Abstract
Purpose
Recovery rate of rod photoreceptor sensitivity (S2 gradient) following a bleach is reduced in age-related macular degeneration (AMD) due to diminished delivery of retinol across a grossly altered Bruch’s membrane. Since triterpenoid saponins are known to improve transport across Bruch’s, we have assessed their possible use for reversing the visual deficits in AMD.
Design
Double-blind, placebo controlled randomised clinical trial.
Methods
Altogether 11 AMD patients and seven age-matched control subjects were recruited to undertake a small proof-of-principle study. Dark adaptation curves were obtained and S2 gradients evaluated using a Humphrey Field Analyser. Following basal determination of S2 gradients, oral supplementation of saponins (200 mg/day) or placebo regime was instigated for a period of 4 months. S2 gradients were re-evaluated at two and four months.
Results
Basal S2 gradients of the AMD cohort were determined as 0.41 ± 0.24 dB/min and those of the control subjects as 1.44 ± 0.1 dB/min. After two months of the saponin treatment, AMD subjects showed improved S2 gradients of 0.92 ± 0.23 dB/min ( P < 0.005) with a further increase to 1.35 ± 0.19 dB/min at four months ( P < 0.01), the latter not being significantly different from control subjects. S2 gradients in placebo subjects were unaltered.
Conclusions
Oral supplementation with saponins results in reversing the reduced S2 gradients in AMD. This improvement in the transport properties of Bruch’s is expected to slow, halt or reverse the progression of AMD.
1
Introduction
Age-related macular degeneration (AMD) is the single largest cause of untreatable blindness in the elderly population with an estimated 67-million affected individuals in the European Union. Epidemiological studies have shown AMD to be a highly complex, multifactorial disease that has both a genetic disposition and considerable gene-environmental interactions, complicated by the additional association of dietary and cardiovascular risk factors. The highest risk factor for the disease is age. Normal aging of Bruch’s membrane results in structural disorganization and deposition of lipid-rich proteinaceous debris that results in an exponential decline in transport (with levels in the elderly reduced by ten-fold) compromising the inward delivery of nutrients and removal of waste products between the photoreceptor–retinal pigment epithelium (RPE) complex and the choroidal circulation. In the advanced aging scenario of AMD, the diffusional transport across Bruch’s membrane is further reduced progressing, with inflammatory intervention, to the death of RPE and photoreceptor cells.
In AMD, the earliest clinical manifestation of abnormal transport across Bruch’s concerns the delivery of retinol. Clinically, this results in abnormal dark adaptation whereby the recovery rate of rod photoreceptor sensitivity following a bright light flash is slowed. Briefly, dark-adapted subjects are exposed to a bright flash that bleaches a large percentage of the rhodopsin and the subsequent recovery in sensitivity is followed for a period of 40–60 min resulting in the typical biphasic dark-adaptation curve shown in Fig. 1 for young control subjects aged 32.9 ± 10.7 years. The curve shows a rapid recovery of cone sensitivity followed by recovery in rod sensitivity; the intersection of the two systems is called the cone-rod break (CRB). The initial rate of rod recovery in sensitivity is calculated as the “S2 gradient”, determined here for young control subjects as 1.97 ± 0.16 dB/min (equivalent to 0.2 ± 0.02 log cd m −2 min −1 ). In AMD, the rod S2 gradient is reduced and may even be absent over the 40–60 min of observation.
Light capture by rhodopsin in photoreceptor outer segments leads to the isomerization of its 11-cis-retinal chromophore to its all-trans form that, on release from the protein, is rapidly reduced to all-trans retinol and transported to the RPE to enter the vitamin A cycle ( Fig. 2 ). After several enzymatic steps, the retinol ester is converted to 11-cis-retinal esters and stored in the RPE retinoid pool. Following rhodopsin bleaching, 11-cis retinal is released from the RPE retinoid pool for delivery to photoreceptors and the rate of this transfer is related to the S2 gradient.
Abnormalities in the sequence of events described above following a light flash offer plausible mechanisms for reduced S2 gradients. Firstly, efficient carrier-mediated transport of the retinoid species between the photoreceptors and RPE together with enzymatic transformation to produce 11-cis-retinal esters is required for regeneration of rhodopsin. Mutations in the members of the enzymatic and transport machinery of the vitamin A cycle are known to result in slowed S2 gradients but none have yet been reported in AMD. Secondly, the level of retinoids in the RPE pool determines the initial rate of transfer of 11-cis retinal to the photoreceptor and hence the S2 gradient. If the vitamin A cycle was 100 % efficient and all released all-trans retinal (from rhodopsin) was converted to 11-cis retinal, then there would be no change in the retinoid pool in the RPE. However, released all-trans retinal can undergo chemical modifications leading to the generation of a variety of toxic bis-retinoids, compromising the retinoid pool. Loss of retinoids also occurs when damaged outer segment disks cannot be degraded adequately by the RPE. This loss in the retinoid pool must be compensated for by delivery of plasma retinol (as a trimeric complex of retinol, retinol-binding-protein and transthyretin, MW 75 kDa) across Bruch’s membrane. In AMD, the severely compromised transport functions of Bruch’s membrane are thought to restrict the entry of retinol, diminishing the retinoid pool in the RPE, leading to deficits in S2 gradients.
