal dysfunction and inhibiting cytotoxicity in induced pluripotent stem cell (iPSC)-RPE cells established from patient-derived iPS cells. However, the ocular retention of HP-β-CyD is low and needs to be improved. Therefore, this study used a viscous agent to develop a sustained-release ophthalmic formulation containing HP-β-CyD. Our results suggest that HP-β-CyD-containing xanthan gum has a considerably higher sustained release capacity than other viscous agents, such as methylcellulose and sodium alginate. In addition, the HP-β-CyD-containing xanthan gum exhibited pseudoplastic behavior. It was less cytotoxic to human retinal pigment epithelial cells compared with HP-β-CyD alone. Furthermore, the slow release of HP-β-CyD from xanthan gum caused a sustained decrease in free intracellular cholesterol. These results suggest that xanthan gum is a useful substrate for the sustained release formulation of HP-β-CyD, and that HP-β-CyD-containing xanthan gum has potential as an eye drop for BCD treatment.
Bietti’s Crystalline Dystrophy (BCD) is an autosomal recessive chorioretinal degenerative disease caused by mutations in the CYP4V2 gene. This disease accounts for approximately 10% of retinal degenerative diseases.1) CYP4V2, which is involved in lipid metabolism, is highly expressed in retinal pigment epithelial (RPE) cells. However, the mechanism underlying BCD pathogenesis remains unclear. Atrophy of RPE cells leads to secondary photoreceptor atrophy and induces chorioretinal degeneration.2) There remains no effective treatment for BCD. Thus, drugs that improve circulation and vitamin supplements are used as symptomatic treatment. Most patients with BCD develop progressive visual dysfunction after the age of 20 years, and blindness occurs after the age of 60 years. Therefore, there is an urgent need for the development of new therapeutic agents.
The therapeutic effects of cyclodextrins (CyDs) have been demonstrated in recent years. Namely, there has been a paradigm shift in their use from pharmaceutical additives to active pharmaceutical ingredients (APIs).3) CyDs have been studied as APIs in Niemann–Pick disease type C (NPC), familial amyloid polyneuropathy, cancer, and septic shock.3) Furthermore, they have been used as adjuvants. The therapeutic effects of CyDs on NPC have been extensively demonstrated. NPC is characterized by the accumulation of free cholesterol or glycolipids such as sphingomyelin and gangliosides in the late endosomal/lysosomal system (endolysosome), owing to abnormalities in the membrane protein NPC1 or the soluble protein NPC2.4) In a 2001 study, intraperitoneal administration of hydroxypropyl-β-cyclodextrin (HP-β-CyD) to NPC model mice reduced free cholesterol contents in the liver and delayed the progression of neurological symptoms.5) This report facilitated further research on the use of HP-β-CyD as a therapeutic agent for various dyslipidemias.
Most recently, we demonstrated that HP-β-CyD exerts a therapeutic effect against BCD by reducing lysosomal dysfunction and inhibiting cytotoxicity in patient-specific induced pluripotent stem cell (iPSC)-RPE cells.6) However, it possesses high water solubility and molecular weight, resulting in low cellular uptake. In addition, the HP-β-CyD used in eye drops for BCD treatment has low retention in the eye and is difficult to deliver to the retina in the posterior region of the eye.
Properties such as improved corneal permeability, prolonged corneal tissue-drug contact time, non-irritation, and appropriate rheological properties improve drug delivery to the eye.7) Thus, attempts have been made to improve these properties using viscous agents, micro-nano particles,8) lipid nanocarriers,9) and other formulations. Viscous agents are often used to increase the viscosity of drug solutions, improve retention on the ocular surface, and improve drug utilization efficiency. Known representative viscous agents include sodium carmellose, methylcellulose, sodium alginate, and xanthan gum. The use of viscous agents prolongs the retention of the active ingredient on the ocular surface and helps to withstand clearance induced by blinking.10) Thus, viscous agents have been widely used to design eye-drop formulations for common low-molecular-weight drugs; however, there have been few reports on the use of viscous agents as eye-drop formulations when utilizing CyD as an API.
In this study, we investigated a sustained-release formulation of HP-β-CyD using viscous agents to construct an ophthalmic formulation containing HP-β-CyD for BCD treatment.
