CASE REPORT OF CHAPERONE THERAPY FOR ADULT TAY-SACHS DISEASE AND COMPARISON TO AN IN-SILICO PHARMACOKINETIC AND CELLULAR SIMULATION John G. (Jack) Keimel and Lawrence Charnas MD PhD, University of Minnesota, Minneapolis, Minnesota ABSTRACT CELLULAR MODELS CELLULAR SIMULATION RESULTS CASE STUDY OF PYRIMETHAMINE IN AN ATSD PATIENT Background: Individuals with Adult Tay-Sachs Disease (ATSD) Model 2: Model 3: Model Calibration: Patient History: Drug Regimen: develop ataxia and dysarthria by early teenage years and later lose Endoplasmic Reticulum Lysosome A single constant calibration factor, “cal”, sets the translation rate of α, A male confirmed with ATSD (alpha mutations: +1IVS12 (G>C) and At the request of the patient and parents, pyrimethamine ability to walk. No therapy has yet been shown clinically effective. α , and β HexA monomers. The factor was chosen such that, for Dimer Formation GM2 GM2 Degradation m G269S ) had been taking a substrate reduction therapy, miglustat, (PYR) 25mg QD was begun for two weeks followed by 75mg ATSD is caused by inadequate β-hexosaminidase-A (HexA) activity normal individuals with no PYR present, the steady state lysosomal for greater than four years, but at age 25 showed accelerated QD for eight weeks. Folic acid (5mg QD) was initiated with the and GM2 ganglioside accumulation in neuronal lysosomes. The concentration of HexA is equal to the above calculated value of 25μM. decline in coordination and leg muscle strength, verified by EMG first dose of PYR to offset partial dihydrofolate reductase most prevalent ATSD mutation, αG269S, is not believed to impact [PYR] [PYR] αβ evaluations. The patient lost the ability to climb stairs and required inhibition caused by PYR. Psychiatric medications, miglustat HexA enzyme function but instead cause post-translation misfolding α Michaelis-Menten Simulation of mutated HexA (αmβ) enzyme the use aids to maintain balance while walking. The patient had a (200mg, tid), and acetylcysteine (10 ml, bid) were continued and reduced enzyme stability. Others have shown in vitro that Saturation Curve 6 V A. 150 mg QD history of periodic psychiatric events requiring hospitalization on the without change. Weekly Hex enzyme assays and monthly MAX αamBβ pyrimethamine (PYR) is a potential pharmacological chaperone (PC) αm αmβ m 5 75 mg QD average of once per year since the age of 18. hematologic tests (CBC) were conducted. The 75 mg QD therapy for improving stability of G269S mutated HexA and / dt [GM2] d was again initiated after being off PYR for eight weeks. ½VMAX increasing its transport to the lysosomes. “Limit of health” ββ 4 25 mg QD Model Description: Computer simulations were done to estimate β CASE STUDY RESULTS PYR pharmacokinetics and GM2 response using three interlinked Km 2Km 3Km 4Km 3 6.25 mg QD Hex A in Leukocytes models: a pharmacokinetics model estimated neuronal PYR [GM2] 50.0 Adult Tay-Sachs Disease Patient (G269S mutation) concentrations; an endoplasmic reticulum model predicted the 2 ) 45.0 relative amount of HexA transported to lysosomes; and a lysosomal 1 44.2 model simulated GM2 degradation and HexA inhibition caused by Monomer Dimer GM2 41.0 Degradation Degradation 40.0 PYR. The simulations also modulated HexA half-life as a function of Degradation 0 0 10 20 30 40 50 60 70 80 90 100 38.5 38.0 accumulated GM2 substrate. Previously published parameter values LYSOSOME Dimer Concentration (uM) TIME (days) Constant Value Units Description 35.0 were used when available. (Ref 5) 33.4 Km 46 μM Michaelis Constant for Hex A ~ GM2 31.2 Case Report: A male confirmed with ATSD and the G269S 30.0 Ki 13 μM Hex A ~ PYR Inhibition Constant Simulation of HexA (αβ) and HexB (ββ) enzyme mutation, had been taking a substrate reduction therapy, miglustat 45 27.1 27.1 6.25 mg QD -1 B. αβaB 25.0 (200mg, tid), for greater than four years, but at age 25 showed kCAT 0.9 hr Hex A ~ GM2 Turnover Number 40 23.9 ββBB accelerated decline in coordination and leg muscle strength, verified k 1.8 μM/hr Influx of GM2 into LYS (see below) 35 21.1 GM2 150 mg QD 20.0 by EMG evaluations. At the request of the patient and parents, PYR 19.3 k f * ln(2)/125 hr-1 αβ degradation rate constant in LYS 30 19.3 25mg QD was begun for two weeks followed by 75mg QD in aB-LYSd 17.2 k f * ln(2)/8 hr-1 α β degradation rate constant in LYS 25 15.0 combination with folic acid (5mg QD) to offset partial PRY amB-LYSd m 13.3 k ln(2)/200 hr-1 ββ degradation rate constant in LYS 20 dihydrofolate