To overcome the diminished retinoid pool in the RPE, megadose retinol supplementation has been considered. In vitamin A deficiency, with a normal Bruch’s membrane, mega-doses of vitamin A can reverse deficits in dark adaptation within a matter of a few days. Transport across Bruch’s in AMD is severely curtailed and short-term high-dose retinol supplementation for a period of 30 days showed only a slight improvement in S2 gradients. Furthermore, mega-doses of retinol are toxic and the intervention cannot be sustained for a long period of time.
Saponins (tri-terpenoid glycosides) are amphipathic molecules that have hydrophilic and hydrophobic domains and can readily partition into lipid structures such as liposomes, micelles, membranes or lipoidal deposits and assist dispersal. These saponins are found in nature as either 4- or 5-membered ring structures with the common sources being ginseng and sea cucumbers ( Fig. 3 A). The incorporation of different chemical groups (sugars, hydroxyl, hydrogen and alkane chains) at the various molecular attachment sites leads to a myriad of compounds, and over 400 have been characterized. Saponins have been shown to remove lipid deposits and denatured matrix proteins from donor human Bruch’s resulting in improved transport functions of the membrane ( Fig. 3 B). Therefore, as a proof-of-principle, we have undertaken a small double-blind placebo-controlled study to assess the potential for saponin-mediated intervention in AMD to improve transport processes across Bruch’s membrane and thereby address the visual deficits associated with reduced S2 gradients.
Measurement of S2 gradients provides information on the rate of delivery of 11-cis retinal from the RPE to the bleached rod photoreceptor cell and this is dependent on adequate transport of retinol across Bruch’s membrane to maintain the retinoid stores for release. Thus, changes in S2 gradients can be unambiguously correlated with transport across Bruch’s membrane.
2
Methods
The CONSORT 2010 guidelines for reporting clinical trials were followed. This double-blind placebo-controlled study was approved by the institutional review board of the Jeonbuk National University Hospital, Korea, permission number CTCF2_2017_AMD and registered in the Clinical Research Information Service (CRiS) Registry (Registration number: KCT0008902). The trial commenced on January 23, 2018 and was completed on December 16, 2020. All participants filled out the informed consent form before participating in the study.
Trial objectives: The primary outcome measures were the change (if any) in the S2 gradients of the dark-adaptation curve of patients on the saponin treatment and the occurrence of adverse events. Secondary outcome measures were changes in macular thickness, best-corrected visual acuity (BCVA) and routine laboratory tests in hematology and clinical chemistry.
2.1
Estimation of saponin dosage and power analysis
In reported animal studies, the bioavailability of saponins was determined as 1.12 ± 1.6 % and the half-life of the measured saponin in plasma as 19.5 h. This, together with the dose-response curves for improvement in transport functions of donor human Bruch’s membrane ( Fig. 3 B), we have calculated that a daily oral dose of 200 mg saponins should achieve a 0.5–1.0-fold improvement in transport across Bruch’s in our study subjects over a period of two to five months . We had assessed the relative efficacies of ginseng (Panax ginseng CA Meyer) and sea cucumber (Apostichopus japonicus) saponins for removing various lipid deposits present in human Bruch’s. This showed that ginseng saponins were more effective at removing hydrophobic constituents (such as cholesterol esters) and sea cucumber for removing hydrophilic deposits (such as phosphatidyl choline). Note that most hydrophobic deposits will be enveloped and stabilized by hydrophilic components. Thus, a combination of the two saponin sources would allow targeting a wider spectrum of deposits in Bruch’s membrane. Since the improvement in transport was much greater with sea cucumber saponins, our 200 mg saponin mixture incorporated 50 mg ginseng saponins and 150 mg sea cucumber saponins.
A pilot assessment was undertaken with six AMD subjects to determine: 1) if the calculated dosage was sufficient to elicit a change in S2 gradients over two months and 2) to enable a power analysis to determine the number of patients that need to be recruited in the present study . The daily 200 mg saponin dosage resulted in a mean S2 difference between basal and two months of 0.49 dB/min in these patients with a standard deviation of 0.22 dB/min giving a large effect size of 2.22. Power analysis was undertaken using G*POWER Statistical Software ( www.gpower.hhu.de ) and at a significance level of 0.05, power at 80 % and effect size of 2.22, the required sample size was determined as four patients. The low number of patients required for statistical significance suggests a marked improvement in the transport properties of Bruch’s leading to efficient delivery of plasma retinol and subsequent elevation of S2 gradients.