Xanthan gum (0.5% (w/v)), sodium alginate (1.0% (w/v)) and methylcellulose (1.0% (w/v))/sodium citrate (0.2 M) solutions containing HP-β-CyD (40% (w/v)) were prepared for the tests. The concentration of viscous agents was set to a concentration that shows viscosity for easy administration and that allows complete dissolution. Dissolution test of HP-β-CyD (40% (w/v)) from the viscous solutions was performed by the Basket method at 32 °C and 50 rpm. To evaluate cholesterol-efflux effects of HP-β-CyD, ARPE-19 cells were incubated for 1, 6, and 12 h with HP-β-CyD (40% (w/v)) viscous solutions. Then, cholesterol level of the supernatant was determined by cholesterol E-Test Wako®. To evaluate cytotoxicity of the viscous solutions, the WST-8 method was conducted. To evaluate intracellular free-cholesterol-reducing effect of released HP-β-CyD, dissolution tests were initially performed using water at 32 °C and 50 rpm. Then, the test solution was collected, and a new solvent was added at 1, 6, and 24 h. The HP-β-CyD in the resulting solution was quantitated using a polarimeter. Moreover, free-cholesterol-reducing effect of released HP-β-CyD in NPC1-null Chinese hamster ovary (CHO) cells was measured by Filipin III assay using a confocal laser microscope. Detailed experimental procedures are described in Supplementary Materials.
The release of HP-β-CyD from each viscous solution was evaluated using the rotating basket method. Initially, three viscous solutions containing HP-β-CyD (40% (w/v)) were prepared using xanthan gum (0.5% (w/v)), sodium alginate (1.0% (w/v)), and methylcellulose (1.0% (w/v))/sodium citrate (0.2 M). The amount of HP-β-CyD released from the viscous solutions was determined using a polarimeter. Approximately 100% of the HP-β-CyD was released from methylcellulose/sodium citrate within 1 h, similar to the HP-β-CyD (40% (w/v)) aqueous solution (Fig. 1). This was possibly because the highly concentrated HP-β-CyD suppressed the temperature-responsive gelation of methylcellulose. However, sodium alginate (1.0% (w/v)) containing HP-β-CyD (40% (w/v)) showed a sustained HP-β-CyD release profile for up to 6 h. Moreover, xanthan gum (0.5% (w/v)) containing HP-β-CyD (40% (w/v)) released HP-β-CyD continuously for up to 30 h. These results suggest that xanthan gum is a suitable viscous agent for the sustained release of HP-β-CyD under these experimental conditions.
Each point represents the mean ± standard error (S.E.) of three experiments. * p < 0.05, compared with HP-β-CyD alone (40% (w/v)). †p < 0.05, compared with methyl cellulose (1.0% (w/v))/sodium citrate (0.2 M). ‡p < 0.05, compared with sodium alginate (1.0% (w/v)).
Loftsson and Másson reported that CyD can form hydrogen bond with hydrophilic polymers such as carboxymethylcellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, and it improves drug solubilizing effect of CyD.11) Therefore, the sustained release profile of HP-β-CyD from xanthan gum is probably due to the entanglement of HP-β-CyD and xanthan gum through hydrogen bond formation and/or the high water-retention effect of xanthan gum.12)
Viscosity of HP-β-CyD/Xanthan GumThe drug solution must exit the container smoothly when administering eye drops. Therefore, the viscosity of the solution is an important factor in the development of eye drop formulations. Xanthan gum exhibits pseudoplastic behavior that facilitates a change in viscosity when an external force is applied. Therefore, the effect of HP-β-CyD on the viscosity of xanthan gum was investigated using a rotational viscometer.
As shown in Supplementary Fig. S1, xanthan gum (0.5% (w/v)) alone exhibited a viscosity of approximately 1.8 Pa·s at a shear rate of 1 rpm. The viscosity decreased with increasing shear rate, indicating a pseudoplastic flow pattern. In contrast, the addition of HP-β-CyD slightly increased the viscosity, probably due to viscosity of HP-β-CyD itself. However, as in the case of xanthan gum alone, the viscosity decreased with increasing shear rate. These results suggest that addition of HP-β-CyD preserved the pseudoplastic flow.
Cholesterol-Efflux Effects of HP-β-CyD/Xanthan GumHP-β-CyD exerts therapeutic effects through cholesterol efflux from iPSC-RPE cells derived from patients with BCD.6) Therefore, we investigated whether HP-β-CyD (40% (w/v))/xanthan gum (0.5% (w/v)) has a cholesterol-efflux effect on the cell plasma membrane. Human retinal pigment epithelial cells (ARPE-19 cells) were seeded in the lower layer of transwells and treated with HP-β-CyD solution or HP-β-CyD/xanthan gum for 1, 6, and 12 h. The culture supernatants were collected, and the cholesterol levels in the supernatant were determined using the cholesterol oxidase and DAOS method (Cholesterol E Test Wako®). Cell viability was measured using a WST-8 assay.
