Antioxidant Studies: Retinal Disease in Patients with the Smith-Lemli-Opitz Syndrome (SLOS) Ellen Roy Elias, MD, FAAP. FACMG Professor, Pediatrics and Genetics Children’s Hospital Colorado

Retinal studies in SLOS Historical reason for interest: While still in Boston, one of my patients with severe SLOS went blind as a teenager and developed severe pigmentary retinopathy, loss of night vision, and markedly abnormal ERG Additional Boston SLOS patients were tested and most found to have abnormal rod cell function Elias ER et al, Arch Ophthalmol 2003, 121(12):1738-1743

Animal Model of the SLOS Retina In Vitro Studies done by Steve Fleisler, in the rat SLO model SLO model: rats treated with AY9944, which causes 7-DHC accumulation AY9944-treated rats exposed to light showed massive loss of photoreceptor cells, and dysfunction of rods and cones on ERG studies (similar findings to that seen in SLO humans). The abnormal retina showed an accumulation of oxysterols, toxic compounds made by the oxidation of 7-DHC The damage caused by light was preventable by pretreatment with the antioxidant, DMTU

Intravitreal Oxysterol Injection Produces Panretinal Degeneration WARNING!! DON’T TRY THIS AT HOME !! Xu L, Liu W, Porter NA, Sheflin LG, Young JE, & Fliesler SJ (ARVO 2011)

The SLOS Protocol at Children’s Hospital Colorado History of Colorado Protocol: Boston protocol brought to CO in 2001, using Cholesterol suspension in Soy oil 2008 introduction of vitamin/antioxidant preparation called AquADEKS in addition to cholesterol. AquADEKS is an antioxidant medicine which has been proven safe to use in pediatric patients. Patients followed by serial Ophthalmology exams/electroretinograms (ERG’s)

SLOS Protocol at Childrens Hospital Colorado The protocol is approved by COMIRB, protocol # 01-410 The protocol was partially supported by the CTRC, as well as a private philanthropic support Up to 100 patients can be enrolled (currently 29) Cholesterol is compounded and shipped by the Ward Rd Pharmacy, and billed to the patient’s insurance The AquADEKS has been provided by the pharmacy at Children’s Hospital Colorado Procedures including ERG/ Eye eval and some labs billed to insurance

SLOS Treatment Protocol Cholesterol Suspension 200 mg/ml (formerly on IND # 101,562) made from pharmaceutical grade Cholesterol in Soy Oil, used at doses ranging from 100-250 mg/kg/day divided three times a day Antioxidant Medication (AQUADEKS) added since 2008. It comes as both a liquid, a chewable tablet and a capsule – dosage based on age and size of patient

SLOS Treatment Protocol AquADEKS is a preparation containing vitamins and minerals with antioxidant properties, chosen as it has been proven safe to use in children. The following substances are found in AquADEKS: Vitamins A, C, E ( also vitamins B, D and K) b-carotene Coenzyme Q10 Zinc Selenium

Antioxidant Treatment in SLOS Patients Effects on the retina in SLOS: Earlier studies had shown two retinal issues: Pigmentary retinopathy Abnormal retinal function on ERG consisting of reduced amplitude of the wave, and an increased latency time; ie. it took a greater amount of light energy a longer period of time to produce a retinal response

The Retina and ERG waves

Results Mild patients: (sterol ratio < 3.5) N=7 patients with at least 1 ERG ERG results: Mild patients showed increased implicit times in rods and cones Rods were affected more than cones: rods showed mild/moderate decrease in amplitude cones showed normal to mild changes. 3 patients with serial studies, all showed improved amplitudes and decreased implicit times. EUA studies: 2 patients showed mild pigmentary retinopathy which improved on follow-up testing No progression of RPE seen in any patient No optic nerve abnormalities seen (1 patient died of unrelated issues, 2 lost to follow-up, 1 recently enrolled)

Results Moderate patients N=10 (Sterol ratio 3.6-17) with at least 1 ERG ERG results All 10 moderate patients showed increased implicit times in rods and cones Amplitudes showed rods affected more than cones rods showed moderate decrease in amplitude cones showed mild to moderate changes. In the 6 patients with serial studies, 4 patients improved and 2 were stable (4 patients more recently enrolled and awaiting follow-up studies) EUA studies: 3 patients showed mild pigmentary retinopathy which improved on follow-up testing No progression of RPE seen in any patient optic nerve hypoplasia seen in 2 patients, and 1 of these also had tortuous vessels