reductase inhibition. Weekly leukocyte HexA assays BB-LYSd Hex A Activity- (nmol MUGS / hr/ mg protein 10.0 and monthly hematology tests (CBC) were conducted. [PYR] -- μM Calculated peripheral [PYR] 15 6.1 5.4 10 5.0 Results: No noticeable side-effects were observed during the first ER Model Assumptions: four weeks of treatment. Additional follow-up and comparisons to the 5 25 mg /day 75 mg /day 0 mg /day 75 mg /day • The α, α (ATSD mutation), and β HexA monomers are translated at 0.0 modeled response will be presented. m 0 the same rate for each viable allele. For ATSD example, k = 0 x cal, 0 10 20 30 40 50 60 70 80 90 100 0123456789101112131415161718192021 α LYSOSOME Dimer Concentration (uM) TIME (days) Time (Weeks) kαm= 1 x cal, and kβ= 2 x cal, with “cal” being a calibration constant. Figure 3. Blood samples were analyzed weekly (Thomas Jefferson University) except following the initiation of the study • The α and β monomers are 10x more stable than the α monomers m and for a period after the end of the first dosing period. Based on the pharmacokinetic and cellular models, it was MODEL OVERVIEW and 2x more stable than the α ~PYR complex. Simulation of GM2 Storage m C. 3500 hypothesized that the response to initiation or termination of the PYR would require approximately three weeks. The No animal models of ATSD exist. The use of pyrimethamine (PYR) • The second order rate constants for αβ, αmβ, and ββ dimer formation GM2(storage) 3000 HexA values used in the analysis in Figure 4 are shown with a green (“OFF”) and blue (“ON”) circles. as a potential pharmacological chaperone therapy for ADTS patients are set to the same value. Note that no αα (HexS) dimers are formed. 6.25 mg QD was therefore simulated using MATLAB® software. Simulations Lysosome Model Assumptions: 2500 were based on three computer models. The models used 19 4.500 • HexA enzyme activity level at 10% of normal is sufficient to maintain 2000 25 mg QD differential equations solved with the MATLAB “ode45” function. health and avoid substrate accumulation ≥ 4 x Km. (Ref 3 & 9) 4.000 1500 • At this “limit of health”, lysosome volume is guessed to be 10% of total 75 mg QD 3.500 tissue volume and is held constant in this model. 1000 PHARMACOKINETIC MODEL 3.000 LYSOSOME GM2 GM2 (uM) LYSOSOME 500 • The short half-life of mutated HexA (αmβ) is a major factor in ATSD The PK model is based on the Weidekamm et al study (Ref 8) with 150 mg QD severity. The Hex A (αβ, α β) dimers have a half-life 125 and 8 hrs, 2.500 repeat measurements taken on 14 normal adults (60 to 85 kg) each m 0 respectively (similar to Ref 2 & 1). HexA is modeled as only 0 10 20 30 40 50 60 70 80 90 100 taking a single dose of 25 mg PYR (mean ± SD): TIME (days) 2.000 degrading when in the “free” state (i.e., no PYR or GM2 in active site). • Elimination half life (95.5 ± 30.6hr) “f” is the free to total enzyme concentration ratio: 1.500 • Time to max concentration (4.2 ± 2.7 hr) f = [E] / [ET] = ([ET] - [ES] - [EI]) / [ET] Simulation of GM2-GM2 Activator Complex Enzyme Activity Enzyme Activity D. 600 1.000 where: [E] ~ free enzyme concentration, vs "Off" "On" Relative • Area under curve (19.1 ± 5.6 mg*hr / L) GM2(aq) [ET] ~ total concentration of enzyme, 0.500 • Volume of central compartment (75.9 ± 28.6 L) 500 6.25 mg QD [ES] ~ enzyme-substrate complex concentration, and OFF ON OFF ON OFF ON OFF ON OFF ON 25 mg QD 0.000 [EI] ~ enzyme-inhibitor complex concentration. 400 Beta-galactosidase Beta-galactosidase Hex Total Hex Total Hex A Model 1: Classical Two Compartment PK Model 4MU-Beta-D-galactoside 4MU-Beta-D-galactoside MUG MUG MUGS Model Calculations: 75 mg QD Leucocytes Plasma Plasma Leucocytes Leucocytes 300 Daily PYR • Calculated GM2 entering lysosomes (assumed to be in complex with OFF (ave) 1.000 1.000 1.000 1.000 1.000 GM2 activator protein) is based on GM2 concentration within normal 150 mg QD ON (ave) 0.976 1.512 1.184 1.842 3.193 GI 200 Oral Dose Derived First Order brain tissue at 20 nmol/g wet weight of tissue (Ref 6), half-life of GM2 Figure 4. Enzyme activity are shown comparing the average for blood samples taken after being on PYR at 75 mg/day Rate Constants for normal fibroblasts at 78 hrs (Ref 7), and all degradation occurring in LYSOSOME GM2 (uM) 100 for at lease three weeks (“ON”) to the average of the samples taken when no PYR was taken for at least the previous ka lysosomes: three weeks (“OFF”). The samples used in the analysis for HexA are noted in Figure 3.