EAE and LA dose effect experiments
The study received approval from the Portland Veterans Affairs Medical Center Institutional Animal Care and Use Committee prior to initiation. These experiments were conducted as described in Marracci et al. [21
]. Pure synthetic LA (without filler) was obtained from Tyler Laboratories, (Gresham, OR). Briefly, female SJL mice were immunized by SC injection with an emulsion containing proteolipid protein (PLP) 139–151 peptide and complete Freund’s adjuvant (CFA) containing 150 μg of peptide and 200 μg of Mycobacterium tuberculosis
in a total volume of 0.2 ml. With this method of immunization, 100% of control mice developed clinical signs of acute EAE 11–14 days after immunization and most mice have one or more spontaneous relapses of EAE after recovering from the first episode. Seven days after immunization and prior to disease onset, mice were randomized to daily SC injections of saline or racemic LA in varying doses (5, 10, 20, 50 and 100 mg/kg/day). Mice were scored for clinical EAE blinded to treatment status. The experiment was conducted as a suppression paradigm, in which mice were randomly assigned to receive 5–100 mg/kg LA or saline each day commencing on day 7 post immunization and prior to disease onset. Daily monitoring and injections continued for 45 days post immunization. The 10-Day Cumulative Disease Score (10-Day CDS) was calculated for each animal by adding the daily disease score on 10 consecutive days commencing on the first day of signs of clinical EAE in the mice receiving saline. The percentage suppression of disease for each dose of LA was calculated using the following formula:
Dosing and PK Sample Collection in Mice
The PK studies were performed in mice using three different doses of SC racemic LA obtained from Sigma-Aldrich. The three doses of SC LA were 100mg/kg, 50mg/kg and 20mg/kg. The PK for an individual dose was performed on a single day. We used one set of naïve and saline control mice, and collected blood at 30 minutes post injection of saline. Blood was collected by cardiac puncture at 1min, 5min, 15min, 30min, 1hr, 2hr and 5hr with three mice at each time point. Serum was recovered and stored at −80C until assay was performed.
The study received approval from the Oregon Health & Science University (OHSU) Institutional Review Board prior to initiation. All subjects gave informed consent before entering the study. To qualify, subjects needed to meet the following inclusion/exclusion criteria: Definite MS by McDonald’s criteria [30
]; EDSS ≤ 7.5 [31
]; age 18–70, inclusive; no clinically significant MS exacerbation within 30 days of the screening; no systemically administered corticosteroids within 30 days of study entry; patient not pregnant or breast-feeding; no LA in previous 2 weeks; patient not on anti-coagulants or aspirin during the study; no significant health problems (e.g. active coronary heart disease, liver disease, pulmonary disease, diabetes mellitus) that could increase risk of patient experiencing adverse events; subject unable to give informed consent. Patients were allowed to receive symptomatic medications while on study. Subjects taking recombinant interferon (IFN)-b or glatiramer acetate were allowed to continue taking these medications.
Lipoic acid in human studies
Viatris® (now called Meda Pharma®) provided a tablet formulation containing 600 mg racemic LA (lot # 4E002-1, expiration date 05/07), referred to as Formulation A. Vital Nutrients® (Middletown, CT, USA) provided gelatin capsules containing 300 mg racemic LA (lot # 5G24 expiration 08/07). Each capsule also contained cellulose powder and small amounts of ascorbyl palmitate and silica. This product is referred to as Formulation B. Pure Encapsulations® (Sudbury, MA, USA) provided vegetable capsules containing 600 mg racemic LA (Lot # 3480504 expiration 11/06) referred to as Formulation C. Each 600 mg LA capsule contained 30 mg of ascorbyl palmitate and pine cellulose plant fiber to add to volume.
Human Study Design
This was a randomized open label trial comparing 1200 mg of three different formulations of oral LA in subjects with MS. Subjects were randomized to one of three treatment arms using 1:1 allocation with a computer-generated, blocked randomization scheme. Subjects came to the Oregon Clinical and Translational Research Institute (OCTRI) clinics at OHSU for all visits. Visit 1 consisted of a screening baseline blood draw, and neurological and physical examinations. Upon enrollment, Visit 2 occurred one week later, at which time the first dose of the study drug was administered and blood samples were collected for the pharmacokinetic study.
LA Dosing and PK Sample Collection in MS subjects
The PK studies were performed in MS subjects using a single oral 1200 mg dose of LA, using the randomly assigned formulations, Formulation A, Formulation B or Formulation C. On the day of PK study, patients had to fast from food and beverages (except for water) overnight for at least 10 hours. Patients received a standardized breakfast in the clinic. Immediately following completion of the meal, each patient received LA 1200 mg orally with 240 mls of water. Blood samples for measurement of LA concentrations in serum were obtained by venipuncture at baseline, and at 5, 10, 15, 30, 60, 90, 120, 180, 240, and 300 minutes after the dose. Blood samples were collected into untreated Vacutainer collection tubes. Serum was immediately separated from the clot by centrifugation, aliquoted (0.5 mL) and stored at −80° C until analyzed.
