A Clinical Trial of an Inhibitor of RAGE-A Interactions in Alzheimer S Disease
A clinical trial of an inhibitor of RAGE-A interactions in Alzheimer’s disease
1. Data from preclinical studies on PF04494700 (data on file at TransTech Pharma)
PF-04494700 is a novel small molecule antagonist of RAGE of MW less than 500. In a binding assay in solution, monitored by fluorescent polarization, PF-04494700 inhibited the interaction of sRAGE with its ligands (IC50 ~ 500 nM) in an ELISA format where one of the RAGE ligands, Aβ, S100b, amphoterin, or carboxymethyl-lysine (CML) is immobilized.
TTP488 readily crosses the blood-brain barrier with a brain:plasma ratio of 12:1 after a single oral dose in rats.
In transgenic mice that overexpress human APP with the London and Swedish mutations, treatment with PF-04494700 starting at 6 months, and continued for 3 months, resulted in reduction of inflammatory markers in the brain (TNF-α, TGF-β and IL-1), decreased amyloid burden measured by image analysis of immunocytochemistry, and behavioral benefits shown by reduction of latency and distance traveled in the Morris water maze. Trough plasma concentrations associated with improvement on these outcome measures ranged from 6 – 51 ng/mL.
In separate experiments, 12 month old transgenic mice expressing the London and Swedish mutations were treated for 3 months with PF-04494700 (compared to vehicle); treatment led to a decreased amyloid burden and improvement on electrophysiological indices related to long-term potentiation (LTP) for treatment relative to vehicle.
Phase 1 studies indicated that oral doses of PF-04494700 ranging from 5-60 mg/day were safe and well-tolerated in healthy volunteers. The half-life (T ½) of the drug was 13-14 days, therefore a loading dose was given in order to shorten the time to steady state in multiple dose studies. Multiple dose studies in young adults evaluated 15/5, 30/10 and 60/20 mg/day (loading dose for 6 days, then steady dosing x 21 days), Mean concentrations achieved after the 6 day load were similar to those achieved after 28 days of dosing. For these 3 dose regimens, mean trough plasma concentrations at steady state were 10, 21 and 62 ng/mL respectively. In the 10 week study in patients with mild AD (6), mean trough levels at steady state were 20 ng/mL for the 30/10 mg group and 42 ng/mL for the 60/20 mg group. These trough levels overlap with the dose levels that were efficacious in transgenic mouse models described above.
2. Detailed description of Clinical Trial outcome measures.
A. Secondary clinical measures:
1. As an index of global function, the Clinical Dementia Rating Sum of boxes (CDR-sb) (10) was administered to informants and subjects. This describes five levels of impairment (0 = normal, 0.5 questionable, 1, 2 and 3 progressively worse) related to 6 categories of cognitive functioning. Scores for each category are summed for a total that ranges from 0 (no impairment) to 18 (severe impairment).
2. The Alzheimer’s Disease Cooperative Study Activities of Daily Living (ADCS-ADL) inventory (11) is a rating scale to assess functional decline, administered by interviewing informants. Informants need to have contact with subjects for at least 3 days of the week. This questionnaire rates 24 activities by structured questions, each of which is scored according to a level of performance, and scores of items are summed, yielding a total that ranges from 0-78 points. Higher scores indicate better function, and change scores associated with worsening are negative.
3. The Neuropsychiatric Inventory (NPI) (12) is an informant interview that rates 10 defined neuropsychiatric symptoms, and typically takes about 15 minutes to administer. Informants need to have contact with subjects for at least 3 days of the week. Features that are rated include delusions, hallucinations, dysphoria, anxiety, agitation/aggression, euphoria, disinhibition, lability, apathy and aberrant motor behavior. Each feature is rated in terms of frequency (1-4, where 1 = occasionally, less than once per week, and 4 = once per day or more). Scores range from 0 (no behavioral symptoms) to 78 (maximal behavioral symptoms).
4. The Mini-Mental State Examination (MMSE) (8) is a brief overall test of cognition, which assesses memory, orientation, language, attention and constructional abilities. Scores range from 0-30.
5. A neuropsychological test battery was administered by psychometrists to study subjects as a detailed assessment of cognitive abilities, and included the following standard tests:
Digit Symbol Substitution Test (13,14): this subtest from the WAIS-R consists of 110 small blank squares (presented in seven rows) each randomly paired with one of 9 numbers (1-9) printed directly above it. Above the row of blank squares is a printed ‘key’ that pairs each of the numbers 1 through 9 with an unfamiliar symbol. Following a short series of practice trials, the participant must use the key to fill in the blank squares in order (working left to right across the rows) with the symbol that is paired with the number above it, working as quickly as possible for 90 seconds. The number of blank squares filled in correctly is scored (maximum raw score = 110).
Forward and Backward Digit Span Test (14): The Digit Span subtest from the WAIS-R requires the subject to repeat sequences of single digit numbers, which are read aloud by the examiner. In the Forward condition, the subject must repeat the digits in the forward order; in the Backward condition, the digits must be repeated in the reverse order. The lengths of digit sequences increase progressively from three to nine digits in the Forward condition, and from two to eight digits in the Backward condition, with two trials presented for each sequence length. Testing is terminate when a subject misses both trials at a given sequence length. A point is awarded for each sequence correctly produced, so the maximum score for each condition is 14 points.
Controlled Oral Word Association Test (COWAT) (15). This is a measure of verbal fluency in which the subject is asked to generate orally as many words as possible that begin with the letters ‘F’, ‘A’, and ‘S’, excluding proper names and different forms of the same word, in one minute for each letter. Performance is measured by the total number of words produced summed across all 3 letters. Perseverations and intrusions are noted.