The amount of cholesterol that leaked at 12 h was comparable between HP-β-CyD alone and HP-β-CyD/xanthan gum, although the cholesterol efflux effects of HP-β-CyD/xanthan gum decreased at 3 and 6 h (Fig. 2). These results suggest that HP-β-CyD/xanthan gum exert cholesterol efflux effects. In contrast, HP-β-CyD/xanthan gum increased the viability of ARPE-19 cells compared to HP-β-CyD alone. This demonstrates its low cytotoxicity, which can be attributed to the sustained release profile of HP-β-CyD from xanthan gum. Nevertheless, it has been reported that HP-β-CyD causes irritation of the eye membrane by instillation at >12.5%.13) As future effort, we should investigate the in vivo corneal toxicity after the instillation of HP-β-CyD/xanthan gum.
ARPE-19 cells were treated with DMEM/F12 containing xanthan gum (0.5% (w/v)) with HP-β-CyD (40% (w/v)) for 1, 6, and 12 h at 37 °C. Cholesterol concentration in the supernatant was determined using the cholesterol E-test (Wako). Cytotoxicity was determined using the WST-8 assay. Each point represents the mean ± S.E. of three experiments. * p < 0.05, compared with HP-β-CyD (40% (w/v)).
HP-β-CyD reduced the accumulation of free cholesterol in the liver of NPC1−/− mice.14) Herein, free cholesterol also accumulates in the lysosomes of patients with BCD. This induces lysosomal dysfunction, resulting in retinal cell death and tissue atrophy.6) Thus, we investigated whether HP-β-CyD released from xanthan gum reduces free cholesterol. First, HP-β-CyD (40% (w/v)) aqueous solution or HP-β-CyD/xanthan gum was placed in a rotating basket, and dissolution tests were performed using distilled water. After 1 h, distilled water was collected and the dissolution test was performed again using new distilled water. This procedure was repeated after 6 and 24 h, and distilled water was collected. The collected solution was then added to NPC-like Chinese hamster ovary-derived cells (NPC1-null CHO cells),15) and the cholesterol levels were observed through Filipin staining at 24 h.
Figure 3 shows the amount of HP-β-CyD in the test solutions collected at each time point. The HP-β-CyD aqueous solution released almost 100% of the HP-β-CyD within 1 h, whereas HP-β-CyD/xanthan gum continuously released HP-β-CyD for up to 24 h. Figure 4 shows the cholesterol levels upon treatment of the NPC1-null CHO cells with each test solution. HP-β-CyD alone reduced the Filipin III-derived fluorescence when the test solution collected at 0–1 h was added to the NPC1-null CHO cells, indicating reduced free cholesterol levels. However, no decrease in the fluorescence intensity was observed in the test solution collected at 1–6 h. This suggests that HP-β-CyD alone does not maintain the free cholesterol-reducing effect because it is immediately released. In contrast, HP-β-CyD/xanthan gum decreased the Filipin III-derived fluorescence intensity in test solutions collected at 0–1 and 1–6 h, suggesting that the free cholesterol-reducing effect was sustained. Therefore, HP-β-CyD/xanthan gum caused a sustained release of HP-β-CyD and enabled sustained free cholesterol reduction.
Each value represents the mean ± S.E. of three experiments.
NPC1-null CHO cells were treated with DMEM/F12 containing HP-β-CyD obtained in the HP-β-CyD release test for 24 h at 37 °C. (A) The cells were washed with PBS and stained with Filipin III for cholesterol. The figure shows a representative image of six experiments. (B) Fluorescence intensity of Filipin III in NPC1-null CHO cells. * p < 0.05, compared to the control. †p < 0.05, compared with 1–6 h HP-β-CyD.
Currently, we examine the reach of HP-β-CyD to the retina and therapeutic effects of this formulation against BCD in vitro and in vivo. Since there are many barriers before HP-β-CyD reaches the retina and the interaction between HP-β-CyD and xanthan gum is considered relatively weak, it is presumed that HP-β-CyD dissociates from xanthan gum before reaching the retina. Hereafter, the release site of HP-β-CyD in vivo should also be examined.
In this study, we developed an ophthalmic formulation capable of sustaining the release of HP-β-CyD using viscous agents. Xanthan gum containing HP-β-CyD showed a higher sustained release profile than other viscous agents. Moreover, HP-β-CyD/xanthan gum showed prolonged free cholesterol reduction compared to HP-β-CyD alone, owing to the sustained release of HP-β-CyD. In the future, more detailed formulation design of HP-β-CyD/xanthan gum eye drops is needed by adjusting pH and osmolality. Aseptic methods also need to be considered. Anyhow, our findings could facilitate the development of HP-β-CyD-containing eye drop formulations.
We would like to thank Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan) for providing HP-β-CyD. We appreciate Prof. Dr. Katsumi Higaki for kind donation of NPC1-null CHO cells. This research was supported by AMED under Grant Number: JP23ek0109504.
The authors declare no conflict of interest.
This article contains supplementary materials.