Lipoic acid analysis
LA and the internal standard, 2-(6-methoxynaphthalen-2-yl) propanoic acid (naprosyn), were obtained from Sigma-Aldrich (St. Louis, MO). HPLC solvents were obtained from Burdick and Jackson (Muskegon, MI) and other solvents and chemicals were from Sigma-Aldrich and were analytical grade. Strata impact protein precipitation 2 ml square filter plates, Strata 96-well collection plates, pierceable sealing mats, and 96-well plate manifold were from Phenomenex (Torrance, CA).
LA levels were determined by liquid chromatrography tandem mass spectrometry (LC/MS/MS) using an adaptation of the method of Chen et al. [32
] in a 96 well plate format. Similar results were obtained using individual micro-centrifuge tubes for sample preparation. Plasma samples were thawed and a 50 μl aliquot was added to 150 μl of acetonitrile that contained 0.33 ng/μl of the internal standard, naprosyn. The samples were allowed to stand for 5 min and then vacuum filtered into the collection plate. A 5 μl sample of the acetonitrile extract was used for analysis. If individual micro-centrifuge tubes were used, the samples were vortexed and the protein precipitate removed as described below for saliva samples. For all patients a baseline plasma sample (i.e. time = 0) was prepared in the absence of naprosyn and spiked with 1000 ng/ml of LA. If the samples contained naprosyn, the amount of LA was calculated from the area of LA alone corrected for the recovery from the spiked sample.
Samples were analyzed using a Thermo TSQ Quantum Discovery triple-quadrupole mass spectrometer (San Jose, CA) equipped with an electrospray ionization source. All mass analyzers were operated at unit mass resolution. The ionization interface was operated in the negative mode using the following settings: spray voltage, 2,000 V; sheath gas flow rate, 25 ml/min; tube lens offset, 35 V; and capillary temperature, 275 °C. LA and naprosyn were monitored by selective reaction monitoring (SRM) with a collision energy of 8 monitoring the transitions of m/z 205.0→171.00 for LA and m/z 229.0→185.0 for naprosyn.
The LC-MS system was composed of an in-line Acella auto-sampler and HPLC pump (ThermoFisher, San Jose, CA). LA and naprosyn were resolved from other plasma components using a 2.1 × 50 mm, 3.5 μm Zorbax SB-C18 and a 5 μm 2.1 × 12.5 mm guard column (Agilent Technology, Wilmington, DE) maintained at 30 °C. The HPLC mobile phase consisted of water with 0.03% acetic acid (solvent A) and methanol with 0.03% acetic acid (solvent B) delivered at a flow rate of 0.3 ml/min. The column was equilibrated with 30% solvent B and then increased to 95% solvent B in 3.0 min, held for 1.5 min and returned to 30% solvent B and equilibrated for 2.4 min. The injection volume was 5 μl. LA and naprosyn eluted at 3.09 and 3.31 min, respectively. A series of standards from 5 to 50000 ng/ml LA were prepared in naïve serum with each sample set. A methanol stock of LA stored at −20 °C was diluted in water to prepare working dilutions on the day of use. Linear least-square regression of the plasma concentrations and measured peak area ratios using a 1/x weighting was used for the quantification. The intra-day coefficient of variation at 5 and 50,000 ng/ml was 8.5% and 3.4%, respectively. The inter-day coefficient of variation was 7%. Data acquisition and quantitative processing were accomplished with Xcalibur software.
Serum LA concentration-time data were analyzed by non-compartmental methods using the WinNonLin software program Version 4.1 (Pharsight Corporation, Mountain View, CA). Maximum serum LA concentration (Cmax) and time to occurrence (Tmax) were the observed values. Area under the serum concentration-time curve from time zero until the last concentration-time point (AUC0-last) was calculated by the linear trapezoidal rule. Terminal area was the quotient of the last concentration divided by the terminal elimination rate constant (λz). This constant was determined from regression analysis of concentration-time points in the terminal elimination phase. AUC from time zero to infinity (AUC0-∞) was the sum of AUC0-last plus terminal area. Terminal elimination half-life (t1/2) was calculated as 0.693/λz, oral clearance (CL/F) was calculated as oral dose/AUC0-∞, and volume of distribution (V) was calculated as CL//λz.
Data were normally distributed and were reported as mean ± S.D. Pearson’s product correlation coefficients were calculated to determine the linear association between patient age, height, weight, and body mass index and LA PK parameters (t1/2, V, CL/F, and AUC0-last). Multiple group comparisons (e.g. Formulation A vs. Formulation B vs. Formulation C) were done using analysis of variance (ANOVA) with Tukey’s post test. Statistical significance was defined as P < 0.05. SPSS for Windows Version 15.0 (SPSS Inc., Chicago, IL) was used for the statistical analyses.