Stroop Color Word interference Test (16): This test examines attention and response inhibition. In the original form of the task, subjects are timed for how quickly they can read words which are the names of colors (i.e., color words), name the colors of ink patches, then finally name the color of the ink in which color words are printed (e.g., say ‘red’ when the word ‘green’ is printed in red ink). The effects of response inhibition are shown by slower response times when naming the color of the ink of non-congruent words than when reading the names of colors or naming the colors of ink patches. A standardized form was used, in which there are 3 pages, each with 100 items, organized in five columns of 20. Page 1 consists of three words ‘RED’, ‘GREEN’ and ‘BLUE’ printed in black ink and randomly arranged on a white piece of paper measuring 8.5 x 11 “. Subjects are asked to read aloud, as fast as they can, down each column; if they finish all 5 columns, they start the first column again. If an error is made, the examiner says ‘no.’ After 45 seconds, the subject is stopped and the number of items correctly named is scored (word reading score). Page 2 has a similar layout, with XXXX printed in different colors in red, green or blue ink. The subject is instructed to name the color of the item, and receive a color-naming score based on 45 seconds (C score). Page 3 consists of the words RED, GREEN and BLUE, printed in the colors of the XXXX items on page 2. The subject is requested to name the color of the words in each column, and receive a score (color-word (CW)) based on 45 seconds. For this trial, a score of (C-CW)/C will be used as a summary measure.
Trail-Making Test (Parts A and B). (from the Halstead Reitan Neuropsychological Test Battery, (17)). Part A consists of 25 circles numbers 1 through 25 semi-randomly distributed over a white sheet of 8.5 x 11 inch paper. The subject is instructed to connect the circles with a pencil line as quickly as possible in numerical order. Part B also consists of 25 circles, which are either numbered 91 through 13) or contain letters (A through L). Now the subject must connect the circles while alternating between numbers and letters in ascending order (e.g.: A-1-B-2-C-3, etc.). Subjects are scored for time taken for Trails A and B. An upper limit is set of 150 seconds for Trails A and 300 seconds for Trails B.
6. The Resource Use Inventory (RUI) (18) collects information on hospitalizations, formal and informal help and hours of paid or voluntary work.
7. The DEMQOL is a standardized and validated measure of quality of life (19). It is given as an interview to caregivers (DEMQUOL-proxy) and to subjects with AD (DEMQOL).
B. Laboratory and Biomarker measurements
1. MRI scans were performed on 1.5T scanners on various platforms across 42 sites. The imaging sequence used for volumetric analyses was a 3D T1-weighted image MPRAGE or SPGR. Minor variations in TR, TE and bandwidth were allowed based on the specific hardware configuration on each scanner. The nominal parameters of the MPRAGE on all other scanners were: coronal plane, TR/TE/TI, 10/4/1000 msec, flip angle 8°, 256x256 in-plane matrix, 1.2 mm slice thickness. Only the Siemens Symphony scanners used a nominal TR of 2800 msec, all other nominal parameters were the same as above. A phantom scan was acquired prior to start of subject enrollment to qualify each scanning site for use and subsequently with each subject scan. Scanner qualification and monitoring used the ADNI phantom and its qualification routine. All images were transferred for storage and processing over a secure FTP internet transfer in DICOM-3 format or sent via CD/DVD via FedEx to CIND, under the direction of Dr. Michael Weiner.
The image processing (20-22) was performed using Freesurfer version 4.5 (http://surfer.nmr.mgh.harvard.edu/). Freesurfer is a fully automated image processing suite that provides cortical thickness, surface area and ROI volume measurements across 116 cortical and subcortical regions. Briefly, this processing includes removal of non-brain tissue using a hybrid watershed/surface deformation procedure, automated Talairach transformation, segmentation of the subcortical white matter and deep gray matter volumetric structures (including hippocampus, amygdala, caudate, putamen, ventricles), intensity normalization, tessellation of the gray matter white matter boundary, automated topology correction, and surface deformation following intensity gradients to optimally place the gray/white and gray/cerebrospinal fluid borders at the location where the greatest shift in intensity defines the transition to the other tissue class. Once the cortical models are complete, a number of deformable procedures were performed for further data processing and analysis including surface inflation, registration to a spherical atlas which utilized individual cortical folding patterns to match cortical geometry across subjects' parcellation of the cerebral cortex into units based on gyral and sulcal structure, and creation of a variety of surface based data including maps of curvature and sulcal depth.
2. Lumbar punctures were performed on subjects who consented. A minimum of 6 mL of CSF was required, and samples were sent to a local laboratory for cell count, protein and glucose measures, and a minimum of 4 mL was divided into 0.5 mL aliquots in polypropylene cryotubes, and shipped to a central laboratory, where it was frozen and stored at -70 degrees until assayed.
3. APO-E genotyping
APOE genotypes were determined at Pfizer, Inc., using standard PCR methods.
4. CSF and plasma biomarker and pK measurements:
Levels of A-beta 1-x, 1-40 and 1-42 in CSF and A-beta 1-40 and 1-x in plasma were measured by ICON Development Solutions, Inc, Manchester, UK, using well-characterized monoclonal antibodies against A-beta sandwich-type immunoassays performed on Gyrolab platform (Gyros AB, Uppsalla, Sweden). Tau and P-tau were measured using the INNOBIA AlzBio 3 kits (Innogenetics, Belgium) on the Luminex XMap multiplex platform, following the manufacturer’s protocol. Plasma levels of A-beta 1-42 were measured using a sandwich-type assay immunoassay performed on MesoScale Discovery (MSD) platform. Plasma levels of PF04494700 were determined using a LC/MS assay, with calibration standards and internal quality control standards within each run.