Participants
Thirty-six subjects with mild-to-moderate probable AD were enrolled at the Mary S. Easton Center for Alzheimer's Disease Research at UCLA. Inclusion criteria were the presence of dementia according to the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) [13], a diagnosis of probable AD by the National Institute of Neurological and Communicative Disorders and Stroke - Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) criteria [14], age greater than 49 years, Mini-Mental Status Examination (MMSE) scores between 17 and 29, English proficiency, and the availability of a study partner to monitor medication administration and adverse effects. Dosage of acetylcholinesterase inhibitors (AchE-I) and memantine had to be stable for one month prior to enrollment. Exclusion criteria included significant systemic illness or recent history of gastrointestinal bleeding. Exclusionary medications included aspirin at doses greater than 325 mg/day, use of non-steroidal anti-inflammatory drugs more than three times a week, coumadin, heparin, gingko biloba, and antioxidant supplements (for example, coenzyme Q10 and alpha-lipoic acid). Concomitant consumption of vitamin E at doses up to 2,000 i.u./day and vitamin C up to 500 mg/day were allowed. To the extent possible, doses of medications and supplements were stable throughout the course of the trial.
A single block of 36 sequential assignments was randomly created and the UCLA research pharmacy packaged and distributed the investigational product accordingly. All study personnel and subjects were blinded to randomization sequence. An unblinded data safety and monitoring board met by teleconference before, and on three occasions during, the study. Subjects or their legally authorized representatives provided written informed consent prior to the initiation of any study procedures. The protocol was approved by the UCLA Institutional Review Board.
Intervention
Subjects were allocated to receive placebo, 2 gm, or 4 gm of Curcumin C3 Complex® per day in two divided doses for 24 weeks in a 1:1:1 ratio. These doses were chosen based on extrapolation from animal studies and allometric scaling. Curcumin C3 Complex® is an orange-yellow crystalline powder plant extract that was encapsulated and provided (as was the matching placebo) by Sabinsa Corporation (Piscataway, NJ, USA). Curcumin C3 Complex® consists of 95% curcuminoids with 70% to 80% comprised by curcumin, 15% to 25% demethoxycurcumin, and 2.5% to 6.5% bisdemethoxycurcumin. This content was independently verified using HPLC by author DDH. Curcumin C3 Complex® or placebo was administered orally as four 500 mg capsules taken twice daily with a fatty meal. At the 24-week visit, all subjects receiving placebo were randomized to one of the two doses of curcumin that was administered for another 24 weeks. Subjects receiving 2 grams or 4 grams of curcumin in the first 24 weeks continued to 48 weeks on the same dose.
Outcome measures
Primary outcomes included tolerability and cognitive measures. At baseline and each of the post-baseline visits (weeks 4, 12, 24, 36, and 48) subjects and caregivers were interviewed regarding adverse events using a checklist. At screening and at each post-baseline visit, vital signs were taken and laboratory value monitoring performed including complete blood count, chemistry panel, lipid profile, thyroid stimulating hormone and thyroxine levels. Bleeding times before and after treatment were also obtained on 24 subjects. The Alzheimer's Disease Assessment Scale, cognitive sub-portion (ADAS-Cog) [15] was administered at baseline and at the 24 and 48 week visits. The current paper describes the results of efficacy outcome measures at 24 weeks and tolerability measures out to 48 weeks.
Secondary clinical outcome measures included the Neuropsychiatric Inventory (NPI) [16] and the Alzheimer's Disease Cooperative Study Activities of Daily Living (ADCS-ADL) [17] instrument which were administered to caregivers at baseline and at the 24 and 48 week visits. The MMSE was also administered at screening and at all post-baseline visits. Plasma and CSF were collected at the baseline visit for biomarker measurements; this was repeated at week 24. All samples were centrifuged and frozen at -80°C. Commercially available kits were used to measure plasma levels of Aβ1-40 and Aβ1-42 (INNO-BIA Plasma Aβ Forms; Innogenetics, Ghent, Belgium) and CSF levels of Aβ1-42, total tau (t-tau), and tau phosphorylated at threonine 181 (p-tau181, INNO-BIA AlzBio3; Innogenetics, Ghent, Belgium) using the Luminex xMAP platform (Luminex, Austin TX, USA). CSF isoprostanes (F2-IsoPs) were quantified using a stable isotope dilution assay with gas chromatography/mass spectrometry and selective ion monitoring as described previously [18]. All plasma and CSF assays were done in a single batch at the end of the 24-week study period.
Pharmacokinetic analysis
Pharmacokinetic analyses were performed in two different manners in two different laboratories.
First, plasma levels of native curcumin at baseline and at 0.5, 1, 2, 3, and 4 hours post-dose at the 24 week visit were measured using HPLC as described previously [19]. The coefficient of variation (CV) for a 20 ng/mL sample was 4.95%. The lower limit of detection using HPLC was 200 ng/mL.
Second, using liquid chromatography/tandem mass spectrometry (LC/MS/MS) plasma (baseline levels and 3 hour post-dose at the 24 week visit) and CSF levels (prior to the first dose and after the dose at the 24 week visit) of native curcumin were measured. Curcumin was extracted from plasma and CSF in 95% ethyl acetate/5% methanol and analyzed by LC/MS/MS, using tetramethoxycurcumin as an internal standard as previously described [6]. The LC/MS/MS system (Varian International, Lake Forest, CA, USA) consisted of the proStar 410 autosampler and pumps and a 310 mass spectrometer with ESI (-) which is able to optimize each ion automatically. Dried plasma/CSF extract samples were re-dissolved in acetonitrile:water (50/50, by volume) in 10 mM ammonium acetate, and an aliquot of the solution (typically 50 ul) was injected onto a reverse phase HPLC column (Atlantis T3, 150 × 2.1 mm; Waters Corporation, Milford, MA, USA). The m/z values of the specific transitions were for curcumin, 367.0, 148.7, 172.4, 216.3, tetrahydrocurcumin, 372.0, 193.5, 236.0 and for tetramethoxycurcumin, 396.4, 149.5, 297.1, 337.6 (the internal standard). Instrument manufacturer-supplied LC/MS/MS Varian software (Varian MS Workstation, Version 6.9.1) was used to calculate each ion according to its qualifier ion. The limit of detection for curcumin and tetrahydrocurcumin in the plasma sample was 1 and 3 ng/mL, respectively. To measure glucuronides, 10 μL of 0.4 M PBS with 2% ascorbic acid and 0.1% ethylenediaminetetraacetic acid (EDTA) (pH 3.6) were added to 0.5 ml plasma along with 30 μL of 0.1 M PBS (pH 6.8). Then, 500 Fishman units of β-glucuronidase (Sigma, St Louis, MO, USA) were added to the sample, which was incubated at 37°C for 45 minutes at pH 5.0, followed by analysis of curcumin and tetrahydrocurcumin using LC/MS/MS.
Statistical analysis
The pre-specified data analysis plan for efficacy measures was repeated measures analyses of variance (ANOVAs) with drug dose as the independent variable and cognitive, behavioral, and biomarker measures as the dependent variables over time. Occurrence of adverse events, changes in vital signs, and safety laboratory results per subject according to the treatment they received over the course of the study were compared between treatment groups by the general linear mixed model.
Analyses of efficacy measures were performed on data from completers due to lack of follow-up biomarker data for subjects who discontinued. Analyses of safety and tolerability measures were performed on all available data from all available subjects. The funding organizations had no role in study design or outcome analysis.