Results Severe patients N=7 (Sterol ratio >17), 6 with at least 1 ERG; (2 patients died of severe disease,1 lost to follow-up, and 1 too medically unstable to undergo procedures under anesthesia) ERG results All 6 severe patients showed increased implicit times in both rods and cones Amplitudes showed rods affected more than cones rods showed moderate/severe decrease in amplitude Cone amplitudes were normal! In the 3 patients with serial studies, 2 patient improved and 1 was stable EUA studies: 3 patients showed stable pigmentary retinopathy No progression of RPE seen in any patient, but also no improvement 1 patient showed marked retinal hypopigmentation

Summary – ERG findings at baseline Patients with Smith-Lemli-Opitz syndrome demonstrate abnormalities of retinal function detected on Electroretinogram (ERG) The abnormalities demonstrated include: Prolonged implicit times in all patients, regardless of severity of disease Decreased wave amplitudes in Rod cells were seen in all patients, with a greater change found as disease severity worsened. Rod cell amplitudes were affected more than cone cells in mild and moderately affected patients Cone cell amplitudes were NORMAL in severely affected patients – unclear why!!

Summary ERG findings on Antioxidant Treatment Serial studies were done on patients treated with both Cholesterol and AquADEKS ERG findings showed: Improvement in amplitude of both rods and cones in all 3 mild patients Improvement in implicit times in all 3 mild patients Improvement in rods and cones in 4/6 moderate patients Improvement in implicit times in 4/6 moderate patients 2/6 moderate patients showed stable results Improvement in rod amplitude in 2/3 severe patients Improvement in implicit time in 2/3 severe patients Cone function remained normal 1 patient showed stable results NO patient showed worsening of retinal function while on treatment

Summary of Ophthalmological Exam under Anesthesia (EUA) findings Pigmentary Retinopathy worsened over time in patients treated with cholesterol alone (historical finding) Pigmentary Retinopathy was only seen in a subset of patients 2/7 mild patients 3/10 moderate patients 3/7 severe patients Pigmentary Retinopathy was present in the periphery and when subtle, required a good exam under anesthesia to document it. On Antioxidant treatment, Pigmentary Retinopathy improved or remained stable 2/7 mild patients with RP – both improved over time 3/10 moderate patients – both improved over time 3/7 severe patients – 2 improved and 1 stable No Patient had worsening Pigmentary Retinopathy while receiving both Cholesterol and Antioxidants

Retinal SLOS Study Conclusions Treatment with the antioxidant preparation AquADEKS plus cholesterol appears to lead to an improvement in retinal function in most patients, as seen by an increase in amplitude and a decrease in latency time on ERG testing. The improvement with AquADEKS is more for rods than for cones Historically, SLOS patients treated with cholesterol alone showed continued worsening of retinal function over time – however it is not known if cholesterol slows this rate, as there is no data available regarding retinal function in SLOS patients prior to the use of cholesterol.

Future plans A collaboration is in place to evaluate oxysterols in plasma of SLOS patients, by Dr Libin Xu in Seattle. Dr Porter’s lab at Vanderbilt is also analyzing the components of AquADEKS to help determine which vitamins are most effective, and doing studies in the parents to develop an easier carrier test.

For the Future This is a small study and it will be important to treat more SLOS patients and follow them for a longer period of time – my goal is to participate in a multi-institutional study to evaluate antioxidant treatment, as well as other possible medications such as bile acids in patients with SLOS Following oxysterol levels may prove to be helpful and can be done more often and easily than ERG’s There may be other benefits of antioxidant treatment, particularly regarding hearing, photosensitivity, ability to fight infections, etc which will require future study.

SLOS Retinal Studies This work has been supported by the CTRC at the Denver Children’s Hospital. MO1-RR00069, General Clinical Research Centers Program, NCRR, NIH I would like to thank my patients, Dr Rebecca Braverman, the Children’s Hospital Colorado Foundation, Ward Rd Pharmacy, and the wonderful CTRC staff at Children’s Hospital Colorado