**5. Functional improvement of Bruch's membrane**

Saponins have been shown to disperse and release lipid deposits, protein aggregates and trapped active MMP enzymes as described earlier. This is akin to reversing the ageing process of Bruch's membrane. The likely impact of these changes on the functional properties of Bruch's has also been assessed by monitoring hydraulic conductivity and diffusional status.

#### **5.1 Hydraulic conductivity of saponin treated human Bruch's**

Hydraulic conductivity measurements were undertaken in 23 preparations of Bruch's membrane from donors over the age range 13-90 years. After determining basal hydraulic conductivity, samples were incubated with saponins (4.6 mg/ml) for 24 hours at 37o C. After a thorough rinse in Tris buffer, the hydraulic conductivity was re-assessed. Treatment with saponins improved the hydraulic conductivity about 2-fold (p < 0.0001) elevating the exponential decay curves upwards, away from the failure threshold (**Figure 9**).

The curves were shifted by 19 years, reversing the ageing decline in fluid transport. Such a shift means that the curves will meet the failure threshold outside the normal human life span. In AMD, this improvement will reduce the risk of pigment epithelial detachments that normally affects 12-20% of these patients. These results were obtained after a single exposure to the saponins. A second subsequent exposure to the saponins (1.15 mg/ml) further increased the improvement in hydraulic conductivity indicative of potential to further elevate the decay curves (**Figure 10**). This is the likely result of continued removal of lipid and protein debris and release of MMP enzymes.

*Saponin-Mediated Rejuvenation of Bruch's Membrane: A New Strategy for Intervention… DOI: http://dx.doi.org/10.5772/intechopen.96818*

#### **Figure 9.**

*Effect of saponins on the hydraulic conductivity of human Bruch's membrane. Saponins improved the hydraulic conductivity by 2-fold across the age range examined (p<0.0001). Decay curves were elevated so that failure threshold was met 19 years later. Modified from reference [43].*

#### **Figure 10.**

*Effect of repeated saponin exposure on the hydraulic conductivity of Bruch's membrane. Initial exposure to saponins improved hydraulic conductivity by over 2-fold. This was further increased by a second exposure.*

#### **5.2 Diffusional status of saponin treated human Bruch's**

Diffusional status of Bruch's was assessed by following the transport of an FITClabelled albumin test molecule (MW 65 kDa, hydrodynamic radius 3.5 nm) across the membrane, at a concentration gradient of 0.1 mM. Bruch's was obtained from 21 human donors (age range 12-92 years) and the basal diffusion rate was first determined. After a thorough wash in Tris buffer for 3 hours to remove all traces of albumin, the samples were incubated with saponin solution (4.6 mg/ml) for 24 hours at 37o C. Following a rinse with Tris buffer, the diffusional status was re-assessed. Exposure to saponins improved the diffusional status of Bruch's membrane by 2-fold (p<0.0001) shifting the ageing decay lines upwards, effectively reversing the ageing process by ~15 years (**Figure 11**).

#### **Figure 11.**

*Effect of saponins on the diffusional status of Bruch's membrane. Saponin incubation improved diffusion (p<0.0001) elevating the decay curves upwards so that they reached failure thresholds 15 years later. Modified from reference [43].*

Saponin mediated improvement in diffusion within Bruch's membrane would allow greater delivery of nutrients, vitamins and anti-oxidants to both the RPE and photoreceptor cells. Reduction in oxidative stress would result in reduced production of A2E and other bis-retinoids minimising the generation of pro-inflammatory mediators. Similarly, the removal of toxic waste products from Bruch's would be facilitated. This would also improve the mobility of TIMP2 and proMMP2 allowing greater activation of the MMP species and thus improved turnover of Bruch's membrane. Furthermore, improved mobility of CFH would minimise the risk of inflammatory involvement. In AMD, these changes are expected to slow the degenerative phase of the disease.

### **6. Conclusions: potential for saponin-mediated therapy in dry AMD**

In-vitro work, using donor human Bruch's preparations has demonstrated the potential for saponin molecules to disperse and remove lipoidal and proteinaceous debris, releasing trapped activated MMP enzymes that can then mediate the normal degradation processes essential for rejuvenation of the membrane. Associated with these changes was a significant improvement in the bi-directional transport properties of Bruch's membrane. In vivo, improved transport would considerably augment the delivery of protective anti-oxidants and related nutrients to the RPE and photoreceptor cells and more importantly stimulate the removal of toxic products that underlie the progression of the disease.

Saponins constitute a large mixture of amphipathic molecules, the diversity being due to the varied nature of sugar residues at the several attachment sites on the aglycone ring structure. Individual species, such as ginsenoside Rb1 or Compound K have been shown to improve the functional status of Bruch's membrane preparations [43]. These compounds could be administered by intra-ocular injection as a potential therapy. But being very difficult to synthesise, reliance would have to be on isolation and purification from natural sources and therefore not likely to be cost-effective.

The alternative would be to utilise a varied mixture of saponins and this has many advantages. Firstly, they can be administered orally and in the Far East have been used as nutritional supplements for centuries. Secondly, and most

#### *Saponin-Mediated Rejuvenation of Bruch's Membrane: A New Strategy for Intervention… DOI: http://dx.doi.org/10.5772/intechopen.96818*

importantly, a mixture can target a broad spectrum of substrates that are normally encountered in the deposits in Bruch's membrane. From the pharmacokinetic data currently available for saponins, and our in-vitro dose response curves, calculations suggest that a 200 mg dose of an appropriate saponin mixture, taken twice daily, should significantly improve the transport characteristics of Bruch's membrane over a period of 4-6 months. Work is in progress to develop a clinical protocol for assessing the usefulness of saponin-mediated therapy for dry AMD.

Unlike other therapeutic interventions in AMD where outcome has been judged by following the progression of the disease over a period of 2-5 years, the saponin intervention can be assessed at 4-6 months using dark-adaptation kinetics. Since the saponin intervention aims at improving the transport systems in Bruch's membrane, the increased delivery of vitamin A would supplement the retinoid stores in the RPE allowing faster delivery of 11-cis retinal to the photoreceptor, and hence faster dark adaptation would be expected as proof of principle of saponin intervention.
