The study protocol was reviewed and approved by the following ethics committees: Medizinische Ethik-Kommission II, der Medizinischen Fakultät Mannheim, Mannheim, Germany; Ethik-Kommission der Medizinischen Fakultät und am Universitätsklinikum Tübingen, Germany; and Regionala Etiksprovningsnamnden Stockholm, Stockholm, Sweden. The study was conducted in accordance with Good Clinical Practice and the 2008 Declaration of Helsinki. All patients provided written, informed consent to participate in the study and, separately, to undergo genetic analysis.
Men and nonfertile women ages 50 to 85 years with a clinical diagnosis of probable mild AD, Mini Mental State Examination (MMSE) score of 20 to 26  or MCI were included. All patients demonstrated a decrease in Aβ1–42 to <550 pg/ml in CSF and increases in total T-tau to >400 pg/ml or phosphorylated tau 181 to >70 pg/ml in CSF, which are indicative of prodromal or mild AD [9, 10]. The term prodromal Alzheimer’s disease is used to describe patients who have a hippocampal type of amnestic disorder and a biomarker indicative of the presence of AD, but who are not functionally impaired and do not meet the criteria for Alzheimer’s dementia [11, 12]. The definition of prodromal AD is similar to that of MCI based on the classification criteria proposed by the National Institute on Aging and Alzheimer’s Association work group .
The inclusion criteria for entry into this trial were that patients had to be in good health, determined on the basis of routine clinical laboratory test screening results, physical examinations, vital signs and 12-lead electrocardiograms (ECGs). Exclusion criteria included a history or evidence of any other CNS disorder that could be interpreted as a cause of dementia; Hachinski Ischemic Scale score >4; a magnetic resonance imaging (MRI) scan not consistent with AD or evidence of any other CNS condition; minimal vascular changes or more than three microhaemorrhagic lesions; and a history of psychiatric illness, cerebral haemorrhage, seizures or strokes during the 3 years preceding the study; and cardiovascular disease or diabetes.
Recruitment of patients into this open-label, single-dose study (protocol number BA1113043, Clinicaltrials.gov Identifier NCT01424436) was carried out from May 2010 to December 2011 at three sites in Germany and two in Sweden. Patients were screened on day −30 prior to dosing and randomised to receive doses of GSK933776. The patients were assigned to receive doses of GSK933776 in accordance with the randomisation schedule produced using RandAll, a web server–based clinical trials system. The doses of GSK933776 were 1 mg/kg, 3 mg/kg or 6 mg/kg (n = 6 per group) administered by intravenous pump infusion for 1 hour. Patients were followed for up to 56 days postdosing.
The primary endpoint was measurement of the levels and temporal changes of Aβ1–42 in CSF after administration of GSK933776 in patients with mild AD and MCI. Secondary endpoints were measurement of the levels and temporal changes following GSK933776 administration of the following: total Aβ4228–42 and Aβ18–35 in plasma and tau and phosphorylated tau 181 in CSF, the pharmacokinetics for GSK933776 in CSF and plasma, the pharmacokinetic–pharmacodynamic (PK–PD) relationship of GSK933776 in CSF and plasma and the safety and tolerability of GSK933776.
Pharmacodynamics sample collection and analysis
For the first four patients who received the lowest dose (1 mg/kg), blood and CSF samples were collected at hourly intervals over a 30-hour time period from approximately 11 hours predosing to 19 hours postdosing. The primary sampling time period for evaluation of baseline levels was 6 to 8 hours predosing, whereby 0 to 5 hours postdosing was considered a run-in period during which we obtained stable values. The time period from 0 to 12 hours postdosing was the focus for the postdosing assessment. This duration was initially based on plasma pharmacodynamics observed in our previous study, that is, the time of onset of lowering of free Aβ (manuscript in submission). After the first four patients completed the protocol, their Aβ levels were reviewed and the sample collection times for CSF and blood for both baseline and postdosing were adjusted as follows. CSF samples were collected hourly (3 ml per sample) from 9 hours predosing to 12 hours postdosing. Blood samples were collected hourly from 6 hours predosing to 12 hours postdosing and at follow-up: once on day 7 and once on day 56 postdosing. Total tau and phosphorylated tau 181 were measured in CSF at 5 hours predosing, at the time of dosing and at 5 hours and 12 hours postdosing. Because of the short-term exposure to GSK933776, no changes in total tau or phosphorylated tau 181 were expected.
Insertion of the lumbar catheter was done according to local hospital protocols. Continuous CSF collection was performed according to the standard operating procedures of the study and carried out by medical staff trained in this procedure. CSF Aβ1–42, total tau and phosphorylated tau 181 were measured using INNOTEST kits (Innogenetics/Fujirebio Europe, Ghent, Belgium). For the INNOTEST assay for Aβ1–42, we used 21F12 as a coating monoclonal antibody, which is specific for the C-terminal epitope 42, and 3D5 as a detection antibody, which is specific to N-terminal epitope 1. The INNOTEST assay for phosphorylated-tau 181 uses HT7 as a coating antibody, which is specific for amino acids 159 to 163, and AT270 as a detection antibody, which is specific to phosphorylated Thr181. All CSF analyses were performed batchwise by board-certified laboratory technicians who were blinded to clinical and treatment data. Intra-assay coefficients of variation were below 10%.