Placebo capsules contained 316.8 mg crystalline cellulose, 2.24 mg Gardenia yellow colorant and 0.96 mg red ginseng flavoring.
2.2
Patient recruitment
Patients were recruited from the AMD clinic at JeonBuk National University Hospital, Korea. Inclusion criteria were AMD subjects over 50 years of age and the absence of geographic atrophy or neovascular lesions in the eye undergoing dark-adaptation testing. Exclusion criteria were the presence of other retinal diseases, local or systemic inflammatory diseases, patients taking steroids or immuno-suppressants, uncontrolled diabetes, allergies to seafood or taking commercially available saponin supplements. The planned protocol was designed to recruit 18 AMD subjects so as to allow nine patients per treatment or placebo group. However, recruitment problems meant that only 11 AMD patients were enrolled (R01-R11). To complete the trial, we enrolled an additional seven age-matched control subjects (R12-R18). The inclusion of control subjects allowed us to assess the extent of deficiencies in S2 gradients in the AMD cohort on their basal visit.
All subjects underwent a routine ophthalmic assessment on their first visit that included BCVA, ocular coherence tomography (OCT) and measurement of macular thickness, color fundus photography (used to grade AMD using the Beckman classification ), fundus autofluorescence imaging, electroretinography, followed by dark-adaptation testing. Routine parameters of hematology and clinical chemistry were also obtained. Basal clinical characteristics are given in Table 1 . Subjects were then started on the daily saponin or placebo intervention (1:1) based on a computer-generated randomization sequence for a period of four months ( http://www.randomizer.org ). All clinical staff, trial managers and analysts remained blinded to the treatment/placebo allocation. All of the above tests were repeated after two and four months of the initiation of the study.
| Patient ID | Age (years) | Sex | Eye for DA | DA threshold (dB) | Basal S2 gradient (dB/min) | Central macular thickness (µm) | AMD classification (Beckman) | Notes |
|---|---|---|---|---|---|---|---|---|
| R01 | 58 | F | Right | 16.05 | ND | 257 | Intermediate | Large drusen, hypopigmentation and hyperpigmentation. |
| R02 | 77 | M | Left | 11.8 | 0 | 245 | Late | Left eye showing presence of hyperfluorescent foci in central macula on FAF. Wet AMD in right eye. |
| R03 | 68 | F | Left | 14.79 | 0.4 | 206 | Intermediate | Large drusen and pigmentary changes in both eyes. Epiretinal membrane in right eye. Underwent macular wrinkle surgery on right eye. |
| R04 | 77 | F | Right | 13.2 | 0 | 162 | Intermediate | Presence of large and confluent drusen together with pigmentary changes. Hyperfluorescent focal spots on FAF. |
| R05 | 75 | M | Right | 15.09 | 0.41 | 273 | Intermediate | Large drusen present. Diabetic. |
| R06 | 81 | M | Left | 13.31 | 0.41 | 294 | Intermediate | Hypo pigmentation and presence of large drusen. Hyperfluorescent foci on FAF. |
| R07 | 73 | M | Left | 13.0 | 0.42 | 216 | Intermediate | Large number of hyperfluorescent foci in central macula on FAF. |
| R08 | 62 | F | Right | 10.57 | 0.55 | 286 | Intermediate | Pigmentary changes and large number of hyperfluorescent foci on FAF. |
| R09 | 78 | F | Right | 12.6 | 0.6 | 271 | Intermediate | Large drusen and hyperfluorescent foci on FAF. Diabetic. |
| R10 | 71 | M | Left | 14.24 | 0.66 | 210 | Late | Geographic atrophy in right eye. Diabetic. |
| R11 | 69 | F | Right | 15.18 | 0.65 | 214 | Intermediate | Extensive presence of large drusen outside central macula. |
| R12 | 62 | M | Right | 19.57 | ND | 270 | Control | Normal aging changes. |
| R13 | 62 | M | Right | 15.29 | 1.45 | 284 | Control | Normal aging changes. Diabetic. |
| R14 | 70 | F | Right | 18.2 | ND | 250 | Control | Normal aging changes. |
| R15 | 63 | M | Right | 19.76 | ND | 275 | Control | Normal aging changes. Diabetic, smoker. |
| R16 | 70 | F | Right | 18.6 | 1.34 | 278 | Control | Normal aging changes. Diabetic. |
| R17 | 73 | F | Right | 11.84 | ND | 245 | Control | Normal aging changes. |
| R18 | 68 | F | Right | 16.86 | 1.53 | 241 | Control | Normal aging changes. |
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