For plasma preparation, blood samples were processed within 30 minutes by centrifugation at 2,000 × g for 15 minutes at 4°C to obtain plasma. A protease inhibitor (0.02 ml; Roche Applied Science, Burgess Hill, UK) was added to 0.5 ml of plasma. Samples were then frozen upright on dry ice or in a freezer at or below −70°C. The specificities of the capture and detection antibodies used in the plasma total Aβ measurements (numbers in brackets indicate the minimal amino acid sequence detected by the plasma Aβ assay) were as follows. The combinations of the capture antibody 6F6 (amino acids 28 to 35, generated by GlaxoSmithKline (GSK)) and detection antibody 5G5 (amino acids 35 to 42; generated by GSK) detected all Aβ fragments containing an intact C terminus, such as Aβ1–42 and Aβ3–42, which is considered indicative of total Aβ4228–42. 6F6 and the detection antibody 4G8 (amino acids 18 to 22) (SIG-39220; Covance, Emeryville, CA, USA) detected Aβ fragments containing the amino acid sequence 18 to 35, including Aβ1–38, Aβ1–40 and Aβ1–42, which are considered indicative of total Aβ18–35. Total assays detected both free and GSK933776-bound Aβ fragments. There was no competition between the capture antibody 6FS and GSK933776, because 6F6 recognises Aβ amino acids 28 to 35 and GSK933776 recognises Aβ amino acids 1 to 5. Total levels of Aβ18–35 and total Aβ4228–42 were measured in plasma only.
Pharmacokinetics sample collection and analysis
Blood samples were collected once prebaseline, once during GSK933776 administration, hourly for 4 hours postdosing and then 6, 8, 10 and 12 hours postdosing as well as at follow-up 7 and 56 days postdosing. CSF samples were collected every hour over a total period of 22 hours, beginning 9 hours predosing and ending 12 hours postdosing. Approximately 0.5 ml of CSF and approximately 3 ml of whole blood were collected at every time point.
Concentrations of GSK933776 were determined in plasma and CSF samples using an immunoassay that detects free GSK933776 antibody. Briefly, the humanized monoclonal antibody drug specific to GSK933776 is captured with a Aβ fragment immobilized on a microtitre plate. The plate was washed removing all nonspecific material and GSK933776 is detected using an anti-human IgG (Fc specific) monoclonal antibody conjugated to a reporter tag which is developed and detected by immunoelectrochemiluminescence, resulting in a quantifiable response.
Pharmacokinetics analyses of plasma and CSF GSK933776 were calculated by standard noncompartmental analysis using WinNonlin Pro 5.2 software (Pharsight, Mountain View, CA, USA) and were based on actual sampling times. The following parameters were measured: the first occurrence of the maximum observed plasma and CSF concentrations determined directly from raw concentration–time data (Cmax); the time at which Cmax was observed, which was determined directly from raw concentration–time data (tmax); the area under the plasma and CSF concentration–time curve to the last quantifiable concentration (AUC0–t); the area under the plasma and CSF concentration–time curve from time 0 to 12 hours postdosing (AUC0–12); the time of the last observed plasma concentration (tlast); and the concentration at tlast (Ct). The following additional parameters were included for analysis of CSF pharmacokinetics: the areas under the CSF concentration–time curve from time 0 to 4 hours postdosing AUC0–4 and from 5 to 12 hours postdosing AUC5–12 and the time prior to the first measurable (that is, nonzero) concentration in CSF (tlag).
Clinical chemistry and haematology tests, vital signs, 12-lead ECG readings and brain MRI were performed at screening, at intervals throughout the study and at follow-up examinations. Adverse events were coded using the Medical Dictionary for Regulatory Activities (http://www.meddra.org/) criteria.
As this was an exploratory experimental medicine study, we did not perform any formal calculations of power or sample size. Sample sizes were chosen based on feasibility to allow preliminary evaluations of the safety and effects GSK933776 on Aβ and tau levels in CSF. No formal statistical analyses were performed.
CSF Aβ parameters were summarised using standard descriptive statistics for a range of summary measures (absolute, absolute change from baseline, absolute percent change from baseline, weighted mean, weighted mean change from baseline and weighted mean percent change from baseline) and are presented in individual participant plots. The weighted means were derived by calculating the AUCs using the trapezoidal rule and then dividing by the actual relevant time interval. CSF parameters (tau and phosphorylated tau 181) and plasma parameters (Aβ18–35 and Aβ4228–42) were summarised (absolute) and are presented in individual participant plots Additional file 1 and Additional file 2, respectively.
Concentrations of GSK933776 in plasma and CSF were summarised using standard summary statistics. Noncompartmental plasma pharmacokinetics parameters were summarised using standard descriptive statistics, which also included coefficients of variation, geometric means and 95% confidence intervals, for the geometric means.